The monokine, cachectin/tumor necrosis factor (TNF) differs from interleukin 1 (IL-1) in primary structure and in recognition by a distinct cellular receptor.It does, however, encode effector functions that are similar to those of IL-1 and characteristic of the host response to inflammation or tissue injury.Accordingly, we examined the possibility that recombinant-generated human TNF regulates hepatic acute-phase gene expression.In picomolar concentrations, TNF mediated reversible, dose-and time-de- pendent increases in biosynthesis of complement proteins factor B and C3, a, antichymotrypsin, and decreases in biosynthesis of albumin and transferrin in human hepatoma cell lines (Hep G2, Hep 3B).Biosynthesis of complement proteins C2 and C4, and a, proteinase inhibitor were not affected by TNF.TNF also increased factor B gene expression, but had no effect on C2 gene expression, in murine fibroblasts transfected with cosmid DNA bearing the human C2 and factor B genes.The effect of TNF on acute-phase protein expression (C3, factor B, albumin) was pre- translational as shown by changes in specific messenger RNA content.

In the classical form of alpha1-antitrypsin (AT) deficiency, a point mutation in AT alters the folding of a liver-derived secretory glycoprotein and renders it aggregation-prone. In addition to decreased serum concentrations of AT, the disorder is characterized by accumulation of the mutant alpha1-antitrypsin Z (ATZ) variant inside cells, causing hepatic fibrosis and/or carcinogenesis by a gain-of-toxic function mechanism. The proteasomal and autophagic pathways are known to mediate degradation of ATZ. Here we show that the autophagy-enhancing drug carbamazepine (CBZ) decreased the hepatic load of ATZ and hepatic fibrosis in a mouse model of AT deficiency-associated liver disease. These results provide a basis for testing CBZ, which has an extensive clinical safety profile, in patients with AT deficiency and also provide a proof of principle for therapeutic use of autophagy enhancers.

In alpha1-AT deficiency, a misfolded but functionally active mutant alpha1-ATZ (alpha1-ATZ) molecule is retained in the endoplasmic reticulum of liver cells rather than secreted into the blood and body fluids. Emphysema is thought to be caused by the lack of circulating alpha1-AT to inhibit neutrophil elastase in the lung. Liver injury is thought to be caused by the hepatotoxic effects of the retained alpha1-ATZ. In this study, we show that several "chemical chaperones," which have been shown to reverse the cellular mislocalization or misfolding of other mutant plasma membrane, nuclear, and cytoplasmic proteins, mediate increased secretion of alpha1-ATZ. In particular, 4-phenylbutyric acid (PBA) mediated a marked increase in secretion of functionally active alpha1-ATZ in a model cell culture system. Moreover, oral administration of PBA was well tolerated by PiZ mice (transgenic for the human alpha1-ATZ gene) and consistently mediated an increase in blood levels of human alpha1-AT reaching 20-50% of the levels present in PiM mice and normal humans. Because clinical studies have suggested that only partial correction is needed for prevention of both liver and lung injury in alpha1-AT deficiency and PBA has been used safely in humans, it constitutes an excellent candidate for chemoprophylaxis of target organ injury in alpha1-AT deficiency.

Autophagy has emerged as a critical lysosomal pathway that maintains cell function and survival through the degradation of cellular components such as organelles and proteins. Investigations specifically employing the liver or hepatocytes as experimental models have contributed significantly to our current knowledge of autophagic regulation and function. The diverse cellular functions of autophagy, along with unique features of the liver and its principal cell type the hepatocyte, suggest that the liver is highly dependent on autophagy for both normal function and to prevent the development of disease states. However, instances have also been identified in which autophagy promotes pathological changes such as the development of hepatic fibrosis. Considerable evidence has accumulated that alterations in autophagy are an underlying mechanism of a number of common hepatic diseases including toxin-, drug- and ischemia/reperfusion-induced liver injury, fatty liver, viral hepatitis and hepatocellular carcinoma. This review summarizes recent advances in understanding the roles that autophagy plays in normal hepatic physiology and pathophysiology with the intent of furthering the development of autophagy-based therapies for human liver diseases.

Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRΔF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein α1-antitrypsin (α1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of α1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of α1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRΔF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, α1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal α1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with α1-ATZ. Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRΔF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein α1-antitrypsin (α1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of α1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of α1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRΔF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, α1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal α1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with α1-ATZ.

Caenorhabditis elegans has been proven to be a useful model organism for investigating molecular and cellular aspects of numerous human diseases. More recently, investigators have explored the use of this organism as a tool for drug discovery. Although earlier drug screens were labor-intensive and low in throughput, recent advances in high-throughput liquid workflows, imaging platforms and data analysis software have made C. elegans a viable option for automated high-throughput drug screens. This review will outline the evolution of C. elegans-based drug screening, discuss the inherent challenges of using C. elegans, and highlight recent technological advances that have paved the way for future drug screens.

Biliary atresia (BA) is the end result of a destructive, inflammatory process that affects intra- and extrahepatic bile ducts, leading to fibrosis and obliteration of the biliary tract with the development of biliary cirrhosis. It is the commonest cause of chronic cholestasis in infants and children, and therefore is the most frequent indication for liver transplantation in this age group. The disease occurs worldwide, affecting an estimated 1 in 8,000 to 12,000 live births. At present, there is no specific therapy for BA; however, sequential surgical therapy begins with creation of a hepatoportoenterostomy (HPE); in those with end-stage liver disease, liver transplantation is indicated. Since most candidates are young children of small size, there is a shortage of size-matched donors for liver transplantation. At present, an increased awareness to ensure early diagnosis and development of methods to prevent progressive fibrosis are needed. These considerations are dependent on detailed studies of the pathogenesis of BA. Recent studies have focused on normal and altered bile duct morphogenesis and the role of various factors (infectious or toxic agents and metabolic insults) in isolation or in combination with a genetic or immunologic susceptibility in the etiology of BA.

Liver injury in PiZZ alpha 1-antitrypsin (alpha 1-AT) deficiency probably results from toxic effects of the abnormal alpha 1-AT molecule accumulating within the ER of liver cells. However, only 12-15% of individuals with this same genotype develops liver disease. Therefore, we predicted that other genetic traits that determine the net intracellular accumulation of the mutant alpha 1-AT molecule would also determine susceptibility to liver disease. To address this prediction, we transduced skin fibroblasts from PiZZ individuals with liver disease or without liver disease with amphotropic recombinant retroviral particles designed for constitutive expression of the mutant alpha 1-AT Z gene. Human skin fibroblasts do not express the endogenous alpha 1-AT gene but presumably express other genes involved in postsynthetic processing of secretory proteins. The results show that expression of human alpha 1-AT gene was conferred on each fibroblast cell line. Compared to the same cell line transduced with the wild-type alpha 1-AT M gene, there was selective intracellular accumulation of the mutant alpha 1-AT Z protein in each case. However, there was a marked delay in degradation of the mutant alpha 1-AT Z protein after it accumulated in the fibroblasts from ZZ individuals with liver disease ("susceptible hosts") as compared to those without liver disease ("protected hosts"). Appropriate disease controls showed that the lag in degradation in susceptible hosts is specific for the combination of PiZZ phenotype and liver disease. Biochemical characteristics of alpha 1-AT Z degradation in the protected hosts were found to be similar to those of a common ER degradation pathway previously described in model experimental cell systems for T-cell receptor alpha subunits and asialoglycoprotein receptor subunits, therefore, raising the possibility that the lag in degradation in the susceptible host is a defect in this common ER degradation pathway. Thus, these data provide evidence that other genetic traits that affect the fate of the abnormal alpha 1-AT Z molecule, at least in part, determine susceptibility to liver disease. These data also validate a system for elucidating the biochemical/genetic characteristics of these traits and for examining the relevance to human disease of pathways for protein degradation in the ER.

During the host response to inflammation/tissue injury there are many changes in intermediary metabolism including a dramatic change in the concentrations of many plasma proteins. Although many of these acute phase proteins are predominantly derived from the liver and the response can be elicited from liver cells incubated in tissue culture with cytokines such as interleukin-6 (IL-6), interleukin-1 (IL-1), tumor necrosis factor-alpha, interferon-gamma, leukemia inhibitory factor, interleukin-11 (IL-11), and oncostatin M, there is now evidence that the response can also be elicited in extrahepatic tissues and cell types. In this study, we show that many of the acute phase plasma proteins are expressed in human intestinal epithelial cell lines Caco2 and T84 and that their expression is induced or regulated by cytokines IL-6, IL-1, interferon, and tumor necrosis factor in a manner characteristic of the acute phase response. In fact, effects of IL-1 and IL-6 which are additive, synergistic, and antagonistic in liver cell lines are also observed in these intestinal epithelial cell lines. Responses to IL-6 and IL-1 are seen at all stages of differentiation of Caco2 cells from crypt-like enterocytes to villus-like enterocytes. Caco2 cells express binding sites for IL-6 at both poles, for IL-1 at the basolateral pole and, to a lesser extent, at the apical pole. T84 cells have IL-1 and IL-6 receptor binding sites only at the basolateral pole. IL-6 and IL-1 also regulate the expression of enterocyte-specific integral membrane proteins as exemplified by down-regulation of sucrase-isomaltase gene expression in response to IL-6. These data raise the possibility that enterocytes are involved in a local response to injury/inflammation at the epithelial surface and establish a model system for examining coordination of the acute phase response in a bipolar cell.

Mutant α1-antitrypsin Z (α1-ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary α1-antitrypsin (α1-AT) deficiency. Previous studies have suggested that efficient intracellular degradation of α1-ATZ is correlated with protection from liver disease in α1-AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of α1-ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of α1-AT deficiency. To provide genetic evidence for autophagy-mediated disposal of α1-ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of α1-ATZ was retarded, and the characteristic cellular inclusions of α1-ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and α1-ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of α1-ATZ mated to the GFP-LC3 mouse, we also found that expression of α1-ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for α1-ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of α1-ATZ. Mutant α1-antitrypsin Z (α1-ATZ) protein, which has a tendency to form aggregated polymers as it accumulates within the endoplasmic reticulum of the liver cells, is associated with the development of chronic liver injury and hepatocellular carcinoma in hereditary α1-antitrypsin (α1-AT) deficiency. Previous studies have suggested that efficient intracellular degradation of α1-ATZ is correlated with protection from liver disease in α1-AT deficiency and that the ubiquitin-proteasome system accounts for a major route, but not the sole route, of α1-ATZ disposal. Yet another intracellular degradation system, autophagy, has also been implicated in the pathophysiology of α1-AT deficiency. To provide genetic evidence for autophagy-mediated disposal of α1-ATZ, here we used cell lines deleted for the Atg5 gene that is necessary for initiation of autophagy. In the absence of autophagy, the degradation of α1-ATZ was retarded, and the characteristic cellular inclusions of α1-ATZ accumulated. In wild-type cells, colocalization of the autophagosomal membrane marker GFP-LC3 and α1-ATZ was observed, and this colocalization was enhanced when clearance of autophagosomes was prevented by inhibiting fusion between autophagosome and lysosome. By using a transgenic mouse with liver-specific inducible expression of α1-ATZ mated to the GFP-LC3 mouse, we also found that expression of α1-ATZ in the liver in vivo is sufficient to induce autophagy. These data provide definitive evidence that autophagy can participate in the quality control/degradative pathway for α1-ATZ and suggest that autophagic degradation plays a fundamental role in preventing toxic accumulation of α1-ATZ. Human α1-antitrypsin (α1-AT), 3The abbreviations used are: α1-AT, α1-antitrypsin; α1-ATZ, mutant α1-antitrypsin Z; BSA, bovine serum albumin; Me2SO, dimethyl sulfoxide; ER, endoplasmic reticulum; ES, embryonic stem; EYFP, enhanced yellow fluorescent protein; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; GFP, green fluorescent protein.3The abbreviations used are: α1-AT, α1-antitrypsin; α1-ATZ, mutant α1-antitrypsin Z; BSA, bovine serum albumin; Me2SO, dimethyl sulfoxide; ER, endoplasmic reticulum; ES, embryonic stem; EYFP, enhanced yellow fluorescent protein; MEF, mouse embryonic fibroblast; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; GFP, green fluorescent protein. a monomeric 394-amino acid glycoprotein, is synthesized and secreted primarily by liver cells. It is a prototypic member of serine protease inhibitor (serpin) superfamily proteins and the most abundant of the circulating serpins. The principal role of α1-AT in serum is to protect lung tissues from destructive proteases (elastase, cathepsin G, and proteinase 3) released by neutrophils during inflammation. Some genetic alterations in α1-AT are responsible for defective secretion and thus cause serum α1-AT deficiency (1.Teckman J.H. Qu D. Perlmutter D.H. Hepatology. 1996; 24: 1504-1516PubMed Google Scholar, 2.Lomas D.A. Mahadeva R. J. Clin. Investig. 2002; 110: 1585-1590Crossref PubMed Scopus (235) Google Scholar, 3.Perlmutter D.H. J. Clin. Investig. 2002; 110: 1579-1583Crossref PubMed Scopus (156) Google Scholar). The most common causal mutation found in Caucasian populations is the replacement of Glu-342 by Lys that characterizes the Z mutant of α1-AT (α1-ATZ). This substitution is sufficient to cause an abnormality in folding early in the secretory pathway with retention of the mutant α1-ATZ molecule in the ER of liver cells. Homozygotes for the α1-ATZ mutation (PIZZ) are characterized by serum levels of α1-AT that are ∼10–15% of those in the general population and are susceptible to two major target organ injuries. Destructive lung disease/emphysema in adults is due to a loss-of-function mechanism. Chronic liver disease often first discovered in childhood, but also affecting adults, is due to a gain-of-toxic-function mechanism in which liver cell injury results from the hepatotoxic effects of retained α1-ATZ. However, only 8–10% of homozygotes develop clinically significant liver disease. This observation has led to the concept that mechanisms by which cells respond to the ER retention of mutant α1-ATZ play a role in determining which of these homozygotes develop liver disease and which are protected from it. Because previous studies have shown that a reduction in α1-ATZ disposal activity correlates with the presence of liver disease among deficient individuals (4.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar), the mechanisms by which α1-ATZ is degraded are thought to be particularly important in determining the liver disease phenotype of patients with α1-AT deficiency.A number of studies have addressed the determinants of the cellular fate of α1-ATZ, including retention in the ER and disposal by the quality control/degradative pathways of the ER. Seminal works by Lomas and co-workers (2.Lomas D.A. Mahadeva R. J. Clin. Investig. 2002; 110: 1585-1590Crossref PubMed Scopus (235) Google Scholar, 5.Sivasothy P. Dafforn T.R. Gettins P.G. Lomas D.A. J. Biol. Chem. 2000; 275: 33663-33668Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) have shown how the Z mutation confers an unstable polymerogenic intermediate conformation on α1-AT so that polymerization is promoted by a reactive loop:β-sheet A linkage reminiscent of the inhibitory interaction between serpins and cognate proteases. Recent studies of the ER-associated degradation system revealed that immature or misfolded glycoproteins are captured by ER chaperone proteins calnexin/calreticulin via the terminal glucose residue on asparagine-linked oligosaccharide side chains, which is reciprocally added or trimmed by UDP-glucose:glycoprotein glucosyltransferase or glucosidase II, respectively, according to the folding status of the glycoprotein (6.Yoshida Y. J. Biochem. (Tokyo). 2003; 134: 183-190Crossref PubMed Scopus (52) Google Scholar, 7.Ellgaard L. Helenius A. Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1656) Google Scholar). Indeed, a stoichiometric interaction was observed between α1-ATZ and calnexin (4.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar, 8.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Terminally misfolded proteins are translocated from ER lumen to the cytoplasm and consequently degraded by the ubiquitin-proteasome system putatively deployed at the cytoplasmic face of ER.Detailed studies of proteasome-mediated α1-ATZ disposal, including cell-free assays using ER-derived microsomes, have suggested the existence of one disposal pathway in which the α1-ATZ-calnexin complex is ubiquitinated on calnexin and subsequently degraded (8.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 9.Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar, 10.Teckman J.H. Burrows J. Hidvegi T. Schmidt B. Hale P.D. Perlmutter D.H. J. Biol. Chem. 2001; 276: 44865-44872Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). It is conceivable that the asparagine-linked Glc1Man8GlcNAc2 on α1-ATZ determines the proteasomal degradation pathway via its physical interaction with calnexin and that diversion to other degradation pathways is brought about by further mannose trimming of the oligosaccharide chain during ER retention (11.Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). An unknown protease activity sensitive to tyrosine phosphatase inhibitors has also been reported as a potential mechanism for nonproteasomal intramicrosomal degradation of α1-ATZ (11.Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar).Autophagy (synonymously used here as macroautophagy) is a major intracellular degradation pathway mediated by proteins of the evolutionarily conserved Atg family unique to this function (12.Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1263) Google Scholar, 13.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar, 15.Mizushima N. Yoshimori T. Ohsumi Y. Int. J. Biochem. Cell Biol. 2003; 35: 553-561Crossref PubMed Scopus (96) Google Scholar, 16.Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1096) Google Scholar, 17.Yoshimori T. Biochem. Biophys. Res. Commun. 2004; 313: 453-458Crossref PubMed Scopus (457) Google Scholar, 18.Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar). Autophagy is characterized by bulk sequestration of cytoplasmic constituents within a double-membrane-bound vesicle, called an autophagosome, and their subsequent degradation upon fusion of this vesicle with lysosome. This process accounts for a major portion of the cellular turnover of long lived proteins and organelles such as ER and mitochondria. In addition to constitutive bulk turnover at steady state, autophagic sequestration is induced by specific physiological perturbations, such as nutrient deprivation. Several lines of evidence have implicated autophagy as a physiological response to cope with accumulation of α1-ATZ in the ER in vivo (19.Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar, 20.Teckman J.H. An J.K. Loethen S. Perlmutter D.H. Am. J. Physiol. 2002; 283: G1156-G1165Crossref PubMed Scopus (77) Google Scholar, 21.Teckman J.H. An J.K. Blomenkamp K. Schmidt B. Perlmutter D. Am. J. Physiol. 2004; 286: G851-G862Crossref PubMed Scopus (172) Google Scholar). The liver lesion in PIZZ patients as well as in the PiZ mouse model of α1-AT deficiency is accompanied by a marked autophagic response as determined by ultrastructural studies. When cells engineered to express α1-ATZ were treated with 3-methyladenine, an inhibitor of autophagy, the degradation of α1-ATZ was attenuated (19.Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar).Traditional methods for monitoring autophagy by means of ultrastructural criteria or the use of chemical inhibitors can be criticized because it is sometimes difficult to discriminate autophagic vacuoles from other organelles especially in degenerating cells, and because 3-methyladenine, a relatively nonspecific inhibitor of autophagy, could potentially suppress degradation pathways other than autophagy (14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar). Here we tried to provide molecular evidence for autophagy-mediated disposal of α1-ATZ by two approaches. First, we used cell lines deleted for the Atg5 gene, a target molecule for the ubiquitin-like Atg12 conjugation that is necessary for the initial steps of autophagic sequestration (12.Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1263) Google Scholar). Second, we used GFP-LC3, a defined marker for autophagosome membrane (13.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 22.Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1892) Google Scholar), for colocalization studies in cell lines and in the liver of novel mouse models of α1-AT deficiency.EXPERIMENTAL PROCEDURESExpression Plasmids, Antibodies, and Reagents—The cDNAs corresponding to α1-ATZ, Atg5, Atg5K130R, and EYFP were subcloned into pCE-neo plasmid. This plasmid is a modified version of pCI-neo (Promega) in which the promoter region was replaced with a fragment containing the cytomegalovirus enhancer and elongation factor promoter of pCE-FL plasmid (a kind gift from Dr. Sumio Sugano). Proteasome sensor vector pcDEF-Ub-G76V-EGFP (23.Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (465) Google Scholar) was kindly provided by Dr. Shigeo Murata (Tokyo Metropolitan Institute of Medical Science). The coding sequence for hemagglutinin-tagged canine Rab7 T22N was inserted into pcDNA3 (Invitrogen). The following antibodies were used: rabbit polyclonal anti-rat LC3 (13.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar); anti-human Atg5 (14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar); anti-α1-AT (DAKO); anti-GFP (Invitrogen); goat polyclonal anti-α1-AT (Research Diagnostics, Inc.); mouse monoclonal anti-α-tubulin (clone B5-1-2; Sigma); and anti-KDEL (clone 10C3; Stressgen SPA-827). Lactacystin (Peptide Institute, Inc.) was prepared as 1 mm stock in distilled water; and MG115, MG132, and epoxomicin (Peptide Institute, Inc.) were prepared as 10 mm stock in Me2SO. Bafilomycin A1 was prepared as 250 μm stock in Me2SO.Cell Culture and Transfection—Wild-type and Atg5-/- ES cells were described previously (14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar). Atg5-/- mouse embryonic fibroblast (MEF) cells were established (24.Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2362) Google Scholar) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mm l-glutamine, and antibiotics in 5% CO2 incubator at 37 °C. Wild-type and Atg5-/- MEF cells were engineered for stable expression of α1-ATZ, Ub-G76V-EGFP, and GFP-LC3, exactly as described previously (25.Lin L. Schmidt B. Teckman J. Perlmutter D.H. J. Biol. Chem. 2001; 276: 33893-33898Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Transient transfection was carried out using Lipofectamine2000 reagent (Invitrogen) according to the manufacturer's protocol.Pulse-Chase Experiments—Cell lines engineered for expression of α1-ATZ either by transient or stable transfection were subjected to pulse-chase studies. The transiently transfected cell lines were studied 24 h after transfection. Separate monolayers were incubated in serum- and methionine/cysteine-free medium for 1 h at 37 °C followed by pulse labeling with 150 μCi/ml of 35S-labeled EasyTag Express protein labeling mix (NEG-772; PerkinElmer Life Sciences) for 2 h at 37°C. Cells were then rinsed with chase medium and chased for several different time periods. Cells were lysed in lysis buffer containing 50 mm Tris-HCl (pH 7.5), 1% Triton X-100, 3 mg/ml BSA, 1 mm PMSF, and protease inhibitor mixture (Roche Applied Science). Cell lysates were subjected to immunoprecipitation using anti-α1-AT polyclonal antibody and protein G-Sepharose 4FF (Amersham Biosciences), followed by analysis with SDS-PAGE (10% gel) and autoradiography using LAS-3000 bioimage analyzer (Fuji Film).Western Blotting—Cells were collected, rinsed with PBS, and lysed in PBS containing 1% Triton X-100, 1 mm PMSF, and protease inhibitor mixture (Roche Applied Science) on ice for 30 min. Triton X-100-soluble and -insoluble fractions were obtained by centrifuging cell lysates at 15,000 rpm for 10 min at 4 °C. Alternatively, cells were rinsed with PBS and directly lysed in SDS sample buffer. Samples were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk in 0.1% Tween 20/Tris-buffered saline and then incubated with primary antibodies. Immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies (The Jackson Laboratories) and luminol solution (1.25 mm luminol, 65 mm Tris-HCl (pH 8.0), 0.2 mm coumaric acid, 0.01% H2O2).Endoglycosidase-H Digestion—Cell lysates were subjected to immunoprecipitation using goat anti-α1-AT polyclonal antibody and protein G-Sepharose 4FF (Amersham Biosciences). Immunoprecipitates were boiled in 1% SDS, 1% 2-mercaptoethanol, and supernatants were added to the reaction containing 50 mm sodium citrate pH 5.0, 1% Triton X-100, 1 mm PMSF, 10 μm pepstatin A, and 0.25 units/ml endoglycosidase-H (Seikagaku Kogyo, Inc.). The reaction was carried out at 37 °C for 16 h.Immunofluorescence Microscopy and Flow Cytometry—For immunofluorescence microscopy, cells cultured on coverslips were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, and blocked with 3% BSA/PBS. Primary antibodies were diluted 1:100, and secondary antibodies were diluted 1:200 in 1% BSA, 0.1% Triton X-100/PBS. Coverslips were successively incubated with primary antibodies, Alexa-conjugated secondary antibodies (Invitrogen), and 1 μg/ml Hoechst 33342/PBS (Sigma) with intervening washes with PBS. Samples were examined by using Olympus FV1000 confocal microscopy. For flow cytometry, cells were collected, rinsed with PBS, and fixed with 4% paraformaldehyde/PBS. Cells in suspension were stained using anti-α1-AT polyclonal antibody and Alexa 488-conjugated secondary antibody as in immunofluorescence microscopy samples and analyzed by BD FACScan.Mice—GFP-LC3 transgenic mice were described previously (22.Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1892) Google Scholar). Z mice, in which expression of human α1-ATZ is induced only in liver parenchymal cells upon removal of doxycycline, were produced by using the Tet-Off gene expression system and TALap 2 mice (26.Hidvegi T. Schmidt B.Z. Hale P. Perlmutter D.H. J. Biol. Chem. 2005; 280: 39002-39015Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). These mice were crossed to produce Z × GFP-LC3 mice. PiZ mice with constitutive expression of α1-ATZ (27.Rudnick D.A. Liao Y. An J.K. Muglia L.J. Perlmutter D.H. Teckman J.H. Hepatology. 2004; 39: 1048-1055Crossref PubMed Scopus (111) Google Scholar) were also crossed to produce PiZ × GFP-LC3 mice. Liver sections were viewed by confocal microscopy. Quantitative morphometry was carried out by counting green vacuoles in 10 cells with many red globules and 10 cells with few or no red globules in three random areas of the liver each from two different liver sections. The results were analyzed using the MetaMorph software program.RESULTSThe Degradation Rate of α1-ATZ Is Attenuated in Atg5-/- Cells—A previous study demonstrated that disruption of the Atg5 gene in an ES cell line resulted in complete abrogation of autophagy, as confirmed by both morphological and biochemical analyses (14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar). To investigate whether α1-ATZ is degraded via the autophagic pathway, we first carried out pulse-chase analysis in wild-type and Atg5-/- ES cells transiently transfected with the α1-ATZ expression plasmid. Twenty four hours after transfection, cells were metabolically radiolabeled for 2 h with [35S]methionine/cysteine and chased for 0, 4, 6, and 8 h. Cell lysates were subjected to immunoprecipitation using anti-α1-AT antibody, and immunoprecipitated samples were resolved by SDS-PAGE followed by autoradiography. A semi-logarithmic plot of α1-ATZ-specific signals against time indicated that there was more than a 2-fold decrease in the degradation rate of α1-ATZ in Atg5-/- cells in comparison with that in wild-type cells (Fig. 1A). Cell culture fluid from these cells was also subjected to immunoprecipitation and autoradiography at the same time, but secreted α1-ATZ was barely detectable in either wild-type or Atg5-/- cells (data not shown), indicating that secretion is not the cause for more rapid disappearance of α1-ATZ in wild-type cells. The intracellular half-life of α1-ATZ was calculated as 122 min (r2 = 0.99) and 274 min (r2 = 0.97) in wild-type and Atg5-/- cells, respectively. The value in wild-type ES cells is well within the range of values that have been described previously in transfected fibroblasts and hepatoma cell lines (8.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 10.Teckman J.H. Burrows J. Hidvegi T. Schmidt B. Hale P.D. Perlmutter D.H. J. Biol. Chem. 2001; 276: 44865-44872Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Next, we examined the fate of α1-ATZ by pulse-chase experiments in wild-type and Atg5-/- MEF cells engineered for stable expression of α1-ATZ (Fig. 1B). The results show that there is a delay in the disappearance of α1-ATZ in the Atg5-/- cells compared with the wild-type MEFs. The difference in degradation of 52-kDa precursor α1-ATZ in the presence and absence of autophagic activity in stable transfected MEFs was almost identical to that in transiently transfected ES cells. A trace amount of mature 55-kDa α1-ATZ was secreted into extracellular fluid. Most interestingly, the abrogation of autophagy was associated with a slight increase in the secretion of α1-ATZ in these cell lines, the significance of which is not yet known.To compare the relative contribution of autophagy and the proteasomal pathway in α1-ATZ degradation in these cells, we performed pulse-chase analysis in the presence of various proteasome inhibitors. Twenty four hours after transient transfection with the α1-ATZ expression plasmid, cells were preincubated with Met/Cys-free medium for 1 h, metabolically labeled for 2 h, and chased for 4 h in the presence of proteasome inhibitors MG115 (20 μm), epoxomicin (10 μm), lactacystin (10 or 30 μm), or vehicle control. The results show that degradation of α1-ATZ was inhibited by all of the proteasome inhibitors in the wild-type cells but not in the Atg5-/- cells. The α1-ATZ degradation rate in wild-type cells treated with proteasome inhibitor was almost same as that in Atg5-/- cells treated with vehicle control (Fig. 1C), indicating that the inhibitory effects of autophagy and the proteasomal pathway were nearly equivalent in these experiments. To exclude the possibility that delayed α1-ATZ degradation in Atg5-/- cells is secondary to proteasome inhibition in these cells, we analyzed proteasome activity by using Ub-G76V-EGFP, a model substrate for the ubiquitin-fusion degradation pathway (23.Dantuma N.P. Lindsten K. Glas R. Jellne M. Masucci M.G. Nat. Biotechnol. 2000; 18: 538-543Crossref PubMed Scopus (465) Google Scholar). Wild-type and Atg5-/- MEF cell lines were engineered for stable expression of Ub-G76V-EGFP and then the cell lines were subjected to flow cytometric analysis for the fluorescent signal. The results showed that there were no differences between the two cell lines in the absence or presence of MG132. Furthermore, transient transfection of the α1-ATZ expression plasmid had no effect on the levels of the proteasomal substrate (Fig. 1D). There was also no difference in the level of polyubiquitinated proteins in the two cell lines as determined by Western blot analysis for ubiquitin (data not shown). These data indicate that expression of α1-ATZ does not inhibit proteasomal activity and furthermore that inhibition of proteasomal activity cannot be an explanation for the delayed degradation in the Atg5-deficient background. Thus, the results of Fig. 1 provide definitive evidence that autophagy can contribute to degradation of α1-ATZ.α1-ATZ Accumulation in Atg5-/- Cells Is Augmented Over Time—Next, we examined steady-state levels of α1-ATZ in wild-type and Atg5-/- cells (Fig. 2A). The results show a significant increase in levels of α1-ATZ in the Atg5-/- cells. α1-ATZ degradation in Atg5-/- cells was restored by cotransfection of wild-type Atg5 but not mutant Atg5K130R (14.Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar), indicating that α1-ATZ disposal requires functional Atg5 that is covalently modified by Atg12 to form autophagosomes and to promote the conversion of LC3-I to faster migrating LC3-II (13.Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 16.Kabeya Y. Mizushima N. Yamamoto A. Oshitani-Okamoto S. Ohsumi Y. Yoshimori T. J. Cell Sci. 2004; 117: 2805-2812Crossref PubMed Scopus (1096) Google Scholar) (Fig. 2A). To examine whether the degradation of any exogenously expressed protein would be inhibited in the autophagy-deficient background, we employed EYFP as a control cytosolic protein. When α1-ATZ and EYFP were cotransfected in wild-type and Atg5-/- cells, α1-ATZ accumulated in Atg5-/- cells, but there was no difference in the amounts of EYFP in wild-type as compared with Atg5-/- cells (Fig. 2B). At least a portion of the increased α1-ATZ levels was found in Triton X-100-insoluble fractions, recapitulating the previous observations that polymerogenic α1-ATZ forms detergent-insoluble aggregates (10.Teckman J.H. Burrows J. Hidvegi T. Schmidt B. Hale P.D. Perlmutter D.H. J. Biol. Chem. 2001; 276: 44865-44872Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 25.Lin L. Schmidt B. Teckman J. Perlmutter D.H. J. Biol. Chem. 2001; 276: 33893-33898Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 28.Le A. Ferrell G.A. Dishon D.S. Le Q.Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar) (Fig. 2B).FIGURE 2α1-ATZ accumulation in Atg5-/- cells relative to that in wild-type (WT) cells is augmented over time. A, wild-type and Atg5-/- ES cells were lysed for Western blot analysis 24 h after transfection withα1-ATZ expression plasmid together with mock vector (+Vec), expression plasmid for wild-type Atg5 (+Atg5), or Atg5K130R (+KR). B, wild-type and Atg5-/- ES cells were lysed 24 h after transfection with expression plasmids for α1-ATZ and EYFP. Cells were lysed in 1% Triton X-100-containing lysis buffer, and insoluble materials were lysed into SDS sample buffer. C, wild-type and Atg5-/- MEF cells were directly lysed in SDS sample buffer for Western blot analysis 24, 48, and 72 h after transient transfection with the α1-ATZ expression plasmid. Equal sample loading was confirmed by Coomassie staining of the blot (not shown). The bar graph in the lower panel shows the relative density units of α1-ATZ-reactive bands from the upper panel. D, Atg5-/- MEF cells were transiently transfected with α1-ATZ expression plasmid, and 24 h later cells were lysed for immunoprecipitation (IP) followed by incubation at 37 °C for 16 h with endoglycosidase-H (Endo-H). WB, Western blot. E, wild-type and Atg5-/- MEF cells were transiently transfected with α1-ATZ, fixed, and stained using anti-α1-AT followed by flow cytometric analysis. For control, mock-transfected cells were processed and analyzed in an

Although there is evidence for specific subcellular morphological alterations in response to accumulation of misfolded proteins in the endoplasmic reticulum (ER), it is not clear whether these morphological changes are stereotypical or if they depend on the specific misfolded protein retained. This issue may be particularly important for mutant secretory protein α 1 -antitrypsin (α 1 AT) Z because retention of this mutant protein in the ER can cause severe target organ injury, the chronic hepatitis/hepatocellular carcinoma associated with α 1 AT deficiency. Here we examined the morphological changes that occur in human fibroblasts engineered for expression and ER retention of mutant α 1 ATZ and in human liver from three α 1 AT-deficient patients. In addition to marked expansion and dilatation of ER, there was an intense autophagic response. Mutant α 1 ATZ molecules were detected in autophagosomes by immune electron microscopy, and intracellular degradation of α 1 ATZ was partially reduced by chemical inhibitors of autophagy. In contrast to mutant CFTRΔF508, expression of mutant α 1 ATZ in heterologous cells did not result in the formation of aggresomes. These results show that ER retention of mutant α 1 ATZ is associated with a marked autophagic response and raise the possibility that autophagy represents a mechanism by which liver of α 1 AT-deficient patients attempts to protect itself from injury and carcinogenesis.

In ␣ 1 -antitrypsin (␣1AT) deficiency, a polymerogenic mutant form of the secretory glycoprotein ␣1AT, ␣1ATZ, is retained in the endoplasmic reticulum (ER) of liver cells.It is not yet known how this results in liver injury in a subgroup of deficient individuals and how the remainder of deficient individuals escapes liver disease.One possible explanation is that the "susceptible" subgroup is unable to mount the appropriate protective cellular responses.Here we examined the effect of mutant ␣1ATZ on several potential protective signaling pathways by using cell lines with inducible expression of mutant ␣1AT as well as liver from transgenic mice with liver-specific inducible expression of mutant ␣1AT.The results show that ER retention of polymerogenic mutant ␣1ATZ does not result in an unfolded protein response (UPR).The UPR can be induced in the presence of ␣1ATZ by tunicamycin excluding the possibility that the pathway has been disabled.In striking contrast, ER retention of nonpolymerogenic ␣1AT mutants does induce the UPR.These results indicate that the machinery responsible for activation of the UPR can distinguish the physical characteristics of proteins that accumulate in the ER in such a way that it can respond to misfolded but not relatively ordered polymeric structures.Accumulation of mutant ␣1ATZ does activate specific signaling pathways, including caspase-12 in mouse, caspase-4 in human, NFB, and BAP31, a profile that was distinct from that activated by nonpolymerogenic ␣1AT mutants.

Chemical Chaperones: A Pharmacological Strategy for Disorders of Protein Folding and Trafficking

Abstract The small-complement C5 activation fragment, C5a, is a potent phlogistic molecule that, on binding to the C5a Receptor (C5aR), mediates contraction of smooth muscle, enhances vascular permeability, and promotes leukocyte functions such as directed chemotaxis, degranulation, mediator release, and production of superoxide anions. Although C5aR expression has traditionally been thought to be limited primarily to myeloid blood cells, including neutrophils, monocytes, macrophages, and eosinophils, we report here that C5aR is expressed by liver and lung cells as well as by cells in several other tissues. By Northern blot analysis, it was determined that mouse liver, baboon liver, human liver, and the human hepatoma-derived cell line HepG2 express a normal size (2.3 kb) C5aR mRNA; in HepG2 cells, the quantity of C5aR mRNA was comparable to that contained in dbcAMP-differentiated U937 cells. HepG2 cells were demonstrated to express the C5aR on their cell surface by flow cytometric and immunofluorescence analyses as well as by 125I-C5a binding assays. The binding data indicated that HepG2 cells express a single class of C5aR with a Kd of 1.18 nM and approximately 28,000 receptors per cell. In vivo expression of C5aR in human liver cells was demonstrated by in situ hybridization and immunohistochemistry analyses. Northern blot analysis of murine and baboon organs shows that, in addition to the liver, other tissues express C5aR mRNA in significant quantities, including the spleen, lung, heart, kidney, and intestine. Moreover, mice treated with LPS show a large increase in C5aR mRNA in all these tissues except the intestine. Immunostaining of human lung tissue demonstrated that bronchial and alveolar epithelial cells, as well as vascular smooth muscle and endothelial cells, also express the C5aR. Collectively, these data indicate that the C5aR is expressed in several different types of cells in liver and lung, and in yet undetermined cell types in spleen, heart, intestine, and kidney. Furthermore, these data suggest that the C5a anaphylatoxin mediates previously unrecognized functions by binding to tissue cells that express the C5aR.

Expression of the alpha 1-proteinase inhibitor (alpha 1PI) gene was studied in human mononuclear cells. Using RNA blot and dot hybridization, alpha 1PI mRNA was detected in human peripheral blood monocytes, bronchoalveolar and breast milk macrophages, but not in B or T lymphocytes. Using incorporation of a radiolabeled amino acid precursor, synthesis and secretion of alpha 1PI were demonstrated in human monocytes and macrophages, but not in lymphocytes. In addition, alpha 1PI was secreted in functionally active form as shown by complexing with serine proteases. Biosynthesis of alpha 1PI by mononuclear phagocytes was greatest during the first 24 hr in culture and progressively decreased over the next 10 days. The reduction in alpha 1PI biosynthesis in vitro involved a mechanism acting at the pretranslational level as alpha 1PI mRNA content also progressively declined over 10 days in culture. The ease of sampling human monocytes and macrophages now permits examination of the biochemical defect in homozygous PiZ and PiS alpha 1PI deficiencies and study of the functional significance of locally produced alpha 1PI in normal tissues and sites of injury or inflammation.

Homozygous, PIZZ alpha(1)-antitrypsin (alpha(1)-AT) deficiency is associated with chronic liver disease and hepatocellular carcinoma resulting from the toxic effects of mutant alpha(1)-anti-trypsin Z (alpha(1)-ATZ) protein retained in the endoplasmic reticulum (ER) of hepatocytes. However, the exact mechanism(s) by which retention of this aggregated mutant protein leads to cellular injury are still unknown. Previous studies have shown that retention of mutant alpha(1)-ATZ in the ER induces an intense autophagic response in hepatocytes. In this study, we present evidence that the autophagic response induced by ER retention of alpha(1)-ATZ also involves the mitochondria, with specific patterns of both mitochondrial autophagy and mitochondrial injury seen in cell culture models of alpha(1)-AT deficiency, in PiZ transgenic mouse liver, and in liver from alpha(1)-AT-deficient patients. Evidence for a unique pattern of caspase activation was also detected. Administration of cyclosporin A, an inhibitor of mitochondrial permeability transition, to PiZ mice was associated with a reduction in mitochondrial autophagy and injury and reduced mortality during experimental stress. These results provide evidence for the novel concept that mitochondrial damage and caspase activation play a role in the mechanism of liver cell injury in alpha(1)-AT deficiency and suggest the possibility of mechanism-based therapeutic interventions.

Formation of the covalently stabilized complex of alpha 1-antitrypsin (alpha 1-AT) with neutrophil elastase, the archetype of serine proteinase inhibitor serpin-enzyme complexes, is associated with structural rearrangement of the alpha 1-AT molecule and hydrolysis of a reactive-site peptide bond. An approximately 4-kDa carboxyl-terminal cleavage fragment is generated. alpha 1-AT-elastase complexes are biologically active, possessing chemotactic activity and mediating increases in expression of the alpha 1-AT gene in human monocytes and macrophages. This suggested that structural rearrangement of the alpha 1-AT molecule, during formation of a complex with elastase, exposes a domain that is recognized by a specific cell surface receptor or receptors. To test this hypothesis, the known three-dimensional structure of alpha 1-AT and comparisons of the primary structures of the serpins were used to select a potentially exteriorly exposed and highly conserved region in the complexed form of alpha 1-AT as a candidate ligand (carboxyl-terminal fragment, amino acids 359-374). We show here that synthetic peptides based on the sequence of this region bind specifically and saturably to human hepatoma cells and human monocytes (Kd = 4.0 X 10(-8) M, 4.5 X 10(5) plasma membrane receptors per cell) and mediate increases in synthesis of alpha 1-AT. Binding of peptide 105Y (Ser-Ile-Pro-Pro-Glu-Val-Lys-Phe-Asn-Lys-Pro-Phe-Val-Tyr-Leu-Ile) is blocked by alpha 1-AT-elastase complexes, antithrombin III (AT III)-thrombin complexes, alpha 1-antichymotrypsin (alpha 1-ACT)-cathepsin G complexes, and, to a lesser extent, complement component C1 inhibitor-C1s complexes, but not by the corresponding native proteins. Binding of peptide 105Y is also blocked by peptides with sequence corresponding to carboxy-terminal fragments of the serpins AT III and alpha 1-ACT, but not by peptides having the sequence of the extreme amino terminus of alpha 1-AT. The results also show that peptide 105Y inhibits binding of 125I-labeled alpha 1-AT-elastase complexes. Thus, these studies demonstrate an abundant, relatively high-affinity cell surface receptor which recognizes serpin-enzyme complexes (SEC receptor). This receptor is capable of modulating the production of at least one of the serpins, alpha 1-AT. Since the ligand specificity is similar to that previously described for in vivo clearance of serpin-enzyme complexes, the SEC receptor may also be involved in the clearance of certain serpin-enzyme complexes.

Liver disease in alpha-1-antitrypsin (alpha1AT) deficiency is caused by a gain-of-toxic function mechanism engendered by the accumulation of a mutant glycoprotein in the endoplasmic reticulum (ER). The extraordinary degree of variation in phenotypical expression of this liver disease is believed to be determined by genetic modifiers and/or environmental factors that influence the intracellular disposal of the mutant glycoprotein or the signal transduction pathways that are activated. Recent investigations suggest that a specific repertoire of signaling pathways are involved, including the autophagic response, mitochondrial- and ER-caspase activation, and nuclear factor kappaB (NFkappaB) activation. Whether activation of these signaling pathways, presumably to protect the cell, inadvertently contributes to liver injury or perhaps protects the cell from one injury and, in so doing, predisposes it to another type of injury, such as hepatocarcinogenesis, is not yet known. Recent studies also suggest that hepatocytes with marked accumulation of alpha1ATZ, globule-containing hepatocytes, engender a cancer-prone state by surviving with intrinsic damage and by chronically stimulating in 'trans' adjacent relatively undamaged hepatocytes that have a selective proliferative advantage. Further, this paradigm may apply to other genetic and infectious liver diseases that are predisposed to hepatocellular carcinoma.

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

The objective of this study was to evaluate the safety, tolerability, and preliminary efficacy of intravenous glutathione in Parkinson's disease (PD) patients. This was a randomized, placebo-controlled, double-blind, pilot trial in subjects with PD whose motor symptoms were not adequately controlled with their current medication regimen. Subjects were randomly assigned to receive intravenous glutathione 1,400 mg or placebo administered three times a week for 4 weeks. Twenty-one subjects were randomly assigned, 11 to glutathione and 10 to placebo. One subject who was assigned to glutathione withdrew from the study for personal reasons prior to undergoing any postrandomization efficacy assessments. Glutathione was well tolerated and there were no withdrawals because of adverse events in either group. Reported adverse events were similar in the two groups. There were no significant differences in changes in Unified Parkinson's Disease Rating Scale (UPDRS) scores. Over the 4 weeks of study medication administration, UPDRS ADL + motor scores improved by a mean of 2.8 units more in the glutathione group (P = 0.32), and over the subsequent 8 weeks worsened by a mean of 3.5 units more in the glutathione group (P = 0.54). Glutathione was well tolerated and no safety concerns were identified. Preliminary efficacy data suggest the possibility of a mild symptomatic effect, but this remains to be evaluated in a larger study.

Alpha-1-antitrypsin (AT) deficiency is the most common genetic cause of liver disease in children. The primary pathological issue is a point mutation that renders an abundant hepatic secretory glycoprotein prone to altered folding and a tendency to polymerize and aggregate. However, the expression of serious liver damage among homozygotes is dependent on genetic and/or environmental modifiers. Several studies have validated the concept that endogenous hepatic pathways for disposal of aggregation-prone proteins, including the proteasomal and autophagic degradative pathways, could play a key role in the variation in hepatic damage and be the target of the modifiers. Exciting recent results have shown that a drug that enhances autophagy can reduce the hepatic load of aggregated protein and reverse fibrosis in a mouse model of this disease.

To prospectively assess the value of serum total bilirubin (TB) within 3 months of hepatoportoenterostomy (HPE) in infants with biliary atresia as a biomarker predictive of clinical sequelae of liver disease in the first 2 years of life.Infants with biliary atresia undergoing HPE between June 2004 and January 2011 were enrolled in a prospective, multicenter study. Complications were monitored until 2 years of age or the earliest of liver transplantation (LT), death, or study withdrawal. TB below 2 mg/dL (34.2 μM) at any time in the first 3 months (TB <2.0, all others TB ≥ 2) after HPE was examined as a biomarker, using Kaplan-Meier survival and logistic regression.Fifty percent (68/137) of infants had TB < 2.0 in the first 3 months after HPE. Transplant-free survival at 2 years was significantly higher in the TB < 2.0 group vs TB ≥ 2 (86% vs 20%, P < .0001). Infants with TB ≥ 2 had diminished weight gain (P < .0001), greater probability of developing ascites (OR 6.4, 95% CI 2.9-14.1, P < .0001), hypoalbuminemia (OR 7.6, 95% CI 3.2-17.7, P < .0001), coagulopathy (OR 10.8, 95% CI 3.1-38.2, P = .0002), LT (OR 12.4, 95% CI 5.3-28.7, P < .0001), or LT or death (OR 16.8, 95% CI 7.2-39.2, P < .0001).Infants whose TB does not fall below 2.0 mg/dL within 3 months of HPE were at high risk for early disease progression, suggesting they should be considered for LT in a timely fashion. Interventions increasing the likelihood of achieving TB <2.0 mg/dL within 3 months of HPE may enhance early outcomes.ClinicalTrials.gov: NCT00061828 and NCT00294684.

In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, "proteotoxic" mechanism. Whereas some ATD patients develop severe liver disease (SLD) that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without SLD could model these personalized variations in hepatic disease phenotypes. Patient-specific iPSCs were generated from ATD patients and a control and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild-type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from SLD patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with SLD.iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that "proteostasis" mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies.

Huntington’s disease (HD) is an inherited neurodegenerative disorder with adult-onset clinical symptoms, but the mechanism by which aging drives the onset of neurodegeneration in patients with HD remains unclear. In this study we examined striatal medium spiny neurons (MSNs) directly reprogrammed from fibroblasts of patients with HD to model the age-dependent onset of pathology. We found that pronounced neuronal death occurred selectively in reprogrammed MSNs from symptomatic patients with HD (HD-MSNs) compared to MSNs derived from younger, pre-symptomatic patients (pre-HD-MSNs) and control MSNs from age-matched healthy individuals. We observed age-associated alterations in chromatin accessibility between HD-MSNs and pre-HD-MSNs and identified miR-29b-3p, whose age-associated upregulation promotes HD-MSN degeneration by impairing autophagic function through human-specific targeting of the STAT3 3′ untranslated region. Reducing miR-29b-3p or chemically promoting autophagy increased the resilience of HD-MSNs against neurodegeneration. Our results demonstrate miRNA upregulation with aging in HD as a detrimental process driving MSN degeneration and potential approaches for enhancing autophagy and resilience of HD-MSNs. Oh et al. modeled age-dependent onset of Huntington’s disease by comparing reprogrammed neurons from pre-symptomatic and symptomatic patients. They found that an age-associated miRNA led to autophagy impairment and neurodegeneration.

HepatologyVolume 35, Issue 6 p. 1297-1304 Concise Review In Mechanisms Of DiseaseFree Access Extrahepatic biliary atresia: A disease or a phenotype? David H. Perlmutter 3705 Fifth Ave., Corresponding Author David H. Perlmutter 3705 Fifth Ave. [email protected] Department of Pediatrics, Washington University School of Medicine; Division of Gastroenterology and Nutrition, St. Louis Children's Hospital; and Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, PAPittsburgh, PA 15213-2583. fax: 412-692-5946.===Search for more papers by this authorRoss W. Shepherd, Ross W. Shepherd Department of Pediatrics, Washington University School of Medicine; Division of Gastroenterology and Nutrition, St. Louis Children's Hospital; and Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, PASearch for more papers by this author David H. Perlmutter 3705 Fifth Ave., Corresponding Author David H. Perlmutter 3705 Fifth Ave. [email protected] Department of Pediatrics, Washington University School of Medicine; Division of Gastroenterology and Nutrition, St. Louis Children's Hospital; and Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, PAPittsburgh, PA 15213-2583. fax: 412-692-5946.===Search for more papers by this authorRoss W. Shepherd, Ross W. Shepherd Department of Pediatrics, Washington University School of Medicine; Division of Gastroenterology and Nutrition, St. Louis Children's Hospital; and Department of Pediatrics, University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh, Pittsburgh, PASearch for more papers by this author First published: 30 December 2003 https://doi.org/10.1053/jhep.2002.34170Citations: 113AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 Carmi R, Magee CA, Neill CA, Karrer FM. 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Temporal changes in the expression and distribution of adhesion molecules during liver development and regeneration. J Cell Biol 1992; 116: 1507– 1515. MEDLINE 47 Terada T. Nakanuma Y. Profiles of expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation: a lectin-histochemical and immunohistochemical study. Hepatology 1994; 20: 388– 397. MEDLINE 48 Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, Qi M, et al. Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1. Nat Genet 1997; 16: 243– 251. MEDLINE 49 Louis AA, Eyken PV, Haber BA, Hicks C, Weinmaster G, Taub R, Rand EB. Hepatic Jagged1 expression studies. Hepatology 1999; 30: 1269– 1275. MEDLINE 50 Rosen EM, Nigam SK, Goldberg ID. Scatter factor and the c-Met receptor: a paradigm for mesenchymal/epithelial interaction. J Cell Biol 1994; 127: 1783– 1787. MEDLINE 51 Huff JL, Jelinek MA, Borgman CA, Lansing TJ, Parsons JT. The proto-oncogene c-Sea encodes a transmembrane protein-tyrosine kinase related to the Met/hepatocyte growth factor/scatter factor receptor. Proc Natl Acad Sci U S A 1993; 90: 6140– 6144. MEDLINE 52 Silveira TR, Salzano FM, Donaldson PT, Mieli-Vergani G, Howard ER, Mowat AP. Association between HLA and extrahepatic biliary atresia. J Pediatr Gastroenterol Nutr 1993; 16: 114– 117. MEDLINE 53 Schreiber RA, Kleinman RE, Barksdale EM, Maganaro TF, Donahue PR. Rejection of murine congenic bile ducts: model for immune-mediated bile duct disease. Gastroenterology 1992; 102: 924– 930. MEDLINE 54 Seidman SL, Duquesnoy RJ, Zeevi A, Fung JJ, Starzl TE, Demetris AJ. Recognition of major histocompatibility complex antigens on cultured human biliary epithelial cells by alloreactive lymphocytes. Hepatology 1991; 13: 239– 246. MEDLINE 55 Dillon P, Belchis D, Tracy T, Cilley R, Hafey I, Krummel T. Increased expression of intracellular adhesion molecules in biliary atresia. Am J Pathol 1994; 145: 263– 267. MEDLINE 56 Smith BM, Laberge J-M, Schreiber R, Weber AM, Blanchard H. Familial biliary atresia in three siblings including twins. J Ped Surg 1991; 26: 1331– 1333. 57 Gunasekaran TS, Hassall EG, Steinbrecher UP, Yong S-L. Recurrence of extrahepatic biliary atresia in two half sibs. Am J Med Genet 1992; 43: 592– 594. MEDLINE 58 Strickland AD, Shannon K, Coln CD. Biliary atresia in two sets of twins. J Pediatr 1985; 107: 418– 419. MEDLINE 59 Moore TC, Hyman PE. Extrahepatic biliary atresia in one human leukocyte antigen identical twin. Pediatrics 1985; 76: 604– 605. MEDLINE Citing Literature Volume35, Issue6June 2002Pages 1297-1304 ReferencesRelatedInformation

alpha 1-Antitrypsin (alpha 1-AT) is an acute phase plasma protein predominantly derived from the liver which inhibits neutrophil elastase. Previous studies have suggested that alpha 1-AT is also expressed in human enterocytes because alpha 1-AT mRNA could be detected in human jejunum by RNA blot analysis, and alpha 1-AT synthesis could be detected in a human intestinal adenocarcinoma cell line Caco2, which spontaneously differentiates into villous-like enterocytes in tissue culture. To definitively determine that the alpha 1-AT gene is expressed in human enterocytes in vivo, we examined tissue slices of human jejunum and ileum by in situ hybridization. The results demonstrate specific hybridization to enterocytes from the bases to the tips of the villi. Although there was no hybridization to enterocytes in most of the crypt epithelium, there was intense specific hybridization in one region of the crypt. Double-label immunohistochemical studies showed that alpha 1-AT and lysozyme co-localized to this region, indicating that it represented Paneth cells. Finally, there was a marked increase in hybridization to alpha 1-AT mRNA in villous enterocytes and Paneth cells in Crohn's disease. The results of this study provide definitive evidence that alpha 1-AT is expressed in human jejunal and ileal enterocytes in vivo, and show that alpha 1-AT is also a product of Paneth cells. Together with the results of other studies, these data raise the possibility that alpha 1-AT detected in fecal alpha 1-AT clearance assays for diagnosing protein-losing enteropathies is predominantly derived from sloughed enterocytes.

Extrahepatic biliary atresia (BA) is a devastating disease of the neonate in which the hepatic and/or common bile duct is obliterated or interrupted. Infants and children with this diagnosis constitute 50% to 60% of the pediatric population that undergoes orthotopic liver transplantation. However, there is still very little known about the etiology and pathogenesis of BA. Several recent studies have demonstrated that anomalies of situs determination are more commonly associated with BA than previously recognized. In this study, we examined the pathogenesis of jaundice in the inv mouse, a transgenic mouse in which a recessive deletion of the inversin gene results in situs inversus and jaundice. The results show that these mice have cholestasis with conjugated hyperbilirubinemia, failure to excrete technetium-labeled mebrofenin from the liver into the small intestine, lack of continuity between the extrahepatic biliary tree and the small intestine as demonstrated by Trypan blue cholangiography, and a liver histological picture indicative of extrahepatic biliary obstruction with negligible inflammation/necrosis within the hepatic parenchyma. Lectin histochemical staining of biliary epithelial cells in serial sections suggests the presence of several different anomalies in the architecture of the extrahepatic biliary system. These results suggest that the inversin gene plays an essential role in the morphogenesis of the hepatobiliary system and raise the possibility that alterations in the human orthologue of inversin account for some of the cases of BA in which there are also anomalies of situs determination.

The liver responds to multiple types of injury with an extraordinarily well orchestrated and tightly regulated form of regeneration. The response to partial hepatectomy has been used as a model system to elucidate the molecular basis of this regenerative response. In this study, we used cyclooxygenase (COX)-selective antagonists and -null mice to determine the role of prostaglandin signaling in the response of liver to partial hepatectomy. The results show that liver regeneration is markedly impaired when both COX-1 and COX-2 are inhibited by indocin or by a combination of the COX-1 selective antagonist, SC-560, and the COX-2 selective antagonist, SC-236. Inhibition of COX-2 alone partially inhibits regeneration whereas inhibition of COX-1 alone tends to delay regeneration. Neither the rise in IL-6 nor the activation of signal transducer and activator of transcription-3 (STAT3) that is seen during liver regeneration is inhibited by indocin or the selective COX antagonists. In contrast, indocin treatment prevents the activation of CREB by phosphorylation that occurs during hepatic regeneration. These data indicate that prostaglandin signaling is required during liver regeneration, that COX-2 plays a particularly important role but COX-1 is also involved, and implicate the activation of CREB rather than STAT3 as the mediator of prostaglandin signaling during liver regeneration.

alpha-1-Antitrypsin (alpha1-AT) deficiency is the most common cause of metabolic pediatric liver disease. Hepatocellular injury is caused by toxicity of the mutant alpha-1-antitrypsin Z (alpha1-ATZ) molecule retained within hepatocytes. In these studies, we used the PiZ transgenic mouse model of alpha1-AT deficiency to examine hepatocellular proliferation in response to chronic liver injury resulting from this metabolic disease. The results showed increased hepatocellular proliferation and caspase 9 activation in male PiZ mice compared with female PiZ and wild-type mice. Hepatic alpha1-AT mRNA and protein expression also were increased in male PiZ mice, suggesting that greater hepatocellular proliferation and caspase activation in males results from increased hepatotoxicity associated with greater intracellular alpha1-ATZ accumulation. Testosterone treatment of female PiZ mice increased alpha1-ATZ expression and hepatocellular proliferation to a level comparable with that in males. In PiZ mice, hepatocytes devoid of intracellular alpha1-AT globules had a proliferative advantage compared with globule-containing hepatocytes. However, this advantage is relative because both globule-containing and globule-devoid hepatocytes exhibited comparable proliferation after partial hepatectomy. In conclusion, these data indicate that intracellular retention of mutant alpha1-ATZ is associated with a regenerative stimulus leading to increased hepatocellular proliferation, that gender-specific signals influence the degree of alpha1-AT expression and associated hepatic injury, and that hepatocytes devoid of alpha1-ATZ have a proliferative advantage over cells that accumulate the mutant protein. This selective proliferation suggests that hepatocellular transplantation may be applicable for treatment of this and other slowly progressive metabolic liver diseases.

Because retention of mutant α1-antitrypsin (α1-AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in α1-AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of α1-ATZ (Qu, D., Teckman, J. H., Omura, S., and Perlmutter, D. H. (1996) J. Biol. Chem. 271, 22791–22795). In more recent studies, Cabral et al.(Cabral, C. M., Choudhury, P., Liu, Y., and Sifers, R. N. (2000) J. Biol. Chem. 275, 25015–25022) found that degradation of α1-ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of α1-ATZ in nonhepatocytic cell types or in cell types with levels of α1-AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of α1-ATZ. In each of these cell lines degradation of α1-ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin β-lactone. Using the inducible expression system to regulate the relative level of α1-ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of α1-ATZ at high and low levels of α1-AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of α1-ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes. Because retention of mutant α1-antitrypsin (α1-AT) Z in the endoplasmic reticulum (ER) is associated with liver disease in α1-AT-deficient individuals, the mechanism by which this aggregated glycoprotein is degraded has received considerable attention. In previous studies using stable transfected human fibroblast cell lines and a cell-free microsomal translocation system, we found evidence for involvement of the proteasome in degradation of α1-ATZ (Qu, D., Teckman, J. H., Omura, S., and Perlmutter, D. H. (1996) J. Biol. Chem. 271, 22791–22795). In more recent studies, Cabral et al.(Cabral, C. M., Choudhury, P., Liu, Y., and Sifers, R. N. (2000) J. Biol. Chem. 275, 25015–25022) found that degradation of α1-ATZ in a stable transfected murine hepatoma cell line was inhibited by tyrosine phosphatase inhibitors, but not by the proteasomal inhibitor lactacystin and concluded that the proteasome was only involved in ER degradation of α1-ATZ in nonhepatocytic cell types or in cell types with levels of α1-AT expression that are substantial lower than that which occurs in hepatocytes. To examine this important issue in further detail, in this study we established rat and murine hepatoma cell lines with constitutive and inducible expression of α1-ATZ. In each of these cell lines degradation of α1-ATZ was inhibited by lactacystin, MG132, epoxomicin, and clasto-lactacystin β-lactone. Using the inducible expression system to regulate the relative level of α1-ATZ expression, we found that lactacystin had a similar inhibitory effect on degradation of α1-ATZ at high and low levels of α1-AT expression. Although there is substantial evidence that other mechanisms contribute to ER degradation of α1-ATZ, the data reported here indicate that the proteasome plays an important role in many cell types including hepatocytes. α1-antitrypsin α1-antitrypsin Z endoplasmic reticulum polyacrylamide gel electrophoresis The classical and most common form of α1-antitrypsin (α1-AT)1deficiency is a relatively unique genetic disease in that it is associated with injury to one tissue, pulmonary emphysema, by a loss-of-function mechanism and injury to another tissue, chronic hepatitis/hepatocellular carcinoma, by a gain-of-function mechanism. Many studies have provided evidence that emphysema results from lack of the anti-elastase activity of α1-AT in the lung (reviewed in Refs. 1Cox D.W. Scriver C.B. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1995: 4125-4158Google Scholar and 2Perlmutter D.H. Schiff E.R. Sorrell M.F. Maddrey W.C. Schiff's Diseases of the Liver. Lippincott-Raven Publishers, Philadelphia, PA1999: 1131-1150Google Scholar). Liver disease is due to toxic effects of aggregated α1-ATZ retained in the ER of liver parenchymal cells. The gain-of-function mechanism is most clearly demonstrated by experiments in mice transgenic for human α1-ATZ. These mice develop liver injury and hepatocellular carcinoma despite the fact that they have their own endogenous anti-elastases (3Dycaico M.J. Grant S.G. Felts K. Nichols W.S. Geller S.A. Hager J.H. Pollard A.J. Kohler S.W. Short H.P. Jirik F.R. Sorge J.A. Science. 1988; 242: 1404-1412Crossref Scopus (93) Google Scholar, 4Carlson J.A. Rogers B.B. Sifers R.N. Hawkins H.K. Finegold M.J. Woo S.L. J. Clin. Invest. 1988; 83: 1183-1190Crossref Scopus (228) Google Scholar, 5Geller S.A. Nichols W.S. Kim S. Tolmachoff T. Lee S. Dycaico M.J. Felts K. Sorge J.A. Hepatology. 1994; 19: 389-397Crossref PubMed Scopus (66) Google Scholar).The mutant Z allele of α1-AT is characterized by a single nucleotide substitution, which results in the replacement of glutamate 342 by a bulky lysine residue (1Cox D.W. Scriver C.B. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1995: 4125-4158Google Scholar, 2Perlmutter D.H. Schiff E.R. Sorrell M.F. Maddrey W.C. Schiff's Diseases of the Liver. Lippincott-Raven Publishers, Philadelphia, PA1999: 1131-1150Google Scholar). The studies of Carrell and Lomas (6Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar, 7Carrel R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar) have shown that this substitution renders the α1-AT molecule more susceptible to polymerization and that highly ordered aggregates accumulate in the ER of liver cells.One interesting observation, arising from unbiased nationwide screening studies of α1-AT deficiency in Sweden, indicates that only 10–15% of deficient individuals develop clinically significant liver disease (8Sveger T. N. Engl. J. Med. 1976; 294: 1316-1321Crossref PubMed Scopus (618) Google Scholar, 9Sveger T. Eriksson S. Hepatology. 1995; 22: 514-517PubMed Google Scholar). In previous studies we tested the hypothesis that this subgroup of deficient individuals is susceptible to liver injury by virtue of additional unlinked genetic traits or environmental factors that delay degradation of the mutant α1-ATZ molecule after it is retained in the ER (10Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar). With the use of fibroblast cell lines from deficient patients with liver disease (susceptible hosts) compared with those from deficient individuals without liver disease (protected hosts), we found that more efficient degradation of retained mutant α1-ATZ in the ER correlated with protection from liver disease. These results, therefore, focused our attention on the mechanism by which α1-ATZ is degraded in the ER. Subsequent studies showed that lactacystin inhibited ER degradation of α1-ATZ in stable transfected human fibroblast cell lines and in a cell-free microsomal system (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 12Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar), indicating that the proteasome and the ubiquitin system were involved in this important quality control mechanism. Degradation of α1-ATZ in stable transfected Chinese hamster ovary cells and in primary cultures of human mononuclear phagocytes was also inhibited by lactacystin (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar). However, Cabral et al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) recently found that lactacystin did not inhibit degradation of α1-ATZ in a stable transfected murine hepatoma cell line. Degradation of α1-ATZ in this cell line was markedly decreased by tyrosine phosphatase inhibitors. These authors concluded that the proteasome was only involved in degradation of α1-ATZ in nonhepatocytic cells or alternatively was only involved in α1-ATZ degradation in cell types with lower levels of α1-AT biosynthesis. Because hepatocytes are the predominant site of synthesis of α1-AT and the cells predominantly affected by the pathobiological process of α1-AT deficiency-associated liver disease, this is a very important issue. In this study we examined hepatocytes in further detail by generating rat and murine hepatoma cell lines that express α1-ATZ. We also examined the role of the relative level of α1-AT biosynthesis by generating hepatoma cell lines with regulated expression of α1-ATZ.DISCUSSIONA detailed elucidation of the mechanisms by which mutant aggregated α1-ATZ is degraded in the ER is essential for understanding how the quality control apparatus of the ER works in general and for understanding the specific issue of how a subgroup of α1-AT-deficient individuals become susceptible to liver injury and carcinogenesis. Previous studies have shown that there is a lag in the disposal of this mutant protein in genetically engineered human fibroblasts from “susceptible” deficient patients (10Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar). Moreover, this lag in the ER disposal/quality control mechanism appears to be specific, i.e. it affected the disposal of two polymerogenic mutants of α1-AT, but not a model unassembled membrane protein (19Teckman J.H. Perlmutter D.H. J. Biol. Chem. 1996; 271: 13215-13220Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Initial studies of the ER degradation of α1-ATZ indicated that the ubiquitin-dependent proteasomal system was involved. Degradation of α1-ATZ in genetically engineered human fibroblast cell lines and in a cell-free microsomal translocation system was inhibited by lactacystin (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Degradation of α1-ATZ in the cell-free system was shown to be dependent on ATP and, more importantly, a polyubiquitinated calnexin-α1-ATZ complex was shown to be a degradative intermediate (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Subsequent studies by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar) have shown that degradation of α1-ATZ is also inhibited by lactacystin in transfected Chinese hamster ovary cells and in primary cultures of human mononuclear phagocytes. Using an experimental approach in which the degradative machinery in the reticulocyte lysate of the cell-free system is fractionated and reconstituted with purified components, we have recently found evidence for at least three different pathways in the degradation of α1-ATZ, including ubiquitin-dependent and -independent proteasomal mechanisms and at least one nonproteasomal mechanism (12Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar). Subsequent studies have suggested that autophagy may constitute one of the nonproteasomal mechanisms (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) and have substantiated the concept that there are multiple pathways involved in ER degradation of α1-ATZ.In the most recent study of this issue, Cabral et al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) found that lactacystin did not inhibit degradation of α1-ATZ in a stable transfected murine hepatoma Hepa1–6 cell line. Degradation of α1-ATZ in this cell line was decreased by tyrosine phosphatase inhibitors. Lactacystin did inhibit degradation of α1-AT Null Hong Kong, a truncated mutant, in a separate stable transfected Hepa1–6 cell line. Taken together, these results indicated that the proteasome did not play a role in degradation of α1-ATZ in hepatoma cell line even though the proteasome was active in these cells, that an entirely separate mechanism for degradation existed, and that this distinct mechanism was specific for α1-ATZ. Taking into consideration the previous results indicating involvement of the proteasome in degradation of α1-ATZ in genetically engineered fibroblasts (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), in primary cultures of human macrophages (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), and in the cell-free microsomal system (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), these authors concluded that the proteasomal mechanism was cell type-specific, either for nonhepatocytic cell types and/or cell types with lower levels of endogenous α1-AT expression than hepatocytes (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar).Because the hepatocyte is an extremely important, if not the most important, site of synthesis of α1-ATZ with respect to the development of liver disease, in this study we sought to examine in further detail the involvement of the proteasome in ER degradation of α1-ATZ in cells of hepatocytic lineage. The results show that lactacystin, MG132, epoxomicin, and clasto-lactacystin β-lactone all inhibit degradation of α1-ATZ in several different types of genetically engineered hepatoma cell lines, including the murine hepatoma Hepa1–6 used by Cabral et al. and a rat hepatoma cell line H11, which has the advantages of being highly differentiated for hepatocytic function, but lacking endogenous expression of α1-AT. Degradation of α1-ATZ in hepatoma cell lines with constitutive and inducible expression of α1-ATZ was blocked to an equivalent extent by proteasomal inhibitors. Finally, studies in HeLa and Hepa1–6 cell lines with inducible expression of α1-ATZ showed that the proteasome was involved in degradation of α1-ATZ at both high and 30–100-fold lower levels of expression. These results suggest to us that the lack of involvement of the proteasome in degradation of α1-ATZ in the Hepa1–6 cell line generated by Cabralet al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) is cell line-specific, perhaps reflecting a type of adaptation. This hypothesis by no means diminishes the importance of the observations of Cabral et al. or the importance of tyrosine phosphatases in the quality control mechanism. There is now ample evidence for multiple mechanisms/pathways in the ER quality control apparatus and for cellular “adaptation.” In fact, gene expression profile analysis has shown marked changes in expression of many genes in yeast cells that accumulate misfolded proteins (20Travers K.J. Patil C.K. Wodlicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar, 21Casagrande R. Stern P. Diehn M. Shamu C. Osario M. Zuniga M. Brown P.O. Ploegh H. Mol. Cell. 2000; 5: 729-735Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 22Ng D.T.W. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar, 23Friedlander R. Jarosch E. Urban J. Volkwin C. Sommer T. Nat. Cell Biol. 2000; 2: 379-384Crossref PubMed Scopus (388) Google Scholar). Moreover, if a cellular adaptation mechanism is truly applicable, then the results of Cabral et al. raise the interesting possibility that the adaptation is specific for α1-ATZ, a polymerogenic mutant, and not for α1-AT Null Hong Kong, a mutant that is truncated and not likely to be polymerogenic.Three other results of this study deserve comment. First, there is a marked increase in the formation of insoluble aggregates of α1-ATZ and two distinct degradation products appear exclusively in the insoluble fraction when the proteasome is inhibited. These degradation products could theoretically be generated in, and/or accumulate in, the ER or the cytoplasm. Several lines of evidence make it more likely that they are generated in and localize to the ER. α1-ATZ has not been detected outside the ER lumen in intact cells (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) or in the supernatant of cell-free mammalian translocation reactions (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) and proteasome inhibitors do not induce aggresomes in cells that express mutant α1-ATZ (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar). Similar degradation products are generated processively in the lumen of microsomal vesicles that have translocated wild type α1-ATZ in a cell-free microsomal translocation reaction (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), suggesting the existence of an endoluminal proteolytic system that recognizes wild type or mutant α1-ATZ when it is retained in the ER for a prolonged period of time. Second, there was no evidence for a significant increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, or HeLa cells in the presence of lactacystin, MG132, epoxomicin, or clasto-lactacystin β-lactone at doses optimal for inhibition of degradation of α1-ATZ. Using the lower doses of lactacystin or longer periods of preincubation with lactacystin described by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), we observed a lesser degree of inhibition of degradation, but no increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, and HeLa cells at low and high levels of expression (data not shown). Third, secretion of α1-ATZ did not increase when the level of its synthesis was decreased. This was shown by modulating the level of synthesis 30–100-fold in tetracycline-regulated cell lines. This result is noteworthy because the rate of polymerization of α1-ATZ decreases at lower concentrations in purified systems (6Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar). The results in this report therefore suggest that other factors working in concert with polymerization play a role in the fate of α1-ATZ when it accumulates in the ER. The classical and most common form of α1-antitrypsin (α1-AT)1deficiency is a relatively unique genetic disease in that it is associated with injury to one tissue, pulmonary emphysema, by a loss-of-function mechanism and injury to another tissue, chronic hepatitis/hepatocellular carcinoma, by a gain-of-function mechanism. Many studies have provided evidence that emphysema results from lack of the anti-elastase activity of α1-AT in the lung (reviewed in Refs. 1Cox D.W. Scriver C.B. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1995: 4125-4158Google Scholar and 2Perlmutter D.H. Schiff E.R. Sorrell M.F. Maddrey W.C. Schiff's Diseases of the Liver. Lippincott-Raven Publishers, Philadelphia, PA1999: 1131-1150Google Scholar). Liver disease is due to toxic effects of aggregated α1-ATZ retained in the ER of liver parenchymal cells. The gain-of-function mechanism is most clearly demonstrated by experiments in mice transgenic for human α1-ATZ. These mice develop liver injury and hepatocellular carcinoma despite the fact that they have their own endogenous anti-elastases (3Dycaico M.J. Grant S.G. Felts K. Nichols W.S. Geller S.A. Hager J.H. Pollard A.J. Kohler S.W. Short H.P. Jirik F.R. Sorge J.A. Science. 1988; 242: 1404-1412Crossref Scopus (93) Google Scholar, 4Carlson J.A. Rogers B.B. Sifers R.N. Hawkins H.K. Finegold M.J. Woo S.L. J. Clin. Invest. 1988; 83: 1183-1190Crossref Scopus (228) Google Scholar, 5Geller S.A. Nichols W.S. Kim S. Tolmachoff T. Lee S. Dycaico M.J. Felts K. Sorge J.A. Hepatology. 1994; 19: 389-397Crossref PubMed Scopus (66) Google Scholar). The mutant Z allele of α1-AT is characterized by a single nucleotide substitution, which results in the replacement of glutamate 342 by a bulky lysine residue (1Cox D.W. Scriver C.B. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1995: 4125-4158Google Scholar, 2Perlmutter D.H. Schiff E.R. Sorrell M.F. Maddrey W.C. Schiff's Diseases of the Liver. Lippincott-Raven Publishers, Philadelphia, PA1999: 1131-1150Google Scholar). The studies of Carrell and Lomas (6Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar, 7Carrel R.W. Lomas D.A. Lancet. 1997; 350: 134-138Abstract Full Text Full Text PDF PubMed Scopus (808) Google Scholar) have shown that this substitution renders the α1-AT molecule more susceptible to polymerization and that highly ordered aggregates accumulate in the ER of liver cells. One interesting observation, arising from unbiased nationwide screening studies of α1-AT deficiency in Sweden, indicates that only 10–15% of deficient individuals develop clinically significant liver disease (8Sveger T. N. Engl. J. Med. 1976; 294: 1316-1321Crossref PubMed Scopus (618) Google Scholar, 9Sveger T. Eriksson S. Hepatology. 1995; 22: 514-517PubMed Google Scholar). In previous studies we tested the hypothesis that this subgroup of deficient individuals is susceptible to liver injury by virtue of additional unlinked genetic traits or environmental factors that delay degradation of the mutant α1-ATZ molecule after it is retained in the ER (10Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar). With the use of fibroblast cell lines from deficient patients with liver disease (susceptible hosts) compared with those from deficient individuals without liver disease (protected hosts), we found that more efficient degradation of retained mutant α1-ATZ in the ER correlated with protection from liver disease. These results, therefore, focused our attention on the mechanism by which α1-ATZ is degraded in the ER. Subsequent studies showed that lactacystin inhibited ER degradation of α1-ATZ in stable transfected human fibroblast cell lines and in a cell-free microsomal system (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 12Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar), indicating that the proteasome and the ubiquitin system were involved in this important quality control mechanism. Degradation of α1-ATZ in stable transfected Chinese hamster ovary cells and in primary cultures of human mononuclear phagocytes was also inhibited by lactacystin (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar). However, Cabral et al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) recently found that lactacystin did not inhibit degradation of α1-ATZ in a stable transfected murine hepatoma cell line. Degradation of α1-ATZ in this cell line was markedly decreased by tyrosine phosphatase inhibitors. These authors concluded that the proteasome was only involved in degradation of α1-ATZ in nonhepatocytic cells or alternatively was only involved in α1-ATZ degradation in cell types with lower levels of α1-AT biosynthesis. Because hepatocytes are the predominant site of synthesis of α1-AT and the cells predominantly affected by the pathobiological process of α1-AT deficiency-associated liver disease, this is a very important issue. In this study we examined hepatocytes in further detail by generating rat and murine hepatoma cell lines that express α1-ATZ. We also examined the role of the relative level of α1-AT biosynthesis by generating hepatoma cell lines with regulated expression of α1-ATZ. DISCUSSIONA detailed elucidation of the mechanisms by which mutant aggregated α1-ATZ is degraded in the ER is essential for understanding how the quality control apparatus of the ER works in general and for understanding the specific issue of how a subgroup of α1-AT-deficient individuals become susceptible to liver injury and carcinogenesis. Previous studies have shown that there is a lag in the disposal of this mutant protein in genetically engineered human fibroblasts from “susceptible” deficient patients (10Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar). Moreover, this lag in the ER disposal/quality control mechanism appears to be specific, i.e. it affected the disposal of two polymerogenic mutants of α1-AT, but not a model unassembled membrane protein (19Teckman J.H. Perlmutter D.H. J. Biol. Chem. 1996; 271: 13215-13220Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar).Initial studies of the ER degradation of α1-ATZ indicated that the ubiquitin-dependent proteasomal system was involved. Degradation of α1-ATZ in genetically engineered human fibroblast cell lines and in a cell-free microsomal translocation system was inhibited by lactacystin (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Degradation of α1-ATZ in the cell-free system was shown to be dependent on ATP and, more importantly, a polyubiquitinated calnexin-α1-ATZ complex was shown to be a degradative intermediate (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Subsequent studies by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar) have shown that degradation of α1-ATZ is also inhibited by lactacystin in transfected Chinese hamster ovary cells and in primary cultures of human mononuclear phagocytes. Using an experimental approach in which the degradative machinery in the reticulocyte lysate of the cell-free system is fractionated and reconstituted with purified components, we have recently found evidence for at least three different pathways in the degradation of α1-ATZ, including ubiquitin-dependent and -independent proteasomal mechanisms and at least one nonproteasomal mechanism (12Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar). Subsequent studies have suggested that autophagy may constitute one of the nonproteasomal mechanisms (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) and have substantiated the concept that there are multiple pathways involved in ER degradation of α1-ATZ.In the most recent study of this issue, Cabral et al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) found that lactacystin did not inhibit degradation of α1-ATZ in a stable transfected murine hepatoma Hepa1–6 cell line. Degradation of α1-ATZ in this cell line was decreased by tyrosine phosphatase inhibitors. Lactacystin did inhibit degradation of α1-AT Null Hong Kong, a truncated mutant, in a separate stable transfected Hepa1–6 cell line. Taken together, these results indicated that the proteasome did not play a role in degradation of α1-ATZ in hepatoma cell line even though the proteasome was active in these cells, that an entirely separate mechanism for degradation existed, and that this distinct mechanism was specific for α1-ATZ. Taking into consideration the previous results indicating involvement of the proteasome in degradation of α1-ATZ in genetically engineered fibroblasts (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), in primary cultures of human macrophages (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), and in the cell-free microsomal system (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), these authors concluded that the proteasomal mechanism was cell type-specific, either for nonhepatocytic cell types and/or cell types with lower levels of endogenous α1-AT expression than hepatocytes (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar).Because the hepatocyte is an extremely important, if not the most important, site of synthesis of α1-ATZ with respect to the development of liver disease, in this study we sought to examine in further detail the involvement of the proteasome in ER degradation of α1-ATZ in cells of hepatocytic lineage. The results show that lactacystin, MG132, epoxomicin, and clasto-lactacystin β-lactone all inhibit degradation of α1-ATZ in several different types of genetically engineered hepatoma cell lines, including the murine hepatoma Hepa1–6 used by Cabral et al. and a rat hepatoma cell line H11, which has the advantages of being highly differentiated for hepatocytic function, but lacking endogenous expression of α1-AT. Degradation of α1-ATZ in hepatoma cell lines with constitutive and inducible expression of α1-ATZ was blocked to an equivalent extent by proteasomal inhibitors. Finally, studies in HeLa and Hepa1–6 cell lines with inducible expression of α1-ATZ showed that the proteasome was involved in degradation of α1-ATZ at both high and 30–100-fold lower levels of expression. These results suggest to us that the lack of involvement of the proteasome in degradation of α1-ATZ in the Hepa1–6 cell line generated by Cabralet al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) is cell line-specific, perhaps reflecting a type of adaptation. This hypothesis by no means diminishes the importance of the observations of Cabral et al. or the importance of tyrosine phosphatases in the quality control mechanism. There is now ample evidence for multiple mechanisms/pathways in the ER quality control apparatus and for cellular “adaptation.” In fact, gene expression profile analysis has shown marked changes in expression of many genes in yeast cells that accumulate misfolded proteins (20Travers K.J. Patil C.K. Wodlicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar, 21Casagrande R. Stern P. Diehn M. Shamu C. Osario M. Zuniga M. Brown P.O. Ploegh H. Mol. Cell. 2000; 5: 729-735Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 22Ng D.T.W. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar, 23Friedlander R. Jarosch E. Urban J. Volkwin C. Sommer T. Nat. Cell Biol. 2000; 2: 379-384Crossref PubMed Scopus (388) Google Scholar). Moreover, if a cellular adaptation mechanism is truly applicable, then the results of Cabral et al. raise the interesting possibility that the adaptation is specific for α1-ATZ, a polymerogenic mutant, and not for α1-AT Null Hong Kong, a mutant that is truncated and not likely to be polymerogenic.Three other results of this study deserve comment. First, there is a marked increase in the formation of insoluble aggregates of α1-ATZ and two distinct degradation products appear exclusively in the insoluble fraction when the proteasome is inhibited. These degradation products could theoretically be generated in, and/or accumulate in, the ER or the cytoplasm. Several lines of evidence make it more likely that they are generated in and localize to the ER. α1-ATZ has not been detected outside the ER lumen in intact cells (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) or in the supernatant of cell-free mammalian translocation reactions (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) and proteasome inhibitors do not induce aggresomes in cells that express mutant α1-ATZ (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar). Similar degradation products are generated processively in the lumen of microsomal vesicles that have translocated wild type α1-ATZ in a cell-free microsomal translocation reaction (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), suggesting the existence of an endoluminal proteolytic system that recognizes wild type or mutant α1-ATZ when it is retained in the ER for a prolonged period of time. Second, there was no evidence for a significant increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, or HeLa cells in the presence of lactacystin, MG132, epoxomicin, or clasto-lactacystin β-lactone at doses optimal for inhibition of degradation of α1-ATZ. Using the lower doses of lactacystin or longer periods of preincubation with lactacystin described by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), we observed a lesser degree of inhibition of degradation, but no increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, and HeLa cells at low and high levels of expression (data not shown). Third, secretion of α1-ATZ did not increase when the level of its synthesis was decreased. This was shown by modulating the level of synthesis 30–100-fold in tetracycline-regulated cell lines. This result is noteworthy because the rate of polymerization of α1-ATZ decreases at lower concentrations in purified systems (6Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar). The results in this report therefore suggest that other factors working in concert with polymerization play a role in the fate of α1-ATZ when it accumulates in the ER. A detailed elucidation of the mechanisms by which mutant aggregated α1-ATZ is degraded in the ER is essential for understanding how the quality control apparatus of the ER works in general and for understanding the specific issue of how a subgroup of α1-AT-deficient individuals become susceptible to liver injury and carcinogenesis. Previous studies have shown that there is a lag in the disposal of this mutant protein in genetically engineered human fibroblasts from “susceptible” deficient patients (10Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (228) Google Scholar). Moreover, this lag in the ER disposal/quality control mechanism appears to be specific, i.e. it affected the disposal of two polymerogenic mutants of α1-AT, but not a model unassembled membrane protein (19Teckman J.H. Perlmutter D.H. J. Biol. Chem. 1996; 271: 13215-13220Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Initial studies of the ER degradation of α1-ATZ indicated that the ubiquitin-dependent proteasomal system was involved. Degradation of α1-ATZ in genetically engineered human fibroblast cell lines and in a cell-free microsomal translocation system was inhibited by lactacystin (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Degradation of α1-ATZ in the cell-free system was shown to be dependent on ATP and, more importantly, a polyubiquitinated calnexin-α1-ATZ complex was shown to be a degradative intermediate (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Subsequent studies by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar) have shown that degradation of α1-ATZ is also inhibited by lactacystin in transfected Chinese hamster ovary cells and in primary cultures of human mononuclear phagocytes. Using an experimental approach in which the degradative machinery in the reticulocyte lysate of the cell-free system is fractionated and reconstituted with purified components, we have recently found evidence for at least three different pathways in the degradation of α1-ATZ, including ubiquitin-dependent and -independent proteasomal mechanisms and at least one nonproteasomal mechanism (12Teckman J.H. Gilmore R. Perlmutter D.H. Am. J. Physiol. 2000; 278: G39-G48Crossref PubMed Google Scholar). Subsequent studies have suggested that autophagy may constitute one of the nonproteasomal mechanisms (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) and have substantiated the concept that there are multiple pathways involved in ER degradation of α1-ATZ. In the most recent study of this issue, Cabral et al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) found that lactacystin did not inhibit degradation of α1-ATZ in a stable transfected murine hepatoma Hepa1–6 cell line. Degradation of α1-ATZ in this cell line was decreased by tyrosine phosphatase inhibitors. Lactacystin did inhibit degradation of α1-AT Null Hong Kong, a truncated mutant, in a separate stable transfected Hepa1–6 cell line. Taken together, these results indicated that the proteasome did not play a role in degradation of α1-ATZ in hepatoma cell line even though the proteasome was active in these cells, that an entirely separate mechanism for degradation existed, and that this distinct mechanism was specific for α1-ATZ. Taking into consideration the previous results indicating involvement of the proteasome in degradation of α1-ATZ in genetically engineered fibroblasts (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), in primary cultures of human macrophages (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), and in the cell-free microsomal system (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), these authors concluded that the proteasomal mechanism was cell type-specific, either for nonhepatocytic cell types and/or cell types with lower levels of endogenous α1-AT expression than hepatocytes (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Because the hepatocyte is an extremely important, if not the most important, site of synthesis of α1-ATZ with respect to the development of liver disease, in this study we sought to examine in further detail the involvement of the proteasome in ER degradation of α1-ATZ in cells of hepatocytic lineage. The results show that lactacystin, MG132, epoxomicin, and clasto-lactacystin β-lactone all inhibit degradation of α1-ATZ in several different types of genetically engineered hepatoma cell lines, including the murine hepatoma Hepa1–6 used by Cabral et al. and a rat hepatoma cell line H11, which has the advantages of being highly differentiated for hepatocytic function, but lacking endogenous expression of α1-AT. Degradation of α1-ATZ in hepatoma cell lines with constitutive and inducible expression of α1-ATZ was blocked to an equivalent extent by proteasomal inhibitors. Finally, studies in HeLa and Hepa1–6 cell lines with inducible expression of α1-ATZ showed that the proteasome was involved in degradation of α1-ATZ at both high and 30–100-fold lower levels of expression. These results suggest to us that the lack of involvement of the proteasome in degradation of α1-ATZ in the Hepa1–6 cell line generated by Cabralet al. (14Cabral C.M. Choudhury P. Liu Y. Sifers R.N. J. Biol. Chem. 2000; 275: 25015-25022Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) is cell line-specific, perhaps reflecting a type of adaptation. This hypothesis by no means diminishes the importance of the observations of Cabral et al. or the importance of tyrosine phosphatases in the quality control mechanism. There is now ample evidence for multiple mechanisms/pathways in the ER quality control apparatus and for cellular “adaptation.” In fact, gene expression profile analysis has shown marked changes in expression of many genes in yeast cells that accumulate misfolded proteins (20Travers K.J. Patil C.K. Wodlicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1570) Google Scholar, 21Casagrande R. Stern P. Diehn M. Shamu C. Osario M. Zuniga M. Brown P.O. Ploegh H. Mol. Cell. 2000; 5: 729-735Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 22Ng D.T.W. Spear E.D. Walter P. J. Cell Biol. 2000; 150: 77-88Crossref PubMed Scopus (273) Google Scholar, 23Friedlander R. Jarosch E. Urban J. Volkwin C. Sommer T. Nat. Cell Biol. 2000; 2: 379-384Crossref PubMed Scopus (388) Google Scholar). Moreover, if a cellular adaptation mechanism is truly applicable, then the results of Cabral et al. raise the interesting possibility that the adaptation is specific for α1-ATZ, a polymerogenic mutant, and not for α1-AT Null Hong Kong, a mutant that is truncated and not likely to be polymerogenic. Three other results of this study deserve comment. First, there is a marked increase in the formation of insoluble aggregates of α1-ATZ and two distinct degradation products appear exclusively in the insoluble fraction when the proteasome is inhibited. These degradation products could theoretically be generated in, and/or accumulate in, the ER or the cytoplasm. Several lines of evidence make it more likely that they are generated in and localize to the ER. α1-ATZ has not been detected outside the ER lumen in intact cells (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar) or in the supernatant of cell-free mammalian translocation reactions (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) and proteasome inhibitors do not induce aggresomes in cells that express mutant α1-ATZ (15Teckman J.H. Perlmutter D.H. Am. J. Physiol. 2000; 279: G961-G974Crossref PubMed Google Scholar). Similar degradation products are generated processively in the lumen of microsomal vesicles that have translocated wild type α1-ATZ in a cell-free microsomal translocation reaction (11Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), suggesting the existence of an endoluminal proteolytic system that recognizes wild type or mutant α1-ATZ when it is retained in the ER for a prolonged period of time. Second, there was no evidence for a significant increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, or HeLa cells in the presence of lactacystin, MG132, epoxomicin, or clasto-lactacystin β-lactone at doses optimal for inhibition of degradation of α1-ATZ. Using the lower doses of lactacystin or longer periods of preincubation with lactacystin described by Novoradovskayaet al. (13Novoradovskaya N. Lee J.H. Yu Z.-X. Ferrans V.J. Brantly M. J. Clin. Invest. 1998; 101: 2693-2701Crossref PubMed Scopus (40) Google Scholar), we observed a lesser degree of inhibition of degradation, but no increase in secretion of α1-ATZ in fibroblasts, hepatoma cells, and HeLa cells at low and high levels of expression (data not shown). Third, secretion of α1-ATZ did not increase when the level of its synthesis was decreased. This was shown by modulating the level of synthesis 30–100-fold in tetracycline-regulated cell lines. This result is noteworthy because the rate of polymerization of α1-ATZ decreases at lower concentrations in purified systems (6Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (886) Google Scholar). The results in this report therefore suggest that other factors working in concert with polymerization play a role in the fate of α1-ATZ when it accumulates in the ER. We are indebted to Marilyn Maksin for preparing the manuscript.

Interleukin-1 (IL-1) is a product of mononuclear phagocytes that mediates changes characteristic of the response to inflammation or tissue injury (the acute-phase response). One of two structurally and functionally homologous major histocompatibility complex (MHC) class III genes encodes a positive acute-phase protein, complement factor B. The closely linked complement C2 gene is not affected during the acute-phase response. Purified human IL-1, pH 7.0, and recombinant-generated murine IL-1, pH 5.0, increased the expression of factor B and other positive acute-phase proteins in human hepatoma cells but decreased the expression of albumin, a negative acute-phase reactant. Furthermore, in a murine fibroblast L-cell line transfected with cosmid DNA bearing the human C2 and factor B genes, IL-1 mediated a reversible dose- and time-dependent increase in factor B expression in the transfected cells. Expression of the C2 gene was not affected by IL-1. The effect of IL-1 on factor B expression involves a mechanism acting at a pre-translational level as demonstrated by an increase in specific messenger RNA content and a corresponding increase in biosynthesis and secretion of factor B. The structural basis and mechanism for selective and independent regulation of these genes provides insight into the molecular control of the inflammatory response.

y-Interferon (IFN-y) is a well characterized lymphokine known to regulate many mononuclear phagocyte functions, including expression of class I and class I1 major histocompatibility complex genes.The second component of complement (C2) and factor B are major histocompatibility complex class I11 gene products synthesized in mononuclear phagocytes.Recombinant IFN-y increased the synthesis of C2 and factor B in primary cultures of human mononuclear phagocytes and in murine fibroblasts transfected with cosmid DNA bearing the human C2 and factor B genes.In both cell types the increases in C2 and factor B protein synthesis were detected at concentrations of IFN-y less than 1 unitlml and the regulation of each was pretranslational.The IFN-y-induced increases in C2 and factor B mRNA did not require new protein synthesis.In primary cultures of human monocytes, the kinetics of induction of C2 and factor B synthesis differed, but in the transfected L-cells the kinetics were similar, suggesting differences in transduction of the IFN-y signal, transcriptional, and/or post-transcriptional events in the two cell types.The small size of the factor B 5' flanking region, which is bounded by the 3' terminus of the IFN-y-regulated C2 gene, provides a well defined region to probe the structural basis for IFN-r regulation of gene expression.The complement system is a set of 20 plasma proteins which is an effector of several functions associated with inflammation, immunologic regulation, and cytotoxicity (1).The genes for two of these proteins, the second component (C2) and factor B, are located in humans within the major histocompatibility complex (MHC') between HLA-DR and HLA-B (2) and are two of the class I11 MHC genes.The C2 Institutes of Health (AM 26609, AI 20032, HD 17461, HL 22487), the

Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.

The cytokine IFN beta 2/IL-6 has recently been shown to regulate the expression of genes encoding hepatic acute phase plasma proteins. INF beta 2/IL-6 has also been shown to be identical to MGI-2, a protein that induces differentiation of bone marrow precursor cells toward mature granulocytes and monocytes. Accordingly, we have examined the effect of IFN beta 2/IL-6 on expression of the IL-1- and tumor necrosis factor-unresponsive acute phase protein alpha 1-antitrypsin (alpha 1 AT) in human hepatoma-derived hepatocytes and in human mononuclear phagocytes. Purified human fibroblast and recombinant IFN beta 2/IL-6 each mediate a specific increase in steady-state levels of alpha 1 AT mRNA and a corresponding increase in net synthesis of alpha 1 AT in primary cultures of human peripheral blood monocytes as well as in HepG2 and Hep3B cells. Thus, the effect of IFN beta 2/IL-6 on alpha 1 AT gene expression in these cells is primarily due to an increase in accumulation of alpha 1 AT mRNA and can be distinguished from the direct, predominantly translational effect of bacterial lipopolysaccharide on expression of this gene in monocytes and macrophages. The results indicate that IFN beta 2/IL-6 regulates acute phase gene expression, specifically alpha 1 AT gene expression, in extrahepatic as well as hepatic cell types.

Although the classical chemotactic receptor for complement anaphylatoxin C5a has been associated with polymorphonuclear and mononuclear phagocytes, several recent studies have indicated that this receptor is expressed on nonmyeloid cells including human endothelial cells, vascular smooth muscle cells, bronchial and alveolar epithelial cells, hepatocytes, and in the human hepatoma cell line HepG2. In this study, we examined the possibility that other members of the chemotactic receptor family are expressed in HepG2 cells and human liver, and the possibility that such receptors mediate changes in acute phase gene expression in HepG2 cells. Using polymerase chain reaction (PCR) amplification of HepG2 mRNA with primers based on highly conserved regions of the chemotactic subgroup of the G protein-coupled receptor family, we identified a PCR fragment from the formyl-methionyl-leucyl-phenylalanine (FMLP) receptor, as well as one from the C5a receptor. Immunostaining with antipeptide antisera to FMLPR confirmed the presence of this receptor in HepG2 cells. Receptor binding studies showed specific saturable binding of a radioiodinated FMLP analogue to HepG2 cells (Kd approximately 2.47 nM; R approximately 6 x 10(3) plasma membrane receptors per cell). In situ hybridization analysis showed the presence of FMLPR mRNA in parenchymal cells of the human liver in vivo. Both C5a and FMLP mediated concentration- and time-dependent changes in synthesis of acute phase proteins in HepG2 cells including increases in complement C3, factor B, and alpha 1-antichymotrypsin, as well as concomitant decreases in albumin and transferrin synthesis. The effects of C5a and FMLP on the synthesis of these acute phase proteins was evident at concentrations as low as 1 nM, and they were specifically blocked by antipeptide antisera for the corresponding receptor. In contrast to the effect of other mediators of hepatic acute phase gene regulation, such as interleukin 6, the effects of C5a and FMLP were reversed by increased concentrations well above the saturation point of the respective receptor. These results suggest that acute phase gene regulation by C5a and FMLP is desensitized at high concentrations, a property that is unique among the several known mechanisms for hepatic acute phase gene regulation.

Acidosis caused by intestinal bacterial d-lactate production occurs in ruminants engorged with carbohydrate. A similar phenomenon was identified in two children who developed recurrent episodes of metabolic acidosis and peculiar neurologic symptoms in response to increased dietary carbohydrate after major small bowel resections. Both children were found to have elevated plasma concentrations of d-lactic acid at the time of each episode. Acid base and neurologic abnormalities responded immediately to neomycin therapy. Among a number of microorganisms isolated from stool cultures of these patients, one anaerobic <i>Lactobacillus acidophilus</i> species produced large amounts of d-lactate in vitro. Reduction in carbohydrate intake in one patient tested led to a fall in d-lactate generation. We believe that excessive d-lactate production by intestinal bacteria, from malabsorbed carbohydrate, may produce metabolic acidosis and neurologic symptoms in children with small bowel resections.

In recent years, we have witnessed several important paradigm shifts in understanding the molecular basis of liver disease in alpha-1-antitrypsin (AT) deficiency. These shifts have become possible as a result of a number of advances in research on the cell biology of aggregation-prone mutant proteins and in research on the pathobiological mechanisms of liver disease in general. Late-breaking research in these areas was the subject of an AASLD/Alpha-1 Foundation Single Topic Conference in Atlanta, Georgia, on January 26 to 28, 2006. The conference was titled "Alpha-1-Antitrypsin Deficiency and Other Liver Diseases Caused by Aggregated Proteins." Investigators from all over the world, representing a broad array of scientific disciplines and perspectives, discussed the pathobiology of AT deficiency, mechanisms of cell injury in diseases associated with aggregation-prone proteins, pathways by which cells respond to protein aggregation and mislocalization, and mechanisms of liver injury in general and in diseases related to AT deficiency. A session of the meeting was devoted to novel therapeutic strategies being developed for AT deficiency as well as to strategies either in development or already being applied to the class of diseases associated with mutant proteins.

Alpha-1-antitrypsin (AT) deficiency is a relatively common autosomal co-dominant disorder, which causes chronic lung and liver disease. A point mutation renders aggregation-prone properties on a hepatic secretory protein in such a way that the mutant protein is retained in the endoplasmic reticulum of hepatocytes rather than secreted into the blood and body fluids where it ordinarily functions as an inhibitor of neutrophil proteases. A loss-of-function mechanism allows neutrophil proteases to degrade the connective tissue matrix of the lung causing chronic emphysema. Accumulation of aggregated mutant AT in the endoplasmic reticulum of hepatocytes causes liver inflammation and carcinogenesis by a gain-of-toxic function mechanism. However, genetic epidemiology studies indicate that many, if not the majority of, affected homozygotes are protected from liver disease by unlinked genetic and/or environmental modifiers. Studies performed over the last several years have demonstrated the importance of autophagy in disposal of mutant, aggregated AT and raise the possibility that predisposition to, or protection from, liver injury and carcinogenesis is determined by the balance of de novo biogenesis of the mutant AT molecule and its autophagic disposal.

As an experimental system, Caenorhabditis elegans offers a unique opportunity to interrogate in vivo the genetic and molecular functions of human disease-related genes. For example, C. elegans has provided crucial insights into fundamental biologic processes, such as cell death and cell fate determinations, as well as pathologic processes such as neurodegeneration and microbial susceptibility. The C. elegans model has several distinct advantages, including a completely sequenced genome that shares extensive homology with that of mammals, ease of cultivation and storage, a relatively short lifespan and techniques for generating null and transgenic animals. However, the ability to conduct unbiased forward and reverse genetic screens in C. elegans remains one of the most powerful experimental paradigms for discovering the biochemical pathways underlying human disease phenotypes. The identification of these pathways leads to a better understanding of the molecular interactions that perturb cellular physiology, and forms the foundation for designing mechanism-based therapies. To this end, the ability to process large numbers of isogenic animals through automated work stations suggests that C. elegans, manifesting different aspects of human disease phenotypes, will become the platform of choice for in vivo drug discovery and target validation using high-throughput/content screening technologies.

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Biliary atresia (BA) is the most common cause of end‐stage liver disease in children and the primary indication for pediatric liver transplantation, yet underlying etiologies remain unknown. Approximately 10% of infants affected by BA exhibit various laterality defects (heterotaxy) including splenic abnormalities and complex cardiac malformations—a distinctive subgroup commonly referred to as the biliary atresia splenic malformation (BASM) syndrome. We hypothesized that genetic factors linking laterality features with the etiopathogenesis of BA in BASM patients could be identified through whole‐exome sequencing (WES) of an affected cohort. DNA specimens from 67 BASM subjects, including 58 patient–parent trios, from the National Institute of Diabetes and Digestive and Kidney Diseases–supported Childhood Liver Disease Research Network (ChiLDReN) underwent WES. Candidate gene variants derived from a prespecified set of 2,016 genes associated with ciliary dysgenesis and/or dysfunction or cholestasis were prioritized according to pathogenicity, population frequency, and mode of inheritance. Five BASM subjects harbored rare and potentially deleterious biallelic variants in polycystic kidney disease 1 like 1 ( PKD1L1 ), a gene associated with ciliary calcium signaling and embryonic laterality determination in fish, mice, and humans. Heterozygous PKD1L1 variants were found in 3 additional subjects. Immunohistochemical analysis of liver from the one BASM subject available revealed decreased PKD1L1 expression in bile duct epithelium when compared to normal livers and livers affected by other noncholestatic diseases. Conclusion: WES identified biallelic and heterozygous PKD1L1 variants of interest in 8 BASM subjects from the ChiLDReN data set; the dual roles for PKD1L1 in laterality determination and ciliary function suggest that PKD1L1 is a biologically plausible, cholangiocyte‐expressed candidate gene for the BASM syndrome.

During the formation of an inhibitory complex with neutrophil elastase, alpha 1 antitrypsin (alpha 1 AT) undergoes a structural rearrangement and the resulting alpha 1 AT-elastase complex becomes endowed with chemoattractant activities, mediates an increase in synthesis of alpha 1 AT, and is rapidly cleared from the circulation. In previous studies we have provided evidence that these biological activities involve the recognition of a conformation-specific domain in the alpha 1 AT molecule by a cell surface receptor on human hepatoma HepG2 cells and human monocytes. The receptor has been termed the serpin-enzyme complex (SEC) receptor because it also recognizes complex of serpins antithrombin III, alpha 1 anti-chymotrypsin, and C1 inhibitor with their cognate enzymes. Because a pentapeptide domain of alpha 1 AT (amino acids 370-374, Phe-Val-Phe-Leu-Met) is sufficient for binding to the SEC receptor and the sequence of this domain is remarkably similar to those of substance P, several other tachykinins, bombesin, and the amyloid-beta peptide, we have examined the possibility that these other ligands bind to the SEC receptor. The results indicate that substance P, several other tachykinins, and bombesin compete for binding to, and cross-linking of, the SEC receptor. The SEC receptor is distinct from the substance P receptor by several criteria. There is no substance P receptor mRNA in HepG2 cells; the SEC receptor is present in much higher density on receptor-bearing cells and binds its ligands at lower affinity than the substance P receptor; the SEC receptor is much less restricted in the specificity with which it recognizes ligand; ligands for the SEC receptor including peptide 105Y (based on alpha 1 AT sequence 359-374), alpha 1 AT-protease complexes, and bombesin do not compete for binding of substance P to a stable transfected cell line expressing the substance P receptor. Finally, we show here that the amyloid-beta peptide competes for binding to the SEC receptor but does not bind to the substance P receptor, therein raising the possibility that the SEC receptor is involved in certain biological activities, including the recently described neurotrophic and neurotoxic effects ascribed to the amyloid-beta peptide.

Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.

A approximately 110-kDa glycoprotein purified from canalicular vesicles by bile acid affinity chromatography has been identified as the canalicular bile acid transport protein. Internal amino acid sequence and chemical and immunochemical characteristics of this protein were found to be identical to a rat liver canalicular ecto-ATPase. In order to definitively determine whether these were two activities of a single polypeptide, we examined the possibility that transfection of cDNA for the ecto-ATPase would confer bile acid transport characteristics, as well as ecto-ATPase activity, on heterologous cells. The results show that transfection of the ecto-ATPase cDNA conferred on COS cells de novo synthesis of a approximately 110-kDa polypeptide, as immunoprecipitated by antibody to the purified canalicular bile acid transport protein and conferred on COS cells the capacity to pump out [3H]taurocholate with efflux characteristics comparable with those previously determined in canalicular membrane vesicles (Km = 100 microM; Vmax = 200 pmol/mg of protein/20 s). A truncated ecto-ATPase cDNA, missing the cytoplasmic tail, was targeted correctly to the cell surface but did not confer bile acid transport activity on COS cells. The results of this study also show that the canalicular ecto-ATPase/bile acid transport protein is phosphorylated on its cytoplasmic tail and that its phosphorylation is stimulated by activation of protein kinase C and inhibited by inhibitors of protein kinase C activation. Moreover, inhibition of protein kinase C activation by staurosporine completely abrogates bile acid transport but does not affect ATPase activity. This study, therefore, demonstrates that the rat liver canalicular ecto-ATPase is also a bile acid transport protein, that the capacity to pump out bile acid can be conferred on a heterologous cell by DNA-mediated gene transfer, and that phosphorylation within the cytoplasmic tail of the transporter is essential for bile acid efflux activity but not for ATPase activity.

In α 1 -antitrypsin (α 1 -AT) deficiency, a mutant form of α 1 -AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant α 1 -AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The α 1 -AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which α 1 -AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant α 1 -AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of ∼110–130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic α 1 -AT mutants that are retained in the ER, indicating that these interactions are not specific for the α 1 -AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that ∼85% of α 1 -AT Z exists in heterogeneous soluble complexes with multiple chaperones and ∼15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of α 1 -AT Z as well as on its residual secretion.

Alpha-1-antitrypsin (AT) deficiency is the most common genetic cause of liver disease in children. In addition to chronic liver inflammation and injury, it has a predilection to cause hepatocellular carcinoma later in life. The deficiency is caused by a mutant protein, ATZ, which is retained in the endoplasmic reticulum (ER) in a polymerized form rather than secreted into the blood in its monomeric form. The histologic hallmark of the disease is ATZ-containing globules in some, but not all, hepatocytes. Liver injury results from a gain-of-toxic function mechanism in which mutant ATZ retained in the ER initiates a series of pathologic events, but little is known about the mechanism by which this leads to carcinogenesis. Several recent observations from my laboratory have led to a novel hypothetical paradigm for carcinogenesis in AT deficiency in which globule-containing hepatocytes are "sick," relatively growth suppressed, but also elaborating trans-acting regenerative signals. These signals are received and transduced by globule-devoid hepatocytes, which, because they are younger and have a lesser load of accumulated ATZ, have a selective proliferative advantage. Chronic regeneration in the presence of tissue injury leads to adenomas and ultimately carcinomas. Aspects of this hypothetical paradigm may also explain the proclivity for hepatocarcinogenesis in other chronic liver diseases, including other genetic diseases, viral hepatitis, and nonalcoholic steatohepatitis.

alpha 1-Antitrypsin (alpha 1-AT) is considered a typical plasma protein and a prototype of the serine proteinase inhibitor (serpin) family. It is synthesized in hepatocytes and, to a lesser extent, in macrophages. In this study we show that the alpha 1-AT gene is also expressed in human intestine and in a human colonic epithelial tumor cell line, Caco2. A single 1.6-kilobase alpha 1-AT-specific mRNA is present in jejunum and in Caco2 cells. It is identical in apparent size to that present in human hepatoma HepG2 cells but slightly smaller than that present in human macrophages, cells in which an alternative upstream transcriptional start site is used. Synthesis and secretion of alpha 1-AT in Caco2 cells is similar to that in HepG2 cells. It is synthesized as an approximately 52-kDa precursor polypeptide, converted to its mature, fully glycosylated 55-kDa form intracellularly, and the native protein is secreted with a half-time of 37 min. Functionally active alpha 1-AT is secreted into the basolateral and apical (luminal) fluid in pulse-chase labeling experiments of Caco2 cells cultured in polarized orientation on collagen-coated nitrocellulose membranes. Expression of alpha 1-AT in Caco2 enterocytes is not affected by soluble factors that regulate expression of alpha 1-AT in macrophages and hepatocytes. However, expression of alpha 1-AT increases markedly in Caco2 cells as they differentiate into enteric villous-type cells.

To determine the basis for low serum concentrations of alpha 1-proteinase inhibitor (alpha 1PI) in individuals with homozygous alpha 1PI deficiency (hereafter referred to as PiZZ), biosynthesis and secretion of alpha 1PI were studied in Xenopus oocytes microinjected with hepatic mRNA and in blood monocytes (an extrahepatic site of alpha 1PI gene expression). Although both the usual alpha 1PI (hereafter referred to as PiMM) and PiZZ alpha 1PI were secreted in functionally active form, the rate of secretion of alpha 1PI was significantly and selectively decreased in Xenopus oocytes injected with PiZZ liver mRNA and in monocytes from PiZZ individuals. The apparent size of alpha 1PI in the intracellular compartment of Xenopus oocytes injected with PiZZ liver mRNA was different from the corresponding intracellular PiMM alpha 1PI in oocytes injected with PiMM liver mRNA. There were also differences in the relative ratio of native and complexed alpha 1PI secreted by monocytes from individuals with PiMM and PiZZ phenotypes.

Alpha-1-antitrypsin (AT) deficiency was first described in the late 1960s in patients with severe pulmonary emphysema . The recognition of AT deficiency as a cause of emphysema then led to what is still the prevailing theory for the pathogenesis of emphysema, the protease-antiprotease theory. Soon it was found that AT deficiency accounted for a significant number of cases of neonatal liver disease that were previously categorized as idiopathic. We now know that AT deficiency is the most common genetic cause of neonatal liver disease and the most frequent diagnosis necessitating liver transplantation. It has also been shown to cause chronic liver disease , cryptogenic cirrhosis , and hepatocellular carcinoma in adults never previously known to have liver disease in infancy or childhood. Observations indicate that genetic traits unlinked to the AT gene or environmental factors predispose to or protect AT-deficient individuals from liver disease.

There is now extensive evidence that amyloid-β peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-β 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of α1-antitrypsin (α1-AT) and other serine protease inhibitors, also recognizes the amyloid-β 25-35 domain. In fact, by recognizing the amyloid-β 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-β peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-β peptide. A series of peptides based on the sequences of amyloid-β peptide and α1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-β peptide 25-35 and amyloid-β peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-β 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric amβ/α1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-β 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-β peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-β peptide, if soluble amyloid-β peptide proves to be an in vivo precursor of the insoluble, toxic peptide. There is now extensive evidence that amyloid-β peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-β 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of α1-antitrypsin (α1-AT) and other serine protease inhibitors, also recognizes the amyloid-β 25-35 domain. In fact, by recognizing the amyloid-β 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-β peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-β peptide. A series of peptides based on the sequences of amyloid-β peptide and α1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-β peptide 25-35 and amyloid-β peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-β 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric amβ/α1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-β 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-β peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-β peptide, if soluble amyloid-β peptide proves to be an in vivo precursor of the insoluble, toxic peptide.

AbstractIn the classical form of alpha-1-antitrypsin (AT) deficiency a point mutation renders aggregation-prone properties on a hepatic secretory protein. The mutant ATZ protein in retained in the endoplasmic reticulum (ER) of liver cells rather than secreted into the blood and body fluids where it ordinarily functions as an inhibitor of neutrophil proteases. A loss-of-function mechanism allows the neutrophil proteases to slowly destroy the connective tissue matrix of the lung, resulting in premature development of pulmonary emphysema as early as the third decade of life. A gain-of-toxic function mechanism is responsible for liver inflammation and carcinogenesis. Indeed this deficiency is the most common genetic cause of liver disease in children in the US. It also causes chronic liver inflammation and carcinoma that manifests itself later in life. However, the majority of affected homozygotes apparently escape liver disease. This last observation has led to the concept that genetic and/or environmental modifiers affect the disposal of mutant ATZ within the ER or affect the protective cellular responses activated by accumulation of ATZ in the ER and, in turn, these modifiers determine which homozygotes develop liver inflammation and carcinoma. In this article I review a series of studies published over the last 6 years showing that autophagy is specifically activated by ER accumulation of ATZ and that autophagy plays a critical role in the disposal of this mutant protein. Further, I review data suggesting that the autophagy is specifically designed for the cellular response to aggregated ATZ and aggregated proteins in general.

The net balance of neutrophil elastase, an enzyme that degrades many components of the extracellular matrix, and its inhibitor, alpha-1-proteinase inhibitor (alpha 1 PI), is thought to be a critical determinant in the development of destructive lung disease, especially in individuals with homozygous alpha 1 PI deficiency. Synthesis and secretion of alpha 1 PI has been recently demonstrated in cells of mononuclear phagocyte lineage, including peripheral blood monocytes and tissue macrophages. In this study we show that alpha 1 PI gene expression in human monocytes and bronchoalveolar macrophages is affected by a novel mechanism, whereby elastase directly regulates the synthesis of its inhibitor. In nanomolar concentrations, neutrophil or pancreatic elastase mediates a dose- and time-dependent increase in steady state levels of alpha 1 PI mRNA and in the rate of synthesis of alpha 1 PI in human monocytes and bronchoalveolar macrophages. Antisera to neutrophil elastase or pretreatment of elastase with the serine proteinase inhibitor diisopropylfluorophosphate abrogates the effect of elastase on alpha 1 PI expression. Elastase also stimulates the synthesis of alpha 1 PI in monocytes from homozygous PiZZ alpha 1 PI-deficient individuals, but has no effect on the rate of secretion; hence, the enzyme mediates an effect on alpha 1 PI that increases the intracellular accumulation of inhibitor and exaggerates the intrinsic defect in secretion of alpha 1 PI that characterizes the homozygous PiZZ alpha 1 PI deficiency.

In the classical form of α₁-antitrypsin deficiency, a mutant protein accumulates in a polymerized form in the endoplasmic reticulum (ER) of liver cells causing liver damage and carcinogenesis by a gain-of-toxic function mechanism. Recent studies have indicated that the accumulation of mutant α₁-antitrypsin Z in the ER specifically activates the autophagic response but not the unfolded protein response and that autophagy plays a critical role in disposal of insoluble α₁-antitrypsin Z. In this study, we used genomic analysis of the liver in a novel transgenic mouse model with inducible expression to screen for changes in gene expression that would potentially define how the liver responds to accumulation of this mutant protein. There was no unfolded protein response. Of several distinct gene expression profiles, marked up-regulation of regulator of G signaling (RGS16) was particularly notable. RGS16 did not increase when model systems were exposed to classical inducers of ER stress, including tunicamycin and calcium ionophore, or when a nonpolymerogenic α₁-antitrypsin mutant accumulated in the ER. RGS16 was up-regulated in livers from patients with α₁-antitrypsin deficiency, and the degree of up-regulation correlated with the hepatic levels of insoluble α₁-antitrypsin Z protein. Taken together, these results indicate that expression of RGS16 is an excellent marker for the distinct form of "ER stress" that occurs in α₁-antitrypsin deficiency, presumably determined by the aggregation-prone properties of the mutant protein that characterizes the deficiency.

The classical form of α1-antitrypsin deficiency (ATD) is an autosomal co-dominant disorder that affects ~1 in 3000 live births and is an important genetic cause of lung and liver disease. The protein affected, α1-antitrypsin (AT), is predominantly derived from the liver and has the function of inhibiting neutrophil elastase and several other destructive neutrophil proteinases. The genetic defect is a point mutation that leads to misfolding of the mutant protein, which is referred to as α1-antitrypsin Z (ATZ). Because of its misfolding, ATZ is unable to efficiently traverse the secretory pathway. Accumulation of ATZ in the endoplasmic reticulum of liver cells has a gain-of-function proteotoxic effect on the liver, resulting in fibrosis, cirrhosis and/or hepatocellular carcinoma in some individuals. Moreover, because of reduced secretion, there is a lack of anti-proteinase activity in the lung, which allows neutrophil proteases to destroy the connective tissue matrix and cause chronic obstructive pulmonary disease (COPD) by loss of function. Wide variation in the incidence and severity of liver and lung disease among individuals with ATD has made this disease one of the most challenging of the rare genetic disorders to diagnose and treat. Other than cigarette smoking, which worsens COPD in ATD, genetic and environmental modifiers that determine this phenotypic variability are unknown. A limited number of therapeutic strategies are currently available, and liver transplantation is the only treatment for severe liver disease. Although replacement therapy with purified AT corrects the loss of anti-proteinase function, COPD progresses in a substantial number of individuals with ATD and some undergo lung transplantation. Nevertheless, advances in understanding the variability in clinical phenotype and in developing novel therapeutic concepts is beginning to address the major clinical challenges of this mysterious disorder.

Abstract Autophagy and apoptosis are cellular processes that regulate cell survival and death, the former by eliminating dysfunctional components in the cell, the latter by programmed cell death. Stress signals can induce either process, and it is unclear how cells ‘assess’ cellular damage and make a ‘life’ or ‘death’ decision upon activating autophagy or apoptosis. A computational model of coupled apoptosis and autophagy is built here to analyze the underlying signaling and regulatory network dynamics. The model explains the experimentally observed differential deployment of autophagy and apoptosis in response to various stress signals. Autophagic response dominates at low-to-moderate stress; whereas the response shifts from autophagy (graded activation) to apoptosis (switch-like activation) with increasing stress intensity. The model reveals that cytoplasmic Ca 2+ acts as a rheostat that fine-tunes autophagic and apoptotic responses. A G-protein signaling-mediated feedback loop maintains cytoplasmic Ca 2+ level, which in turn governs autophagic response through an AMP-activated protein kinase (AMPK)-mediated feedforward loop. Ca 2+ /calmodulin-dependent kinase kinase β (CaMKKβ) emerges as a determinant of the competing roles of cytoplasmic Ca 2+ in autophagy regulation. The study demonstrates that the proposed model can be advantageously used for interrogating cell regulation events and developing pharmacological strategies for modulating cell decisions.

Abstract Operando X-ray micro-computed tomography (µCT) provides an opportunity to observe the evolution of Li structures inside pouch cells. Segmentation is an essential step to quantitatively analyzing µCT datasets but is challenging to achieve on operando Li-metal battery datasets due to the low X-ray attenuation of the Li metal and the sheer size of the datasets. Herein, we report a computational approach, batteryNET, to train an Iterative Residual U-Net-based network to detect Li structures. The resulting semantic segmentation shows singular Li-related component changes, addressing diverse morphologies in the dataset. In addition, visualizations of the dead Li are provided, including calculations about the volume and effective thickness of electrodes, deposited Li, and redeposited Li. We also report discoveries about the spatial relationships between these components. The approach focuses on a method for analyzing battery performance, which brings insight that significantly benefits future Li-metal battery design and a semantic segmentation transferrable to other datasets.

α 1 -Antitrypsin (α 1 -AT) deficiency causes severe liver injury in a subgroup of patients. Liver injury is thought to be caused by retention of a polymerized mutant α 1 -ATZ molecule in the endoplasmic reticulum (ER) of hepatocytes and is associated with an intense autophagic response. However, there is limited information about what physiologic stressors might influence liver injury. In this study, we examined the effect of fasting in the PiZ mouse model of α 1 -AT deficiency, because fasting is a well-characterized physiological stressor and a known stimulus for autophagy. Results show that there is a marked increase in fat accumulation and in α 1 -AT-containing globules in the liver of the PiZ mouse induced by fasting. Although fasting induced a marked autophagic response in wild-type mice, the autophagic response was already activated in PiZ mice and did not further increase with fasting. PiZ mice also had a significantly decreased tolerance for prolonged fasting compared with wild-type mice (PiZ mice 0% survival of 72-h fast; wild-type 100% survivial). These results demonstrate an altered response to stress in the α 1 -AT-deficient liver, including inability to further increase an activated autophagic response, a developmental state-specific increase in α 1 -AT-containing globules, and increased mortality.

It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein α1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of α1 antitrypsin (α1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of α1 antitrypsin Z (α1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of α1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with α1-AT deficiency. It is now well known that the addition and trimming of oligosaccharide side chains during post-translational modification play an important role in determining the fate of secretory, membrane, and lysosomal glycoproteins. Recent studies have suggested that trimming of oligosaccharide side chains also plays a role in the degradation of misfolded glycoproteins as a part of the quality control mechanism of the endoplasmic reticulum (ER). In this study, we examined the effect of several inhibitors of carbohydrate processing on the fate of the misfolded secretory protein α1 antitrypsin Z. Retention of this misfolded glycoprotein in the ER of liver cells in the classical form of α1 antitrypsin (α1-AT) deficiency is associated with severe liver injury and hepatocellular carcinoma and lack of its secretion is associated with destructive lung disease/emphysema. The results show marked alterations in the fate of α1 antitrypsin Z (α1-ATZ). Indeed, one glucosidase inhibitor, castanospermine (CST), and two mannosidase inhibitors, kifunensine (KIF) and deoxymannojirimycin (DMJ), mediate marked increases in secretion of α1-ATZ by distinct mechanisms. The effects of these inhibitors on secretion have interesting implications for our understanding of the quality control apparatus of the ER. These inhibitors may also constitute models for development of additional drugs for chemoprophylaxis of liver injury and emphysema in patients with α1-AT deficiency. endoplasmic reticulum α1 antitrypsin α1 antitrypsin Z kifunensine deoxymannojirimycin castanospermine N-methyldeoxynojirimycin 1,4-dideoxy-1,4-imino-d-mannitol hydrochloride endoglycosidase H N-glycosidase F polyacrylamide gel electrophoresis Dulbecco's modified Eagle's medium extracellular fluid cell lysate(s) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid phenylmethylsulfonyl fluoride phenylbutyric acid Recent studies have provided further evidence that asparagine-linked oligosaccharide side chains play an important role in intracellular transport of glycoproteins. For one, mannose-6-phosphate modification is a key determinant of sorting to the lysosome (1.Kornfeld S. Mellman I. Annu. Rev. Cell Biol. 1989; 5: 483-525Crossref PubMed Scopus (1235) Google Scholar). Second, a number of studies have now shown that transport of secretory and membrane glycoproteins from the ER1 to their appropriate destination depends on the interaction of the innermost glucose residue of the oligosaccharide side chains with the resident ER molecular chaperones calnexin and calreticulin (2.Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 3.Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). This means that trimming of the N-glycan by glucosidases I and II and interaction with calnexin and calreticulin facilitate the proper folding and translocation of wild type glycoproteins. There is also evidence that trimming of glucose residues by glucosidases and of mannose residues by ER mannosidases is involved in the degradation of misfolded, unassembled, or mutant glycoproteins (4.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 6.Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (303) Google Scholar, 7.Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 8.Moore S. Spiro R. J. Biol. Chem. 1993; 268: 3809-3813Abstract Full Text PDF PubMed Google Scholar, 9.Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar, 10.Vierhoeven A.J.M. Neve B.P. Jansen H. Biochem. J. 1999; 337: 133-140Crossref PubMed Google Scholar). Third, Nichols et al. (11.Nichols W.C. Seligsohn U. Zivelin A. Terry V.H. Hertel C.E. Wheatley M.A. Moussalli M.J. Hauri H.-P. Ciavarella N. Kaufman R.J. Ginsburg D. Cell. 1998; 93: 61-70Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar) have shown that ERGIC-53, a lectin which is specifically localized in the ER-Golgi intermediate compartment, is mutated in the combined deficiency of coagulation factors V and VIII. These results suggest that a lectin-like mechanism involving the interaction of carbohydrate side chains with ERGIC-53 is required for secretion of factors V and VIII. In the classic type of α1 antitrypsin (α1-AT) deficiency, the most common genetic cause of emphysema in adults and of liver disease in children, the mutant glycoprotein α1-ATZ is retained in the ER of liver cells rather than secreted into the extracellular fluid (12.Teckman J. Qu D. Perlmutter D.H. Hepatology. 1996; 24: 1504-1516PubMed Google Scholar). The mutant α1-ATZ molecule is characterized by a single amino acid substitution, which results in its polymerization in the ER (13.Lomas D.A. Evans D.L. Finch J.J. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (899) Google Scholar, 14.Yu M.-H. Lee K.N. Kim J. Nat. Struct. Biol. 1995; 2: 363-367Crossref PubMed Scopus (141) Google Scholar). However, the mutant protein retains ∼80% of the functional activity of its wild type counterpart, inhibition of neutrophil elastase (15.Bathurst I.C. Travis J. George P.M. Carrell R.W. FEBS Lett. 1984; 177: 179-183Crossref PubMed Scopus (46) Google Scholar,16.Ogushi F. Fells G.A. Hubbard R.C. Straus S.D. Crystal R.G. J. Clin. Invest. 1987; 89: 1366-1374Crossref Scopus (159) Google Scholar). Because of the lack of this elastase inhibitor in the lung, deficient individuals often develop destructive lung disease/emphysema (12.Teckman J. Qu D. Perlmutter D.H. Hepatology. 1996; 24: 1504-1516PubMed Google Scholar). A subgroup of α1-AT-deficient individuals, predominantly infants and children, also develop chronic liver disease apparently because of the hepatotoxic effect of the mutant α1-ATZ molecule retained in the ER (12.Teckman J. Qu D. Perlmutter D.H. Hepatology. 1996; 24: 1504-1516PubMed Google Scholar). Recent studies have indicated that this subgroup is “susceptible” to liver injury by virtue of a lag in ER degradation resulting in greater accumulation of the misfolded hepatotoxic α1-ATZ molecule in liver cells (17.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (234) Google Scholar). There are at least two and perhaps more pathways responsible for degradation of α1-ATZ in the ER. One pathway involves stable binding of α1-ATZ to calnexin, conjugation of ubiquitin to the cytoplasmic tail of complexed calnexin, and degradation of the α1-ATZ-polyubiquitinated calnexin complex by the proteasome (18.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). There also appears to be a ubiquitin-independent proteasomal mechanism for degradation of α1-ATZ in the ER (19.Teckman, J. H., Gilmore, R., and Perlmutter, D. H. (1999) Am. J. Physiol., in pressGoogle Scholar). In the current study, we used glucosidase and mannosidase inhibitors to examine the role of oligosaccharide side chain trimming in the fate of α1-ATZ. Previous studies have shown that glucosidase and mannosidase inhibitors inhibit secretion of wild type α1-AT (20.Gross V. Tran-Thi T.-A. Schwarz R.T. Elbein A.D. Decker K. Heinrich P.C. Biochem. J. 1986; 236: 853-860Crossref PubMed Scopus (36) Google Scholar). Studies of another mutant α1-AT molecule which is retained and degraded in the ER, α1-ATHONG KONG, have shown that glucosidase and mannosidase inhibitors alter ER degradation—accelerated by glucosidase inhibitors and delayed by mannosidase inhibitors (4.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). However, there are no previous reports of the effect of these inhibitors on mutant α1-ATZ. The results show that there are effects on ER degradation but, to our surprise, several glucosidase and mannosidase inhibitors mediated an increase in secretion of α1-ATZ. Because the mutant α1-ATZ molecule partially retains functional activity (15.Bathurst I.C. Travis J. George P.M. Carrell R.W. FEBS Lett. 1984; 177: 179-183Crossref PubMed Scopus (46) Google Scholar, 16.Ogushi F. Fells G.A. Hubbard R.C. Straus S.D. Crystal R.G. J. Clin. Invest. 1987; 89: 1366-1374Crossref Scopus (159) Google Scholar) and because clinical studies have suggested that only partial correction is needed for prevention of both liver and lung injury in α1-AT deficiency (17.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (234) Google Scholar, 21.Campbell E.J. Campbell M.A. Boukedes S.S. Owen C.A. J. Clin. Invest. 1999; 104: 337-344Crossref PubMed Scopus (100) Google Scholar), the effects of this class of drugs are of therapeutic as well as pathobiologic interest. Materials—Neutrophil elastase was obtained from Athens Research and Technology, Inc. (Athens, GA). Kifunensine (KIF), castanospermine (CST), and N-methyldeoxynojirimycin (MDNJ) were obtained from Toronto Research Chemicals (Ontario, Canada).N-butyldeoxynojirimycin (BDNJ) was a generous gift from Dr. G. Jacob (Monsanto, St. Louis, MO). 1,4-Dideoxy-1,4-imino-d-mannitol hydrochloride (DIM) was obtained from Sigma. Deoxymannojirimycin (DMJ) was obtained from Calbiochem. Endoglycosidase H (Endo H) and N-glycosidase F (PNGase F) were obtained from Roche Molecular Biochemicals. The human fibroblast cell line CJZ12B engineered for stable expression of mutant α1-ATZ by transduction of amphotropic recombinant retroviral particles has been previously described (17.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (234) Google Scholar). CJZ12B cells were preincubated for 1 h at 37 °C in serum-free DMEM with or without drug. Cells were then subjected to a pulse-chase protocol in the absence or presence of drug. The pulse was 1.5 h using 350–400 μCi TRAN35S label. Extracellular fluid (EC) and cell lysates (IC) were clarified, immunoprecipitated, and analyzed by 8–10% SDS-PAGE exactly as described previously (17.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (234) Google Scholar). Aliquots of each sample were subjected to trichloroacetic acid precipitation to ensure that there were equivalent levels of incorporation of radiolabel in the absence or presence of drug. The gels were visualized and quantitated on a PhosphorImager (Molecular Dynamics). To exclude the possibility that drug altered the viability of CJZ12B cells during the pulse-chase protocol, total protein synthesis was examined at the end of a simulated pulse-chase protocol (in the absence of TRAN35S label, but in the absence or presence of drug). At the 0-, 3-, and 6-h chase time points, cells were washed to remove drug and then labeled with 200 μCi TRAN35S for 30 min. Total incorporated counts were determined by TCA precipitation. Cell viability was also assessed by trypan blue exclusion. The method of Qu et al. (18.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) was used. Purified α1-ATZ mRNA was used to program the reticulocyte lysate cell free system supplemented with canine pancreatic microsomes for translation over 60 min at 30 °C. The microsomal vesicles were then isolated by centrifugation and incubated in a proteolysis-primed lysate at 37 °C for the indicated time intervals. Aliquots were taken at the indicated time intervals, and the samples were resolved on 10% SDS-PAGE gels. Co-immunoprecipitation with anti-GRP78 antibody was done using a nondenaturing buffer of 1% CHAPS in 50 mm HEPES, pH 7.5, with 200 mm NaCl (HBS) and apyrase 50 units/ml. Samples were immunoprecipitated with antibody to α1-AT, and the immunoprecipitates were resuspended in 20 μl of 50 mmTris, pH 6.8, 0.5% SDS, and 0.1 m β-mercaptoethanol. Samples were heated at 90–100 °C for 5 min. For Endo H digestion, the 20-μl samples were diluted with 17 μl of 0.15 mNaOAc, pH 6, and 0.5 μl of 0.1 m PMSF. Endo H was added using 3 μl of a 1 munits/μl solution. For N-glycosidase F digestion, the resuspended samples were added to 8 μl of 1m NaPO4 buffer, pH 8.3, 5 μl of 14% Nonidet P-40 detergent, 7.5 μl of 250 munits/μl N-glycosidase F, and 0.5 μl of 0.1 m PMSF. All samples were incubated overnight at 37 °C and then dissolved in sample buffer for SDS-PAGE. Cells were subjected to a pulse-chase protocol, and the EC medium was harvested after 6 h of the chase period. This medium was incubated for 30 min at 37 °C with neutrophil elastase, in amounts from 1 ng to 5 μg in 0.5 ml of 50 mm Tris buffer, pH 8.0, and then subjected to immunoprecipitation with anti-α1-AT for SDS-PAGE. First, we examined the effect of several glucosidase inhibitors, CST and MDNJ, on the relative electrophoretic migration of α1-ATZ (Fig. 1 A). In the absence of drug, α1-ATZ migrates at ∼ 52 kDa. Previous studies have shown that this 52-kDa polypeptide represents a biosynthetic intermediate with high-mannose-type oligosaccharide side chains (17.Wu Y. Whitman I. Molmenti E. Moore K. Hippenmeyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (234) Google Scholar). In the presence of CST or MDNJ, α1-ATZ migrates slightly more slowly, reflecting inhibition of glucose removal. Next we examined the effect of CST on the fate of α1-ATZ in pulse-chase experiments (Fig.1 B). The results show that in the absence of CST, the 52-kDa α1-ATZ polypeptide is retained for 1 h but begins to disappear from IC between 2 and 4 h, with only trace amounts of the 55-kDa mature α1-AT polypeptide appearing in EC. In the presence of CST, the untrimmed α1-ATZ polypeptide also disappears from IC between 2 and 4 h of the chase period, but in this case there is a marked increase in the amount of mature 55 kDa α1-ATZ secreted into the EC medium. By 6 h into the chase period, 31 ± 2% of the newly synthesized α1-ATZ was secreted into the medium in the presence of CST as compared with 17 ± 3% in the absence of CST. The half-times for disappearance of α1-ATZ from IC were 0.92 ± 0.35 h in the absence and 0.90 ± 0.26 h in the presence of CST. These results indicate that there is increased secretion of α1-ATZ in the presence of CST. We also examined the effect of the glucosidase inhibitor MDNJ on the fate of α1-ATZ in CJZ12 cells (Fig. 1 C). The results also show that the untrimmed α1-ATZ generated in the presence of MDNJ disappears from IC more rapidly but, in contrast to CST, there was no increase in the secretion of α1-ATZ. These results indicate that the effect of CST on secretion of α1-ATZ is specific. Experiments with a third glucosidase inhibitor, BDNJ, show that it also does not increase secretion of α1-ATZ (data not shown). It is not clear at this time why CST, but not DNJ or MDNJ, mediates increased secretion. Previous studies have suggested that there may be differences in the efficiency of the glucosidase inhibition and in the number of glucose residues on the three oligosaccharide side chains of wild type α1-AT after treatment with CST, DNJ, and MDNJ (20.Gross V. Tran-Thi T.-A. Schwarz R.T. Elbein A.D. Decker K. Heinrich P.C. Biochem. J. 1986; 236: 853-860Crossref PubMed Scopus (36) Google Scholar, 22.Kang M.H. Liu P.S. Bernotas R.C. Harry B.S. Sunkara P.S. Glycobiology. 1995; 5: 147-152Crossref PubMed Scopus (18) Google Scholar). Alternatively, CST may mediate the increased secretion of α1-ATZ through a secondary effect or through an effect that is unrelated to glucosidase inhibition. Next, we examined the effect of CST on ER degradation of α1-ATZ. CST is known to prevent the interaction of glycoproteins with calnexin (2.Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar,3.Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Our previous studies have indicated that interaction with calnexin is, at least in part, required for proteasomal degradation of α1-ATZ and that it is the α1-ATZ-calnexin complex which is targeted by the ubiquitin system and the proteasome (18.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). Although careful accounting for radioactivity in the pulse-chase experiments in intact cells in Fig. 1 B suggests that there is no difference in the disappearance of α1-ATZ from IC in the presence of CST, we chose to more specifically determine the effect of CST on degradation of α1-ATZ here using the cell-free microsomal translocation system. Previous studies have indicated that α1-ATZ is specifically degraded in this cell free system by a mechanism which involves the proteasome and which closely recapitulates the degradation of α1-ATZ in intact cells (18.Qu D. Teckman J.H. Omura S. Perlmutter D.H. J. Biol. Chem. 1996; 271: 22791-22795Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar). In Fig. 2 A,left panel, the 52-kDa α1-ATZ polypeptide begins to disappear between 15–30 min and is completely degraded between 30–45 min of the chase period. When the reaction is preincubated with CST (center panel), the untrimmed slower migrating α1-ATZ polypeptide is degraded slightly more slowly, beginning 30 min and only completed by 45–90 min of the chase period. When the CST is only added during the chase period (right panel), a condition which does not interfere with initial trimming by glucosidase but does prevent dissociation of glycoproteins from calnexin (23.Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar), the untrimmed 52-kDa α1-ATZ polypeptide is degraded at a rate which is not slowed but rather is very close to that in control with disappearance beginning between 15–30 min and completed between 30–45 min. In Fig. 2 B, microsomal vesicles from these reactions were homogenized under nondenaturing conditions and subjected to immunoprecipitation with antibodies to calnexin and GRP78/BiP. This protocol was designed to determine whether α1-ATZ could be co-immunoprecipitated by anticalnexin and/or anti-BiP and, if so, to determine the kinetics of α1-ATZ-calnexin and α1-ATZ-BiP association and dissociation. The results show that trimmed α1-ATZ is co-immunoprecipitated by anticalnexin early in the chase period but this complex disappears between 30–45 min of the chase period (center panel, control). The kinetics of disappearance of the α1-ATZ-calnexin complex is similar to those of α1-ATZ in reactions that have not been subjected to immunoprecipitation (left panel, control). Very little α1-ATZ is co-precipitated by anticalnexin in reactions that were preincubated with CST (center panel, CST). Although very little α1-ATZ is co-precipitated with anti-BiP under control conditions (right panel, control), some untrimmed α1-ATZ is immunoprecipitated with anti-BiP after preincubation with CST (right panel, CST). This α1-ATZ-BiP complex is detected at the beginning of the chase period and very rapidly disappears. Previous studies have shown that misfolded proteins may aggregate in association with BiP when glucose trimming is inhibited (24.Zhang J.-X. Braakman I. Matlack K.E.S. Helenius A. Mol. Biol. Cell. 1997; 8: 1943-1954Crossref PubMed Scopus (171) Google Scholar). This data suggests that in the presence of CST there is less, or negligible, interaction of untrimmed α1-ATZ with calnexin, that a certain proportion of the untrimmed α1-ATZ has bound to BiP, and that the untrimmed α1-ATZ-BiP complex is rapidly dissociated or degraded. Thus, when CST inhibits glucose trimming and, in turn, interaction with calnexin, α1-ATZ is degraded rapidly by an alternative degradative pathway that involves an untrimmed α1-ATZ-BiP complex. This alternative degradative pathway appears to also involve the proteasome because it is significantly abrogated by the proteasomal inhibitors MG132 in the cell free system (data not shown) as well as in CJZ12B cells (Fig. 3). This alternative degradative mechanism also probably explains the relatively modest inhibition of α1-ATZ degradation in the cell free system after preincubation with CST. Taking into consideration differences in fluorographic exposure time for the panels in Fig.2 B, we estimate that only 5–10% of total α1-ATZ is co-immunoprecipitated with anti-BiP antibody in the presence of CST. This implies that one or more additional mechanisms are involved in degradation of α1-ATZ under these conditions. Several recent studies have indicated that ER mannosidases play a role in the processing of carbohydrate side chains of some glycoproteins in the ER (4.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Here (Fig.4) we examined the effect of a mannosidase I inhibitor, KIF, a mannosidase II inhibitor, DIM, and an inhibitor of both mannosidases I and II, DMJ (25.Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar). The effect of these inhibitors on the relative electrophoretic mobility of α1-ATZ is shown in Fig. 4 A. There is slightly slower electrophoretic migration in the presence of KIF and DMJ but not in the presence of DIM. Next, we examined the effect of these inhibitors on the fate of α1-ATZ in pulse-chase experiments (Fig. 4,B–D). In the presence of KIF and DMJ, the untrimmed α1-ATZ polypeptide is retained IC for a longer period of time than in control, and there is a marked increase in the amount of α1-ATZ that appears EC. The α1-ATZ polypeptide that is secreted in the presence of KIF and DMJ is 52 kDa as compared with the 55-kDa polypeptide secreted in the presence of CST. In contrast, the mannosidase II inhibitor DIM has no effect on degradation or secretion of α1-ATZ (Fig. 4 D). DIM is active in these experiments as evidenced by the fact that the small amount of α1-ATZ that reaches the EC fluid has a more rapid relative electrophoretic mobility at ∼53 kDa. These data indicate that DMJ and KIF mediate a marked decrease in degradation and an increase in secretion of α1-ATZ. The effect of DMJ and KIF on secretion of α1-ATZ probably involves a different mechanism than the effect of CST because the α1-ATZ that is secreted in each of these cases has a different electrophoretic mobility. The data also implicate ER mannosidase I but not ER mannosidase II in playing an important role in the fate of the misfolded α1-ATZ molecule. To determine whether α1-ATZ secreted in the presence of CST or KIF has high mannose- or complex-type oligosaccharide side chains, we examined the effect of Endo H and PNGase F (Fig. 5 A). In HepG2 cells, the 55-kDa wild type α1-AT in the extracellular fluid was resistant to digestion with Endo H but cleaved to an ∼46-kDa polypeptide by PNGase F as expected (left panel). In CJZ12B cells treated with CST, the 55-kDa α1-ATZ polypeptide was partially resistant and partially sensitive to Endo H (center panel). A 46-kDa Endo H-sensitive polypeptide probably represents the cleavage product of α1-ATZ with high-mannose carbohydrate. The ∼48- and ∼52-kDa partially Endo H-sensitive polypeptides probably represent cleavage products of α1-ATZ with high-mannose carbohydrates on one or two of its three carbohydrate side chains. The 55-kDa Endo H-resistant polypeptide probably represents α1-ATZ with complex carbohydrates at all three of its side chains. This pattern was not because of incomplete digestion because it did not change with higher concentrations of Endo H (data not shown). An identical result has previously been observed for the 55-kDa wild type α1-AT polypeptide secreted by hepatocytes in the presence of CST (20.Gross V. Tran-Thi T.-A. Schwarz R.T. Elbein A.D. Decker K. Heinrich P.C. Biochem. J. 1986; 236: 853-860Crossref PubMed Scopus (36) Google Scholar). Maturation of one, two, or all three carbohydrate side chains to the complex type in the presence of CST is probably a result of the cell type-specific expression in Golgi of endo-α-d-mannosidase which can deglucosylate glycoproteins during inhibition of glucosidases (26.Karaivanova V.K. Luan P. Spiro R.G. Glycobiology. 1998; 8: 725-730Crossref PubMed Scopus (43) Google Scholar). In CJZ12B cells treated with CST, the 55-kDa α1-ATZ polypeptide was cleaved to 46 kDa by PGNase F as expected (center panel). In CJZ12B cells treated with KIF, the 52-kDa α1-ATZ polypeptide was completely Endo H-sensitive being cleaved to 46 kDa (right panel), indicating that it is an intermediate with high-mannose carbohydrate side chains. We examined the possibility that α1-ATZ secreted in the presence of CST, KIF, or DMJ could form an SDS-stable complex with neutrophil elastase (Fig. 5 B). The results show that wild type α1-AT from HepG2 cells forms ∼66- and ∼75-kDa high molecular mass complexes with elastase (left panel). Complexes begin to form at 0.1 μg of elastase added, with complete conversion by 0.5 μg of elastase added. The ∼66-kDa band probably represents complexes that have undergone partial hydrolysis during the reaction or during processing/gel electrophoretic analysis. The ∼ 51-kDa band represents cleaved α1-ATZ. The α1-ATZ secreted from CST-treated CJZ12B cells also forms high molecular mass complexes with elastase that migrate to ∼75 kDa. Here, however, complexes start to become apparent at 0.5 μg of elastase added, and complete conversion to the complex form requires 2 μg of elastase. The α1-ATZ secreted from KIF-treated or DMJ-treated CJZ12B also forms complexes with elastase. In this case, the complex migrates at ∼70 kDa. Because α1-ATZ secreted in the presence of KIF or DMJ is ∼52 kDa, the ∼70-kDa complex probably corresponds to the ∼75 kDa complex formed with wild type α1-AT from HepG2 cells. The α1-ATZ from KIF-treated or DMJ-treated CJZ12B cells only starts to form complexes when 0.5 μg of elastase is added and only at 2 μg of elastase is it completely converted to the complex form. These data indicate that α1-ATZ secreted in the presence of CST, KIF, or DMJ is functionally active although, in each case, it is apparently slightly lower in activity than wild type α1-AT secreted by HepG2 cells. Similar results have been described for wild type human α1-AT secreted with high-mannose type carbohydrate side chains by Saccharomyces cerevisiaeand methylotrophic yeasts (27.Kang H.A. Sohn J.-H. Choi E.-S. Chung B.H., Yu, M.-H. Rhee S.-K. Yeast. 1998; 14: 371-381Crossref PubMed Scopus (58) Google Scholar, 28.Kwon K.-S. Yu M.-H. Biochim. Biophys. Acta. 1997; 1335: 265-272Crossref PubMed Scopus (55) Google Scholar). Taken together, the results of these studies indicate that the glucosidase inhibitor CST mediates increased secretion of α1-ATZ and that the mannosidase inhibitors KIF and DMJ mediate both decreased degradation and increased secretion of α1-ATZ. The results, therefore, suggest that there are mechanisms, at least two, by which steps in oligosaccharide side chain trimming can be circumvented and a cohort of misfolded glycoproteins permitted to traverse the secretory pathway. In one previous study, CST has been shown to increase secretion of carboxyl-terminal truncated fragments of influenza virus hemagglutinin (24.Zhang J.-X. Braakman I. Matlack K.E.S. Helenius A. Mol. Biol. Cell. 1997; 8: 1943-1954Crossref PubMed Scopus (171) Google Scholar), but there are no previous reports of a similar effect for KIF or DMJ. It is not clear at this time whether the alterations in the structure of the oligosaccharide side chain prevent the side chain from interacting with molecules responsible for its retention, permit the side chain to interact with molecules that can facilitate the folding of the mutant α1-ATZ protein, or result in alterations in the conformation of α1-ATZ in such a way that its folding is facilitated. In the case of α1-ATZ generated in the presence of mannosidase inhibitors KIF and DMJ, the ERGIC-53 cycling pathway is an excellent candidate for mediating increased secretion. Mousalli et al. (29.Moussalli M. Pipe S.W. Hauri H.-P. Nichols W.C. Ginsburg D. Kaufman R.J. J. Biol. Chem. 1999; 274: 32539-32542Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar) have recently shown that ERGIC-53 mediates secretion of coagulation factors V and VIII by specific recognition of fully glucose-trimmed, mannose 9 oligosaccharide side chains. Interaction with the ERGIC-53 cycling pathway would not, however, provide an explanation for the delay in degradation that also occurs in the presence of KIF and DMJ. Several previous studies have suggested the possibility that uncharacterized “carbohydrate-binding chaperones” are responsible for stabilization of untrimmed secretory and membrane proteins during treatment with mannosidase inhibitors (6.Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (303) Google Scholar, 9.Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar). Nevertheless, comparison of different studies in the literature (4.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 5.Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 6.Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (303) Google Scholar, 7.Kearse K.P. Williams D.B. Singer A. EMBO J. 1994; 13: 3678-3686Crossref PubMed Scopus (111) Google Scholar, 8.Moore S. Spiro R. J. Biol. Chem. 1993; 268: 3809-3813Abstract Full Text PDF PubMed Google Scholar, 9.Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (202) Google Scholar, 10.Vierhoeven A.J.M. Neve B.P. Jansen H. Biochem. J. 1999; 337: 133-140Crossref PubMed Google Scholar, 20.Gross V. Tran-Thi T.-A. Schwarz R.T. Elbein A.D. Decker K. Heinrich P.C. Biochem. J. 1986; 236: 853-860Crossref PubMed Scopus (36) Google Scholar, 21.Campbell E.J. Campbell M.A. Boukedes S.S. Owen C.A. J. Clin. Invest. 1999; 104: 337-344Crossref PubMed Scopus (100) Google Scholar, 22.Kang M.H. Liu P.S. Bernotas R.C. Harry B.S. Sunkara P.S. Glycobiology. 1995; 5: 147-152Crossref PubMed Scopus (18) Google Scholar, 23.Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 24.Zhang J.-X. Braakman I. Matlack K.E.S. Helenius A. Mol. Biol. Cell. 1997; 8: 1943-1954Crossref PubMed Scopus (171) Google Scholar, 25.Weng S. Spiro R.G. Arch. Biochem. Biophys. 1996; 325: 113-123Crossref PubMed Scopus (70) Google Scholar) suggests that there are species-specific and substrate-specific differences in the effects of glucosidase and mannosidase inhibitors and that there may also be differences in their effects depending on whether the protein substrate is membrane-bound, secretory, wild type, unassembled, or mutant. The results also provide further evidence that physiologic and pharmacologic perturbations have separate and independent effects on ER degradation and secretion of the misfolded α1-ATZ molecule. Glycerol and 4-phenylbutyric acid (PBA) mediate increases in secretion of α1-ATZ without affecting its degradation (30.Burrows, J. A. J., Willis, L. K., and Perlmutter, D. H. (2000) Proc. Natl. Acad. Sci. U. S. A., in pressGoogle Scholar). Lactacystin, cycloheximide, and lowering of temperature to 27 °C are associated with a decrease in degradation of α1-ATZ without any change in secretion (30.Burrows, J. A. J., Willis, L. K., and Perlmutter, D. H. (2000) Proc. Natl. Acad. Sci. U. S. A., in pressGoogle Scholar). KIF, DMJ (Fig. 4), and elevation of temperature to 42 °C are associated with decreased degradation and increased secretion (30.Burrows, J. A. J., Willis, L. K., and Perlmutter, D. H. (2000) Proc. Natl. Acad. Sci. U. S. A., in pressGoogle Scholar). Because the mutant α1-ATZ retains functional activity and because clinical studies have suggested that only partial correction of the secretory defect is needed for prevention of both liver and lung disease in α1-AT deficiency, any drug which enhances secretion is a candidate for chemoprophylaxis in patients with this deficiency. Our recent studies have shown that a chemical chaperone PBA is an excellent candidate (30.Burrows, J. A. J., Willis, L. K., and Perlmutter, D. H. (2000) Proc. Natl. Acad. Sci. U. S. A., in pressGoogle Scholar). The current study suggests that CST may be another potential chemoprophylactic agent. Indeed, a compound based on CST, 6-O-butanoyl CST is currently in clinical trials as an adjuvant treatment for HIV infection (31.Jacob G.S. Curr. Opin. Struct. Biol. 1995; 5: 605-611Crossref PubMed Scopus (278) Google Scholar). The mannosidase inhibitors KIF and DMJ are also less attractive drugs for chemoprophylaxis because they delay degradation of α1-ATZ as well as increase its secretion. Thus, drugs based on these compounds have the potential to prevent lung injury but to increase the likelihood of liver injury in α1-AT-deficient patients. We are indebted to Mary Pichler for preparing this manuscript.

We have theorized that a subset of PiZZ α1-antitrypsin (α1-AT)-deficient individuals is more susceptible to liver injury by virtue of second inherited trait(s) or environmental factor(s), which exaggerate the accumulation of mutant α1-AT Z within the endoplasmic reticulum (ER) of liver cells. Using a complementation approach in which cell lines from PiZZ individuals with liver disease ("susceptible" hosts) and from PiZZ individuals without liver disease ("protected" hosts) are transduced with the mutant α1-AT Z gene, we have recently shown that there is a delay in ER degradation of mutant α1-AT Z protein that is only present in cell lines from susceptible hosts and correlates with the liver disease phenotype. In the present study we examined the specificity of this ER degradation pathway to determine if it is responsible for degrading other misfolded mutants of α1-AT and/or for unassembled membrane proteins. The S mutant of α1-AT and H2a subunit of the asialoglycoprotein receptor (ASGPR H2a) were expressed in skin fibroblast cell lines from susceptible and protected hosts. The results showed in both susceptible and protected hosts that α1-AT S was associated with a delay in secretion as compared with wild type α1-AT. The α1-AT S mutant was retained in ER, albeit to a lesser extent than the α1-AT Z mutant. There was, however, a significant increase in retention of α1-AT S in the ER of susceptible as compared with protected host cells. The same host cell lines were transduced to express an unassembled membrane protein, ASGPR H2a. There was no difference in the kinetics of ER degradation of ASGPR H2a in susceptible as compared with protected hosts. Taken together, the results show that α1-AT S is associated with a defect in biogenesis, intracellular retention, which is similar to but milder than α1-AT Z. Like α1-AT Z, α1-AT S is degraded by a pathway in the ER, which is relatively inefficient in PiZZ individuals with the liver disease phenotype. However, this pathway appears to be different from that previously described for a model unassembled membrane protein.

Homozygous PIZZ alpha 1-antitrypsin deficiency, which has an incident of 1 in 1600 to 1 in 2000 live births, is the most common genetic cause of liver disease in children. It is also associated with chronic liver disease and hepatocellular carcinoma in adults. It is a well-known cause of pulmonary emphysema. Although emphysema is due to uninhibited proteolytic destruction of the connective tissue backbone of the lung, liver disease is thought to result from the toxic effects of the mutant alpha 1AT molecule retained within the endoplasmic reticulum of liver cells. Screening studies done by Sveger in Sweden have shown that only 10 to 15% of the PIZZ population develop clinically significant liver disease over the first 20 years of life. Recent studies have suggested that a subgroup of PIZZ individuals are predisposed to liver injury because of an inefficient degradation of mutant alpha 1ATZ within the endoplasmic reticulum. Altered migration of the abnormal alpha 1ATZ molecule in isoelectric focussing gels is the basis of the diagnosis of alpha 1AT deficiency. Treatment of alpha 1AT deficiency-associated liver disease is mostly supportive. Liver replacement therapy has been used successfully for severe liver injury. An increasing number of patients with severe emphysema have undergone lung transplantation.

HepatologyVolume 13, Issue 1 p. 172-185 Special ArticleFree Access The cellular basis for liver injury in α1-antitrypsin deficiency Dr. David H. Perlmutter, Corresponding Author Dr. David H. Perlmutter Departments of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine; and the Division of Gastroenterology and Nutrition, Children's Hospital, St. Louis, Missouri 63110Associate Professor of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine, St. Louis Children's Hospital, 400 S. Kingshighway, St. Louis, MO 63110===Search for more papers by this author Dr. David H. Perlmutter, Corresponding Author Dr. David H. Perlmutter Departments of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine; and the Division of Gastroenterology and Nutrition, Children's Hospital, St. Louis, Missouri 63110Associate Professor of Pediatrics, Cell Biology and Physiology, Washington University School of Medicine, St. Louis Children's Hospital, 400 S. Kingshighway, St. Louis, MO 63110===Search for more papers by this author First published: January 1991 https://doi.org/10.1002/hep.1840130125Citations: 45AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume13, Issue1January 1991Pages 172-185 ReferencesRelatedInformation

The serpin-enzyme complex (SEC) receptor mediates catabolism of alpha 1-antitrypsin (alpha 1-AT)-elastase complexes and increases in synthesis of alpha 1-AT in cell culture. The SEC receptor recognizes a pentapeptide domain on alpha 1-AT-elastase complexes (alpha 1-AT 370-374), and the same domain in several other serpins, amyloid-beta peptide, substance P, and other tachykinins. Thus, it has also been implicated in the biological properties of these ligands, including the neurotoxic effect of amyloid-beta peptide. In this study, we examined the possibility that the SEC receptor mediates the previously described neutrophil chemotactic activity of alpha 1-AT-elastase complexes, and whether the other ligands for the SEC receptor have neutrophil chemotactic activity. The results show that 125I-peptide 105Y (based on alpha 1-AT 359-374) binds specifically and saturably to human neutrophils, and the characteristics of this binding are almost identical to that of monocytes and hepatoma-derived hepatocytes. Peptide 105Y and amyloid-beta peptide mediate chemotaxis for neutrophils with maximal stimulation at 1-10 nM. Mutant or deleted forms of peptide 105Y, which do not bind to the SEC receptor, have no effect. The neutrophil chemotactic effect of alpha 1-AT-elastase complexes is blocked by antiserum to peptide 105Y and by antiserum to the SEC receptor, but not by control antiserum. Preincubation of neutrophils with peptide 105Y or substance P completely blocks the chemotactic activity of amyloid-beta peptide, but not that of FMLP. These results, therefore, indicate that the SEC receptor can be modulated by homologous desensitization and raise the possibility that pharmacological manipulation of this receptor will modify the local tissue response to inflammation/injury and the neuropathologic reaction of Alzheimer's disease.

alpha 1 Proteinase inhibitor (PI) is the principle inhibitor of neutrophil elastase, an enzyme that degrades many components of the extracellular matrix. Expression and regulation of alpha 1 PI, therefore, affects the delicate balance of elastase and antielastase, which is critical to turnover of connective tissue during homeostasis, tissue injury, and repair. In this study we show that expression of alpha 1 PI in human monocytes and macrophages is regulated during activation by LPS. LPS mediates a concentration- and time-dependent increase in the rate of synthesis of alpha 1 PI in mononuclear phagocytes. There is a 4.5-8.7-fold increase in functionally active inhibitor delivered to the cell culture fluid of monocytes. The effect of LPS is specific in that it is neutralized by an mAb to the lipid A moiety. The increase in expression of alpha 1 PI mediated by LPS occurs in the context of other specific changes in the expression of serine proteinase inhibitor genes in mononuclear phagocytes. There is an increase in the rate of synthesis of C1 inhibitor and a decrease in synthesis of alpha 2 macroglobulin. Regulation of alpha 1 PI by LPS is distinctive in that it is largely determined by a change in the efficiency of translation of alpha 1 PI mRNA. LPS has no effect on the rate of posttranslational processing and/or secretion of alpha 1 PI and, therein, causes greater intracellular accumulation of alpha 1 PI in mononuclear phagocytes from individuals with homozygous PiZZ alpha 1 PI deficiency.

Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1⁺ endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized ¹²⁵I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (<i>K_d</i>) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Abstract Up to 1 in 3000 individuals in the United States have α-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.

α1-Antitrypsin deficiency (ATD) is a common genetic disorder that can lead to end-stage liver and lung disease. Although liver transplantation remains the only therapy currently available, manipulation of the proteostasis network (PN) by small molecule therapeutics offers great promise. To accelerate the drug-discovery process for this disease, we first developed a semi-automated high-throughput/content-genome-wide RNAi screen to identify PN modifiers affecting the accumulation of the α1-antitrypsin Z mutant (ATZ) in a Caenorhabditis elegans model of ATD. We identified 104 PN modifiers, and these genes were used in a computational strategy to identify human ortholog-ligand pairs. Based on rigorous selection criteria, we identified four FDA-approved drugs directed against four different PN targets that decreased the accumulation of ATZ in C. elegans. We also tested one of the compounds in a mammalian cell line with similar results. This methodology also proved useful in confirming drug targets in vivo, and predicting the success of combination therapy. We propose that small animal models of genetic disorders combined with genome-wide RNAi screening and computational methods can be used to rapidly, economically and strategically prime the preclinical discovery pipeline for rare and neglected diseases with limited therapeutic options.

Endoplasmic-reticulum associated degradation (ERAD) is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY*) was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.

The classical form of α1-antitrypsin deficiency (ATD) is associated with hepatic fibrosis and hepatocellular carcinoma. It is caused by the proteotoxic effect of a mutant secretory protein that aberrantly accumulates in the endoplasmic reticulum of liver cells. Recently we developed a model of this deficiency in C. Elegans and adapted it for high-content drug screening using an automated, image-based array scanning. Screening of the Library of Pharmacologically Active Compounds identified fluphenazine (Flu) among several other compounds as a drug which reduced intracellular accumulation of mutant α1-antitrypsin Z (ATZ). Because it is representative of the phenothiazine drug class that appears to have autophagy enhancer properties in addition to mood stabilizing activity, and can be relatively easily re-purposed, we further investigated its effects on mutant ATZ. The results indicate that Flu reverses the phenotypic effects of ATZ accumulation in the C. elegans model of ATD at doses which increase the number of autophagosomes in vivo. Furthermore, in nanomolar concentrations, Flu enhances the rate of intracellular degradation of ATZ and reduces the cellular ATZ load in mammalian cell line models. In the PiZ mouse model Flu reduces the accumulation of ATZ in the liver and mediates a decrease in hepatic fibrosis. These results show that Flu can reduce the proteotoxicity of ATZ accumulation in vivo and, because it has been used safely in humans, this drug can be moved rapidly into trials for liver disease due to ATD. The results also provide further validation for drug discovery using C. elegans models that can be adapted to high-content drug screening platforms and used together with mammalian cell line and animal models.

The classical form of alpha‐1‐antitrypsin deficiency (A1ATD) is known to cause liver disease in children and adults, but there is relatively little information about the risk of severe, progressive liver disease and the need for liver transplantation. To better understand how newly evolving pharmacological, genetic, and cellular therapies may be targeted according to risk for progressive liver disease, we sought to determine the age distribution of A1ATD as a cause of severe liver disease, as defined by the need for liver transplantation. Using 3 US liver transplantation databases for the period 1991‐2012, we found 77.2% of 1677 liver transplants with a reported diagnosis of A1ATD were adults. The peak age range was 50‐64 years. Using 2 of the databases which included specific A1AT phenotypes, we found that many of these adults who undergo liver transplantation with A1ATD as the diagnosis are heterozygotes and have other potential causes of liver disease, most notably obesity and ethanol abuse. However, even when these cases are excluded and only ZZ and SZ phenotypes are considered, severe liver disease requiring transplantation is more than 2.5 times as likely in adults. The analysis also showed a markedly increased risk for males. In the pediatric group, almost all of the transplants are done in children less than 5 years of age. In conclusion, A1ATD causes progressive liver disease most commonly in adults with males in the highest risk category. In the pediatric group, children less than 5 years of age are highest in risk. These results suggest that A1ATD most commonly causes liver disease by mechanisms similar to age‐dependent degenerative diseases and more rarely in children by powerful modifiers. Liver Transplantation 22 886–894 2016 AASLD

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-l-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-l-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-l-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-l-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a β-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-l-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-l-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-l-lysine. Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-l-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-l-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-l-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-l-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a β-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-l-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-l-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-l-lysine. Receptor-mediated gene transfer has great appeal as a strategy for gene therapy because of its specificity and low toxicity in vivo but has drawbacks, including low level and transient gene expression (1Cotten M. Wagner E. Zatloukal K. Phillips D.T. Curiel S. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6094-6098Crossref PubMed Scopus (306) Google Scholar, 2Ferkol T. Perales J.C. Mularo F. Hanson R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 101-105Crossref PubMed Scopus (159) Google Scholar, 3Ferkol T. Kaetzel C.S. Davis P.B. J. Clin. Invest. 1992; 92: 2394-2400Crossref Scopus (119) Google Scholar, 4Michael S.I. Curiel D.T. Gene Ther. 1994; 1: 223-232PubMed Google Scholar, 5Perales J.C. Ferkol T. Beegen H. Ratnoff O.D. Hanson R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4086-4090Crossref PubMed Scopus (297) Google Scholar, 6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar, 7Wagner E. Zenke M. Cotten M. Beug H. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3410-3414Crossref PubMed Scopus (659) Google Scholar, 8Wu G.Y. Wu C.H. J. Biol. Chem. 1987; 262: 4299-4432Google Scholar, 9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). Targeting the serpin (serineprotease inhibitor) enzyme complex receptor (SEC-R) 1The abbreviations SEC-Rserpin enzyme complex receptorpoly Kpoly-l-lysineAFMatomic force microscopysulfo-LC SPDPsulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoatekbkilobasesCMVcytomegalovirus 1The abbreviations SEC-Rserpin enzyme complex receptorpoly Kpoly-l-lysineAFMatomic force microscopysulfo-LC SPDPsulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoatekbkilobasesCMVcytomegalovirusmight transfect many cell types that are potentially interesting for gene therapy, including hepatocytes, macrophages, and neurons (10Bu G. Morton P.A. Schwartz A.L. J. Biol. Chem. 1992; 267: 15595-15602Abstract Full Text PDF PubMed Google Scholar, 11Perlmutter D.H. Glover G.I. Meheryar M. Schasteen C.S. Fallon R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3753-3757Crossref PubMed Scopus (156) Google Scholar, 12Perlmutter D.H. Joslin G. Nelson P. Schasteen C. Adams S.P. Fallon R.J. J. Biol. Chem. 1990; 265: 16713-16716Abstract Full Text PDF PubMed Google Scholar, 13Joslin G. Fallon R. Bullock J. Adams S.P. Perlmutter D.H. J. Biol. Chem. 1991; 266: 11282-11288Abstract Full Text PDF PubMed Google Scholar, 14Perlmutter D.H. Pediatr. Res. 1994; 36: 271-277Crossref PubMed Scopus (13) Google Scholar). Ligand-conjugated poly-l-lysine (poly K)-DNA complexes directed at this receptor deliver reporter genes, specifically, to receptor-bearing cells. The synthetic ligands are based in sequence on amino acids 346–374 of human α1-antitrypsin (9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). We undertook a systematic study of the contribution of the protein portion of the complex to intensity and duration of gene expression.Prior studies of the composition of the protein portion of receptor-directed gene transfer complexes have focused on the degree of substitution (6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar, 15Erbacher P. Roche A.C. Monsigny M. Midoux P. Bioconjugate Chem. 1995; 6: 401-410Crossref PubMed Scopus (139) Google Scholar). One study did not find a consistent relationship between chain length and gene expression in vitro (6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar). No study has examined the relation between the chain length of poly K used to condense DNA and duration of expression. Moreover, it is not clear whether modifications worked out in an in vitro model can be applied to the in vivo situation (16Ferkol T. Lindberg G.L. Perales J.C. Chen J. Ratnoff O.D. Hanson R.W. FASEB J. 1993; 7: 1081-1091Crossref PubMed Scopus (50) Google Scholar, 17Ferkol T. Perales J.C. Kaetzel C.S. Eckman E. Hanson R.W. Davis P.B. J. Clin. Invest. 1995; 95: 493-502Crossref PubMed Google Scholar).Our system has several advantages for studies of this type, including the ability to determine the extent of lysine substitution by linker and ligand, even at very low levels, with NMR. Furthermore, the same ligands bind to the receptors of human, mouse, and rat, thus allowing parallel examination in vitro in human cells and in vivo in animal models. We used this system to investigate the influence of chain length of poly K used on the intensity and duration of gene expression in vitro and in vivo. Using our in vitro model, we compared the optimally substituted poly K molecules of average chain length 36 with those of average chain length 256. Longer chain poly K molecules gave significantly longer duration of expression both in vitro and in vivothan shorter chain poly K molecules for a given ligand and degree of substitution.Manipulation of the protein portion of receptor-targeted DNA complexes strongly influenced the duration and intensity of gene expression. This demonstration, in vitro and in vivo, addresses some of the limitations of the system and provides a new basis for improvement of this molecular conjugate for therapeutic purposes.DISCUSSIONThe composition of the protein portion of receptor-targeted gene transfer protein-DNA complexes profoundly affects the intensity and duration of gene expression, both in vitro and in vivo. In vitro the number of ligands/DNA molecule, the identity of the ligand, and the chain length of poly K all have strong effects on intensity and duration of gene expression, but the most striking effect was that of poly K chain length on the duration of expression. Using the ligand and number of ligands/DNA molecule found to be optimal in the cell culture system, we tested the effect of poly K chain length on in vivo expression. Complexes made with long chain poly K produced much more protracted gene expression in vivo than complexes prepared with short chain poly K.Optimization of the SEC-R-directed gene transfer for in HuH7 cells resulted in greatly increased reporter gene expression (relative to Lipofectin control) compared with our prior report (9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). Activity in the best complexes tested in the present study was about 20-fold higher than activity produced by the same amount of plasmid DNA delivered with the Lipofectin reagent; in our previous report maximal activity was less than the Lipofectin control. Increased peak activity indicates increased efficiency, which has been a problem for most nonviral strategies. At the same time, duration of activity could be greatly prolonged in vitro by informed selection of the specific receptor ligand (C105Y versus C1315), the number of ligands/plasmid DNA molecule (over 2–40 ligands), and the chain length of poly K used to condense the DNA. It is likely that the number of ligands and the identity of the ligand entrains routing within the cell, with the best complexes promoting routing into the endosomal compartment rather than to the lysosomal degradative pathway. For another receptor, the epidermal growth factor receptor, high number of ligands promotes lysosomal trafficking, whereas fewer ligands promote endosomal recycling (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar), so fewer ligands might well entrain a more favorable trafficking pattern, delaying the destruction of delivered DNA. In addition, different ligands for some receptors, such as the transferrin receptor, may be trafficked differently (28Scidmore M.A. Ficher E.R. Hackstadt T. J. Cell Biol. 1996; 134: 363-374Crossref PubMed Scopus (128) Google Scholar). A similar mechanism may account for the differences in duration of expression for complexes containing the two SEC-R ligands, which have very similar affinity for the receptor and very similar initial transgene expression.In vitro, the expression of the firefly luciferase reporter gene persisted (within a log maximum) in HuH7 cells for at least 40 days for some complexes that contained the long chain poly K-ligand conjugates. Because the half-life of the luciferase protein in mammalian cells is 3 h (29Brubaker J.O. Thompson C.M. Morrison L.A. Knipe D.M. Siber G.R. Finberg R.W. J. Immunol. 1996; 157: 1598-1604PubMed Google Scholar) and we observed rapid decline in luciferase activity in HuH7 cells transfected using Lipofectin (thus, HuH7 cells do not retain high levels of luciferase activity, however the gene is delivered), protracted luciferase expression from complexes made with long chain poly K probably represents continuing transcription of plasmid DNA. In an in vitro assay, we did not observe transcription from plasmid DNA compacted with either short or long chain poly K, so it is likely that in vivo, plasmid DNA separates from poly K before transcription. These considerations suggest that the prolonged expression results from retention of plasmid DNA complexed with poly K for a longer period of time for complexes containing long chain poly K than for the short chain. Condensation with poly K protects DNA against degradation (4Michael S.I. Curiel D.T. Gene Ther. 1994; 1: 223-232PubMed Google Scholar), and better protection is afforded by long chain poly K than short chain in in vitro assays. Thus, we speculate that plasmid DNA is retained within the cell for both complexes (because both have better persistence of expression than Lipofectin) with gradual degradation, which is resisted longer by complexes containing long chain poly K. Whether the complexes reside in membrane-bound cytoplasmic compartments, the cytoplasm, or the nucleus is not yet clear. It is also possible that the less tightly compacted complexes with the shorter poly K allow better access for the RNA polymerase to the DNA and accounts for the higher initial expression. In such a scenario, transgene expression would depend on a dynamic relationship between the availability of the DNA for transcription and the rate of its subsequent degradation.Transgene activity persists better in vitro (40 days) thanin vivo (less than 30 days), even for genes delivered using complexes containing long chain poly K. There are probably several reasons for this difference. In vitro, only processes within the transfected cell account for decay of transgene activity, whereasin vivo, immune processes can be recruited to destroy cells expressing proteins not recognized as “self.” Bacterial β-galactosidase has been shown, in and of itself, to incite cytotoxic lymphocyte responses in vivo, and therefore, cells expressing this protein are preferentially targeted and destroyed (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar). This is probably one mechanism of extinguishing β-galactosidase activity. In addition, the HuH7 cells transfected in vitroare immortal but fail to grow and divide after about 12–14 days under our culture conditions if they are not subcultured. In vivo, normal cell turnover of transfected cells may limit duration of expression.Complexes containing a minimal number of ligands that specifically deliver exogenous DNA to receptor bearing cells might be less immunogenic than more heavily substituted complexes. DNA (30Ebner R. Derynck R. Cell Regul. 1991; 2: 599-612Crossref PubMed Scopus (207) Google Scholar) and poly-l-amino acids (31Maurer P.H. J. Immunol. 1962; 88: 330-338PubMed Google Scholar) have been found to be relatively nonimmunogenic. Thus, possible immunogenicity of receptor-targeted complexes in vivo may depend on the ligand portion and, in part, its abundance. The abundance of SEC-R in lung, liver, and brain (14Perlmutter D.H. Pediatr. Res. 1994; 36: 271-277Crossref PubMed Scopus (13) Google Scholar), all of which might be potential target tissues for therapeutic gene transfer in common inherited (e.g.α1-antitrypsin deficiency) or acquired (e.g.Alzheimer's disease) disorders, has made it a desirable target for receptor-mediated gene therapy. The development of optimal complexes that produce high level gene expression for short (9.7-kDa poly K conjugates) or longer (53.7-kDa poly K conjugates) periods of time will be useful in achieving optimal therapeutic effects. Receptor-mediated gene transfer has great appeal as a strategy for gene therapy because of its specificity and low toxicity in vivo but has drawbacks, including low level and transient gene expression (1Cotten M. Wagner E. Zatloukal K. Phillips D.T. Curiel S. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6094-6098Crossref PubMed Scopus (306) Google Scholar, 2Ferkol T. Perales J.C. Mularo F. Hanson R.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 101-105Crossref PubMed Scopus (159) Google Scholar, 3Ferkol T. Kaetzel C.S. Davis P.B. J. Clin. Invest. 1992; 92: 2394-2400Crossref Scopus (119) Google Scholar, 4Michael S.I. Curiel D.T. Gene Ther. 1994; 1: 223-232PubMed Google Scholar, 5Perales J.C. Ferkol T. Beegen H. Ratnoff O.D. Hanson R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4086-4090Crossref PubMed Scopus (297) Google Scholar, 6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar, 7Wagner E. Zenke M. Cotten M. Beug H. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3410-3414Crossref PubMed Scopus (659) Google Scholar, 8Wu G.Y. Wu C.H. J. Biol. Chem. 1987; 262: 4299-4432Google Scholar, 9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). Targeting the serpin (serineprotease inhibitor) enzyme complex receptor (SEC-R) 1The abbreviations SEC-Rserpin enzyme complex receptorpoly Kpoly-l-lysineAFMatomic force microscopysulfo-LC SPDPsulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoatekbkilobasesCMVcytomegalovirus 1The abbreviations SEC-Rserpin enzyme complex receptorpoly Kpoly-l-lysineAFMatomic force microscopysulfo-LC SPDPsulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoatekbkilobasesCMVcytomegalovirusmight transfect many cell types that are potentially interesting for gene therapy, including hepatocytes, macrophages, and neurons (10Bu G. Morton P.A. Schwartz A.L. J. Biol. Chem. 1992; 267: 15595-15602Abstract Full Text PDF PubMed Google Scholar, 11Perlmutter D.H. Glover G.I. Meheryar M. Schasteen C.S. Fallon R.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3753-3757Crossref PubMed Scopus (156) Google Scholar, 12Perlmutter D.H. Joslin G. Nelson P. Schasteen C. Adams S.P. Fallon R.J. J. Biol. Chem. 1990; 265: 16713-16716Abstract Full Text PDF PubMed Google Scholar, 13Joslin G. Fallon R. Bullock J. Adams S.P. Perlmutter D.H. J. Biol. Chem. 1991; 266: 11282-11288Abstract Full Text PDF PubMed Google Scholar, 14Perlmutter D.H. Pediatr. Res. 1994; 36: 271-277Crossref PubMed Scopus (13) Google Scholar). Ligand-conjugated poly-l-lysine (poly K)-DNA complexes directed at this receptor deliver reporter genes, specifically, to receptor-bearing cells. The synthetic ligands are based in sequence on amino acids 346–374 of human α1-antitrypsin (9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). We undertook a systematic study of the contribution of the protein portion of the complex to intensity and duration of gene expression. serpin enzyme complex receptor poly-l-lysine atomic force microscopy sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate kilobases cytomegalovirus serpin enzyme complex receptor poly-l-lysine atomic force microscopy sulfosuccinimidyl 6-[3′(2-pyridyldithio)-propionamido] hexanoate kilobases cytomegalovirus Prior studies of the composition of the protein portion of receptor-directed gene transfer complexes have focused on the degree of substitution (6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar, 15Erbacher P. Roche A.C. Monsigny M. Midoux P. Bioconjugate Chem. 1995; 6: 401-410Crossref PubMed Scopus (139) Google Scholar). One study did not find a consistent relationship between chain length and gene expression in vitro (6Wagner E. Cotten M. Foisner R. Birnstiel M.L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4255-4259Crossref PubMed Scopus (481) Google Scholar). No study has examined the relation between the chain length of poly K used to condense DNA and duration of expression. Moreover, it is not clear whether modifications worked out in an in vitro model can be applied to the in vivo situation (16Ferkol T. Lindberg G.L. Perales J.C. Chen J. Ratnoff O.D. Hanson R.W. FASEB J. 1993; 7: 1081-1091Crossref PubMed Scopus (50) Google Scholar, 17Ferkol T. Perales J.C. Kaetzel C.S. Eckman E. Hanson R.W. Davis P.B. J. Clin. Invest. 1995; 95: 493-502Crossref PubMed Google Scholar). Our system has several advantages for studies of this type, including the ability to determine the extent of lysine substitution by linker and ligand, even at very low levels, with NMR. Furthermore, the same ligands bind to the receptors of human, mouse, and rat, thus allowing parallel examination in vitro in human cells and in vivo in animal models. We used this system to investigate the influence of chain length of poly K used on the intensity and duration of gene expression in vitro and in vivo. Using our in vitro model, we compared the optimally substituted poly K molecules of average chain length 36 with those of average chain length 256. Longer chain poly K molecules gave significantly longer duration of expression both in vitro and in vivothan shorter chain poly K molecules for a given ligand and degree of substitution. Manipulation of the protein portion of receptor-targeted DNA complexes strongly influenced the duration and intensity of gene expression. This demonstration, in vitro and in vivo, addresses some of the limitations of the system and provides a new basis for improvement of this molecular conjugate for therapeutic purposes. DISCUSSIONThe composition of the protein portion of receptor-targeted gene transfer protein-DNA complexes profoundly affects the intensity and duration of gene expression, both in vitro and in vivo. In vitro the number of ligands/DNA molecule, the identity of the ligand, and the chain length of poly K all have strong effects on intensity and duration of gene expression, but the most striking effect was that of poly K chain length on the duration of expression. Using the ligand and number of ligands/DNA molecule found to be optimal in the cell culture system, we tested the effect of poly K chain length on in vivo expression. Complexes made with long chain poly K produced much more protracted gene expression in vivo than complexes prepared with short chain poly K.Optimization of the SEC-R-directed gene transfer for in HuH7 cells resulted in greatly increased reporter gene expression (relative to Lipofectin control) compared with our prior report (9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). Activity in the best complexes tested in the present study was about 20-fold higher than activity produced by the same amount of plasmid DNA delivered with the Lipofectin reagent; in our previous report maximal activity was less than the Lipofectin control. Increased peak activity indicates increased efficiency, which has been a problem for most nonviral strategies. At the same time, duration of activity could be greatly prolonged in vitro by informed selection of the specific receptor ligand (C105Y versus C1315), the number of ligands/plasmid DNA molecule (over 2–40 ligands), and the chain length of poly K used to condense the DNA. It is likely that the number of ligands and the identity of the ligand entrains routing within the cell, with the best complexes promoting routing into the endosomal compartment rather than to the lysosomal degradative pathway. For another receptor, the epidermal growth factor receptor, high number of ligands promotes lysosomal trafficking, whereas fewer ligands promote endosomal recycling (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar), so fewer ligands might well entrain a more favorable trafficking pattern, delaying the destruction of delivered DNA. In addition, different ligands for some receptors, such as the transferrin receptor, may be trafficked differently (28Scidmore M.A. Ficher E.R. Hackstadt T. J. Cell Biol. 1996; 134: 363-374Crossref PubMed Scopus (128) Google Scholar). A similar mechanism may account for the differences in duration of expression for complexes containing the two SEC-R ligands, which have very similar affinity for the receptor and very similar initial transgene expression.In vitro, the expression of the firefly luciferase reporter gene persisted (within a log maximum) in HuH7 cells for at least 40 days for some complexes that contained the long chain poly K-ligand conjugates. Because the half-life of the luciferase protein in mammalian cells is 3 h (29Brubaker J.O. Thompson C.M. Morrison L.A. Knipe D.M. Siber G.R. Finberg R.W. J. Immunol. 1996; 157: 1598-1604PubMed Google Scholar) and we observed rapid decline in luciferase activity in HuH7 cells transfected using Lipofectin (thus, HuH7 cells do not retain high levels of luciferase activity, however the gene is delivered), protracted luciferase expression from complexes made with long chain poly K probably represents continuing transcription of plasmid DNA. In an in vitro assay, we did not observe transcription from plasmid DNA compacted with either short or long chain poly K, so it is likely that in vivo, plasmid DNA separates from poly K before transcription. These considerations suggest that the prolonged expression results from retention of plasmid DNA complexed with poly K for a longer period of time for complexes containing long chain poly K than for the short chain. Condensation with poly K protects DNA against degradation (4Michael S.I. Curiel D.T. Gene Ther. 1994; 1: 223-232PubMed Google Scholar), and better protection is afforded by long chain poly K than short chain in in vitro assays. Thus, we speculate that plasmid DNA is retained within the cell for both complexes (because both have better persistence of expression than Lipofectin) with gradual degradation, which is resisted longer by complexes containing long chain poly K. Whether the complexes reside in membrane-bound cytoplasmic compartments, the cytoplasm, or the nucleus is not yet clear. It is also possible that the less tightly compacted complexes with the shorter poly K allow better access for the RNA polymerase to the DNA and accounts for the higher initial expression. In such a scenario, transgene expression would depend on a dynamic relationship between the availability of the DNA for transcription and the rate of its subsequent degradation.Transgene activity persists better in vitro (40 days) thanin vivo (less than 30 days), even for genes delivered using complexes containing long chain poly K. There are probably several reasons for this difference. In vitro, only processes within the transfected cell account for decay of transgene activity, whereasin vivo, immune processes can be recruited to destroy cells expressing proteins not recognized as “self.” Bacterial β-galactosidase has been shown, in and of itself, to incite cytotoxic lymphocyte responses in vivo, and therefore, cells expressing this protein are preferentially targeted and destroyed (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar). This is probably one mechanism of extinguishing β-galactosidase activity. In addition, the HuH7 cells transfected in vitroare immortal but fail to grow and divide after about 12–14 days under our culture conditions if they are not subcultured. In vivo, normal cell turnover of transfected cells may limit duration of expression.Complexes containing a minimal number of ligands that specifically deliver exogenous DNA to receptor bearing cells might be less immunogenic than more heavily substituted complexes. DNA (30Ebner R. Derynck R. Cell Regul. 1991; 2: 599-612Crossref PubMed Scopus (207) Google Scholar) and poly-l-amino acids (31Maurer P.H. J. Immunol. 1962; 88: 330-338PubMed Google Scholar) have been found to be relatively nonimmunogenic. Thus, possible immunogenicity of receptor-targeted complexes in vivo may depend on the ligand portion and, in part, its abundance. The abundance of SEC-R in lung, liver, and brain (14Perlmutter D.H. Pediatr. Res. 1994; 36: 271-277Crossref PubMed Scopus (13) Google Scholar), all of which might be potential target tissues for therapeutic gene transfer in common inherited (e.g.α1-antitrypsin deficiency) or acquired (e.g.Alzheimer's disease) disorders, has made it a desirable target for receptor-mediated gene therapy. The development of optimal complexes that produce high level gene expression for short (9.7-kDa poly K conjugates) or longer (53.7-kDa poly K conjugates) periods of time will be useful in achieving optimal therapeutic effects. The composition of the protein portion of receptor-targeted gene transfer protein-DNA complexes profoundly affects the intensity and duration of gene expression, both in vitro and in vivo. In vitro the number of ligands/DNA molecule, the identity of the ligand, and the chain length of poly K all have strong effects on intensity and duration of gene expression, but the most striking effect was that of poly K chain length on the duration of expression. Using the ligand and number of ligands/DNA molecule found to be optimal in the cell culture system, we tested the effect of poly K chain length on in vivo expression. Complexes made with long chain poly K produced much more protracted gene expression in vivo than complexes prepared with short chain poly K. Optimization of the SEC-R-directed gene transfer for in HuH7 cells resulted in greatly increased reporter gene expression (relative to Lipofectin control) compared with our prior report (9Ziady A.G. Perales J.C. Ferkol T. Gerken T. Beegen H. Perlmutter D.H. Davis P.B. Am. J. Phys. 1997; 273: G545-G552PubMed Google Scholar). Activity in the best complexes tested in the present study was about 20-fold higher than activity produced by the same amount of plasmid DNA delivered with the Lipofectin reagent; in our previous report maximal activity was less than the Lipofectin control. Increased peak activity indicates increased efficiency, which has been a problem for most nonviral strategies. At the same time, duration of activity could be greatly prolonged in vitro by informed selection of the specific receptor ligand (C105Y versus C1315), the number of ligands/plasmid DNA molecule (over 2–40 ligands), and the chain length of poly K used to condense the DNA. It is likely that the number of ligands and the identity of the ligand entrains routing within the cell, with the best complexes promoting routing into the endosomal compartment rather than to the lysosomal degradative pathway. For another receptor, the epidermal growth factor receptor, high number of ligands promotes lysosomal trafficking, whereas fewer ligands promote endosomal recycling (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar), so fewer ligands might well entrain a more favorable trafficking pattern, delaying the destruction of delivered DNA. In addition, different ligands for some receptors, such as the transferrin receptor, may be trafficked differently (28Scidmore M.A. Ficher E.R. Hackstadt T. J. Cell Biol. 1996; 134: 363-374Crossref PubMed Scopus (128) Google Scholar). A similar mechanism may account for the differences in duration of expression for complexes containing the two SEC-R ligands, which have very similar affinity for the receptor and very similar initial transgene expression. In vitro, the expression of the firefly luciferase reporter gene persisted (within a log maximum) in HuH7 cells for at least 40 days for some complexes that contained the long chain poly K-ligand conjugates. Because the half-life of the luciferase protein in mammalian cells is 3 h (29Brubaker J.O. Thompson C.M. Morrison L.A. Knipe D.M. Siber G.R. Finberg R.W. J. Immunol. 1996; 157: 1598-1604PubMed Google Scholar) and we observed rapid decline in luciferase activity in HuH7 cells transfected using Lipofectin (thus, HuH7 cells do not retain high levels of luciferase activity, however the gene is delivered), protracted luciferase expression from complexes made with long chain poly K probably represents continuing transcription of plasmid DNA. In an in vitro assay, we did not observe transcription from plasmid DNA compacted with either short or long chain poly K, so it is likely that in vivo, plasmid DNA separates from poly K before transcription. These considerations suggest that the prolonged expression results from retention of plasmid DNA complexed with poly K for a longer period of time for complexes containing long chain poly K than for the short chain. Condensation with poly K protects DNA against degradation (4Michael S.I. Curiel D.T. Gene Ther. 1994; 1: 223-232PubMed Google Scholar), and better protection is afforded by long chain poly K than short chain in in vitro assays. Thus, we speculate that plasmid DNA is retained within the cell for both complexes (because both have better persistence of expression than Lipofectin) with gradual degradation, which is resisted longer by complexes containing long chain poly K. Whether the complexes reside in membrane-bound cytoplasmic compartments, the cytoplasm, or the nucleus is not yet clear. It is also possible that the less tightly compacted complexes with the shorter poly K allow better access for the RNA polymerase to the DNA and accounts for the higher initial expression. In such a scenario, transgene expression would depend on a dynamic relationship between the availability of the DNA for transcription and the rate of its subsequent degradation. Transgene activity persists better in vitro (40 days) thanin vivo (less than 30 days), even for genes delivered using complexes containing long chain poly K. There are probably several reasons for this difference. In vitro, only processes within the transfected cell account for decay of transgene activity, whereasin vivo, immune processes can be recruited to destroy cells expressing proteins not recognized as “self.” Bacterial β-galactosidase has been shown, in and of itself, to incite cytotoxic lymphocyte responses in vivo, and therefore, cells expressing this protein are preferentially targeted and destroyed (27Lai W.H. Cameron P.H. Doherty II, I.W.J.-J. Kay D.G. Posner B.I. Bergeron J.J.M. J. Cell Biol. 1989; 109: 2741-2749Crossref PubMed Scopus (81) Google Scholar). This is probably one mechanism of extinguishing β-galactosidase activity. In addition, the HuH7 cells transfected in vitroare immortal but fail to grow and divide after about 12–14 days under our culture conditions if they are not subcultured. In vivo, normal cell turnover of transfected cells may limit duration of expression. Complexes containing a minimal number of ligands that specifically deliver exogenous DNA to receptor bearing cells might be less immunogenic than more heavily substituted complexes. DNA (30Ebner R. Derynck R. Cell Regul. 1991; 2: 599-612Crossref PubMed Scopus (207) Google Scholar) and poly-l-amino acids (31Maurer P.H. J. Immunol. 1962; 88: 330-338PubMed Google Scholar) have been found to be relatively nonimmunogenic. Thus, possible immunogenicity of receptor-targeted complexes in vivo may depend on the ligand portion and, in part, its abundance. The abundance of SEC-R in lung, liver, and brain (14Perlmutter D.H. Pediatr. Res. 1994; 36: 271-277Crossref PubMed Scopus (13) Google Scholar), all of which might be potential target tissues for therapeutic gene transfer in common inherited (e.g.α1-antitrypsin deficiency) or acquired (e.g.Alzheimer's disease) disorders, has made it a desirable target for receptor-mediated gene therapy. The development of optimal complexes that produce high level gene expression for short (9.7-kDa poly K conjugates) or longer (53.7-kDa poly K conjugates) periods of time will be useful in achieving optimal therapeutic effects. We thank John Kim for crucial assistance with the in vivo studies, Dr. Thomas Gerken for assistance with NMR analysis, Helga Beegen for technical assistance with electron microscopy, and Dr. Roger Marchant for assistance with AFM microscopy.

An elevation in the circulating level of the squamous-cell carcinoma antigen (SCCA) can be a poor prognostic indicator in certain types of squamous-cell cancers. Total SCCA in the circulation comprises 2 nearly identical, approximately 45 kDa proteins, SCCA1 and SCCA2. Both proteins are members of the high-molecular weight serine proteinase inhibitor (serpin) family with SCCA1 paradoxically inhibiting lysosomal cysteine proteinases and SCCA2 inhibiting chymotrypsin-like serine proteinases. Although SCCA1 and SCCA2 are detected in the cytoplasm of normal squamous epithelial cells, neither serpin is detected normally in the serum. Thus, their presence in the circulation at relatively high concentrations suggests that malignant epithelial cells are re-directing serpin activity to the fluid phase via an active secretory process. Because serpins typically inhibit their targets by binding at 1:1 stoichiometry, a change in the distribution pattern of SCCA1 and SCCA2 (i.e., intracellular to extracellular) could indicate the need of tumor cells to neutralize harmful extracellular proteinases. The purpose of our study was to determine experimentally the fate of SCCA1 and SCCA2 in squamous carcinoma cells. Using subcellular fractionation, SCCA-green fluorescent fusion protein expression and confocal microscopy, SCCA1 and SCCA2 were found exclusively in the cytosol and were not associated with nuclei, mitochondria, lysosomes, microtubules, actin or the Golgi. In contrast to previous reports, metabolic labeling and pulse-chase experiments showed that neither non-stimulated nor TNFalpha/PMA-stimulated squamous carcinoma cells appreciably secreted these ov-serpins into the medium. Collectively, these data suggest that the major site of SCCA1 and SCCA2 inhibitory activity remains within the cytosol and that their presence in the sera of patients with advanced squamous-cell carcinomas may be due to their passive release into the circulation.

α1-Antitrypsin (α1AT), the archetype of the Serpin supergene family, is the principal blood-borne inhibitor of destructive neutrophil proteases including elastase, cathepsin G, and proteinase 3 (reviewed in ref. 1). This glycoprotein, is secreted by liver cells and is considered an acute-phase reactant because its plasma levels increase during the host response to inflammation/tissue injury. The classical form of α1AT deficiency, which affects 1 in 1,800 live births in Northern European and North American populations, is associated with a mutant molecule termed α1ATZ, which is retained as a polymer in the endoplasmic reticulum (ER) of liver cells (reviewed in refs. 2, 3). Homozygotes are predisposed to premature development of pulmonary emphysema by a loss-of-function mechanism in which lack of α1AT in the lung permits uninhibited proteolytic damage to the connective tissue matrix (4, 5). Cigarette smoking markedly increases the risk and rate of development of emphysema (6). One mechanism for this environmental risk factor involves the functional inactivation of residual α1AT by phagocyte-derived active oxygen intermediates (4, 5). However, a growing body of evidence suggests that other environmental factors and genetic traits affect the incidence and severity of lung disease among α1AT-deficient individuals (7). It is still not entirely clear whether heterozygotes for α1ATZ are predisposed to lung disease. Homozygotes for α1ATZ (“PIZZ” individuals) are also at risk for liver disease. In fact, α1AT deficiency is the most common genetic cause of liver disease in children (2, 3), and it predisposes adults to chronic liver disease and hepatocellular carcinoma (8). However, in contrast to the pathobiology of lung disease, liver injury in this deficiency appears to involve a gain-of-function mechanism whereby retention of the mutant α1ATZ molecule in the ER triggers a series of events that are eventually hepatotoxic. The strongest evidence for a gain-of-function mechanism comes from studies in which mice transgenic for mutant human α1ATZ develop liver injury with many of the histopathologic hallmarks of the human condition (9, 10). Because there are normal levels of anti-elastases in these mice, as directed by endogenous genes, the liver injury cannot be attributed to a loss of function. Landmark nationwide prospective screening studies done by Sveger in Sweden have documented an extraordinary variation in the phenotypic expression of liver disease among homozygotes. In these studies, only 10–15% of the PIZZ population developed clinically significant liver disease over the first 20 years of life (11, 12). These data indicate that other genetic traits and/or environmental factors predispose a subgroup of PIZZ individuals to liver injury. Because only a subgroup of homozygotes develop liver disease and because there is an inherent bias in ascertainment in other clinical studies of α1AT deficiency, it has been very difficult to determine whether heterozygous (“PIMZ”) individuals are at increased risk for liver disease.

The classical form of α1-antitrypsin (α1-AT) deficiency is associated with a mutant α1-ATZ molecule that polymerizes in the endoplasmic reticulum (ER) of liver cells. A subgroup of individuals homozygous for the protease inhibitor (PI) <i>Z</i> allele develop chronic liver injury and are predisposed to hepatocellular carcinoma. In this study we evaluated the primary structure of α1-AT in a family in which three affected members had severe liver disease associated with α1-AT deficiency. We discovered that one sibling was a compound heterozygote with one PI <i>Z</i> allele and a second allele, the PI <i>Z</i> + <i>saar</i> allele, bearing the mutation that characterizes α1-ATZ as well as the mutation that characterizes α1-AT Saarbrucken (α1-AT saar). The mutation in PI<i>saar</i> introduces a premature termination codon resulting in an α1-AT protein truncated for 19 amino acids at its carboxyl terminus. Studies of a second sib with severe liver disease and other living family members did not reveal the presence of the α1-AT saar mutation and therefore do not substantiate a role for this mutation in the liver disease phenotype of this family. However, studies of α1-AT saar and α1-ATZ + saar expressed in heterologous cells show that there is prolonged intracellular retention of these mutants even though they do not have polymerogenic properties. These results therefore have important implications for further understanding the fate of mutant α1-AT molecules, the mechanism of ER retention, and the pathogenesis of liver injury in α1-AT deficiency.

The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.

Using metabolic labeling techniques in human intestinal epithelial cell lines in tissue culture and <i>in situ</i> hybridization techniques in normal and inflamed (Crohn's) intestine, recent studies have shown that there is synthesis of acute phase proteins in enterocytes. Moreover, these studies have shown that acute phase protein biosynthesis in enterocytes is regulated by inflammatory cytokines in a manner characteristic of the physiologic acute phase response. In the course of these studies it was noticed that one inflammatory cytokine, interleukin-6 (IL-6), mediated selective down-regulation of the enterocyte-specific, differentiation-dependent integral membrane protein sucrase-isomaltase (SI) in the Caco2 intestinal epithelial cell line. In the current study we examined the effect of several other inflammatory cytokines interleukin-1 (IL-1β), tumor necrosis factor α (TNFα), and interferon _γ (IFN_γ) on synthesis of SI in Caco2 cells, examined the possibility that inflammatory cytokines affect the synthesis of other enterocyte integral membrane proteins using lactase as a prototype, and examined the possibility that SI gene expression was down-regulated in villous enterocytes <i>in vivo</i> during the local inflammatory response of Crohn's disease. The results show that IL-6 and IFN_γ each mediate a decrease and TNFα mediates an increase in synthesis of SI in Caco2 cells. The magnitude of down-regulation by IL-6 and IFN_γ is significantly greater than the up-regulation by TNFα. IL-1β has no effect on synthesis of SI. Synthesis of lactase is not affected by any of the cytokines. There is a marked specific decrease in SI gene expression in villous enterocytes in acutely inflamed Crohn's ileum as compared to adjacent uninflamed ileum and normal ileum. Taken together, these data show that inflammatory cytokines have specific and selective effects on the expression of the brush border hydrolase SI in tissue culture and <i>in vivo</i> and provide evidence for a previously unrecognized mechanism for disaccharidase deficiency in intestinal inflammation.

alpha 1-Antitrypsin (alpha 1 AT) is plasma glycoprotein that constitutes the principle inhibitor of neutrophil elastase in tissue fluids. It has been considered a prototype for liver-derived acute phase proteins in that its concentration in plasma increases three- to fourfold during the host response to inflammation/tissue injury. However, recent studies have shown that alpha 1 AT is expressed in several types of extrahepatic cells, including mononuclear phagocytes and enterocytes, and that there are distinct transcriptional units used in hepatocytes and at least one extra-hepatic cell type, blood monocytes. In this study, we have used a combination of ribonuclease protection assays, primer elongation analysis, and transcriptional run-on assays to further characterize mechanisms of basal and modulated alpha 1 AT gene expression in hepatocytes, enterocytes, and macrophages. The hepatoma cell line HepG2, intestinal epithelial cell line Caco2, and primary cultures of human peripheral blood monocytes were used as examples of the cell types. The results indicate that there are three macrophage-specific transcriptional initiation sites upstream from a single hepatocyte-specific transcriptional initiation site. Macrophages use these sites during basal and modulated expression. Hepatoma cells use the hepatocyte-specific transcriptional initiation site during basal and modulated expression but also switch on transcription from the upstream macrophage transcriptional initiation sites during modulation by the acute phase mediator interleukin 6 (IL-6). Caco2 cells use the hepatocyte-specific transcriptional initiation site during basal expression. There is a marked increase in the use of this site and an increase in the rate of transcriptional elongation of alpha 1 AT mRNA during differentiation of Caco2 cells from crypt-type to villous-type enterocytes. Caco2 cells also switch on transcription from the upstream macrophage transcriptional initiation sites during modulation by IL-6. These results provide further evidence that there are differences in the mechanisms of constitutive and regulated expression of the alpha 1 AT gene in at least three different cell types, HepG2-derived hepatocytes, Caco2-derived enterocytes and mononuclear phagocytes.

Four adolescents with achalasia were treated with nifedipine. All the patients' symptoms improved dramatically. On manometric evaluation, following oral nifedipine, the lower esophageal sphincter pressure decreased approximately 50%. No change in esophageal peristaltic activity was noted. Side effects were minimal; two patients had mild headache initially. Nifedipine, which is commonly used in adult patients with achalasia, may be beneficial for short-term symptomatic relief in children until more definitive therapy can be performed.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read More

Individuals who are homozygous for the protease inhibitor phenotype Z (PiZ) genetic variant of alpha 1-antitrypsin (alpha 1-AT) have reduced plasma concentrations of alpha 1-AT, and are susceptible to premature development of pulmonary emphysema. A subset of this population develops chronic liver disease. The reduction in plasma concentrations of alpha 1-AT results from a selective defect in secretion as the abnormal PiZ alpha 1-AT protein accumulates within the cell. It has recently been shown in several experimental systems that the heat shock/stress response, a response characterized by the synthesis of a family of highly evolutionarily conserved proteins during thermal or chemical stress, may also be activated by the presence of abnormal proteins within the cell. Therefore, we predicted that the heat shock/stress response would be induced in the absence of thermal or chemical stress in alpha 1-AT-synthesizing cells of PiZZ individuals. In the following study, however, we show that net synthesis of proteins in the heat shock/stress gene family (SP90, SP70, ubiquitin) is increased only in a subset of the population, PiZZ individuals with liver disease. It is not significantly increased in PiZZ individuals with emphysema or in those without apparent tissue injury. Net synthesis of stress proteins is not increased in individuals with another variant of the alpha 1-AT gene (PiS alpha 1-AT) and is not increased in individuals with severe liver disease but a normal alpha 1-AT haplotype (PiM alpha 1-AT). These results demonstrate that the synthesis of stress proteins is increased in a subset of individuals with homozygous PiZZ alpha 1-AT deficiency, those also having liver disease.

When taken together, three conceptual paradigms have led to major advances in understanding the clinical manifestations of protein misfolding and recently have led to novel therapeutic strategies. First, disorders caused by misfolded proteins are now classified according to the mechanism by which they cause clinical effects, either loss-of-function or toxic gain-of-function. Loss-of-function is the result of mutations that specifically alter folding, such that the function of the protein is impaired or such that the protein does not reach the cellular destination where its function is required, or both. Cystic fibrosis is the prototype disease caused by a loss-of-function mechanism in that all of the disease manifestations arise from lack of chloride transport. The CFTRΔF508 variant does not reach the apical surface of epithelial cells predominantly because of misfolding in the endoplasmic reticulum (ER) and rapid degradation by the proteasome. The small amount of CFTRΔF508 that does reach the cell surface is unstable and this probably also contributes to loss of chloride transport activity at epithelia. Toxic gain-of-function mechanisms are attributable to the pathologic activity of the mutant protein itself or to the effect of its mislocalization or both. This type of mechanism is implicated when the mutant protein produces a toxic effect in a cell line or live animal model. Huntington disease and early-onset forms of Alzheimer disease are prototypes of the gain-of-function mechanism as protein misfolding leads to degeneration of neurons. Diseases with childhood onset also fit into the paradigm, including conditions as diverse as respiratory failure in the newborn 1 Whittsett J.A. Wert S.E. Weaver T.E. Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu Rev Med. 2010; 61: 105-119 Crossref PubMed Scopus (312) Google Scholar and early-onset diabetes, 2 Liu M. Hodish I. Haataja L. Lara-Lomus R. Rajpal G. Wright J. et al. Proinsulin misfolding and diabetes: mutant INS gene-induced diabetes of youth. Trends Endocrinol Metab. 2010; 21: 652-659 Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar among many others.

The accumulation of serpin oligomers and polymers within the endoplasmic reticulum (ER) causes cellular injury in patients with the classical form α1-antitrypsin deficiency (ATD). To better understand the cellular and molecular genetic aspects of this disorder, we generated transgenic C. elegans strains expressing either the wild-type (ATM) or Z mutant form (ATZ) of the human serpin fused to GFP. Animals secreted ATM, but retained polymerized ATZ within dilated ER cisternae. These latter animals also showed slow growth, smaller brood sizes and decreased longevity; phenotypes observed in ATD patients or transgenic mouse lines expressing ATZ. Similar to mammalian models, ATZ was disposed of by autophagy and ER-associated degradation pathways. Mutant strains defective in insulin signaling (daf-2) also showed a marked decrease in ATZ accumulation. Enhanced ATZ turnover was associated with the activity of two proteins central to systemic/exogenous (exo)-RNAi pathway: the dsRNA importer, SID-1 and the argonaute, RDE-1. Animals with enhanced exo-RNAi activity (rrf-3 mutant) phenocopied the insulin signaling mutants and also showed increased ATZ turnover. Taken together, these studies allude to the existence of a novel proteostasis pathway that mechanistically links misfolded protein turnover to components of the systemic RNAi machinery.

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy. Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.

Aging is a common risk factor in neurodegenerative disorders. Investigating neuronal aging in an isogenic background stands to facilitate analysis of the interplay between neuronal aging and neurodegeneration. Here we perform direct neuronal reprogramming of longitudinally collected human fibroblasts to reveal genetic pathways altered at different ages. Comparative transcriptome analysis of longitudinally aged striatal medium spiny neurons (MSNs) in Huntington’s disease identified pathways involving RCAN1, a negative regulator of calcineurin. Notably, RCAN1 protein increased with age in reprogrammed MSNs as well as in human postmortem striatum and RCAN1 knockdown rescued patient-derived MSNs of Huntington’s disease from degeneration. RCAN1 knockdown enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, leading to TFEB’s nuclear localization by dephosphorylation. Furthermore, G2-115, an analog of glibenclamide with autophagy-enhancing activities, reduced the RCAN1–calcineurin interaction, phenocopying the effect of RCAN1 knockdown. Our results demonstrate that targeting RCAN1 genetically or pharmacologically can increase neuronal resilience in Huntington’s disease. To analyze neuronal aging in Huntington’s disease, Lee et al. perform direct neuronal reprogramming of longitudinally aged human fibroblasts, uncovering RCAN1 as a therapeutic target to promote neuronal resilience through chromatin reconfiguration.

A delay in intracellular degradation of the mutant alpha(1)-antitrypsin (alpha(1)AT)Z molecule is associated with greater retention within the endoplasmic reticulum (ER) and susceptibility to liver disease in a subgroup of patients with alpha(1)AT deficiency. Recent studies have shown that alpha(1)ATZ is ordinarily degraded in the ER by a mechanism that involves the proteasome, as demonstrated in intact cells using human fibroblast cell lines engineered for expression of alpha(1)ATZ and in a cell-free microsomal translocation assay system programmed with purified alpha(1)ATZ mRNA. To determine whether the ubiquitin system is required for proteasomal degradation of alpha(1)ATZ and whether specific components of the ubiquitin system can be implicated, we have now used two approaches. First, we overexpressed a dominant-negative ubiquitin mutant (UbK48R-G76A) by transient transfection in the human fibroblast cell lines expressing alpha(1)ATZ. The results showed that there was marked, specific, and selective inhibition of alpha(1)ATZ degradation mediated by UbK48R-G76A, indicating that the ubiquitin system is at least in part involved in ER degradation of alpha(1)ATZ. Second, we subjected reticulocyte lysate to DE52 chromatography and tested the resulting well-characterized fractions in the cell-free system. The results showed that there were both ubiquitin-dependent and -independent proteasomal mechanisms for degradation of alpha(1)ATZ and that the ubiquitin-conjugating enzyme E2-F1 may play a role in the ubiquitin-dependent proteasomal mechanism.

A prospective nationwide screening study initiated more than 20 years ago in Sweden has shown that clinically significant liver disease develops in only 10% to 15% of alpha1-antitrypsin (AT)-deficient children. This study provides information about 85% to 90% of those children, many of whom had elevated serum transaminases in infancy but have no evidence of liver injury by age 18 years. However, there is relatively limited information about the course of alpha1-AT-deficient children who have cirrhosis or portal hypertension. Based on several anecdotal experiences, we have been impressed by the relatively slow progression and stable course of the liver disease in some of these children.We reviewed the course of patients with homozygous PIZZ alpha1-antitrypsin deficiency seen at this institution since establishing a patient database 16 years ago.Of 44 patients with alpha1-AT deficiency, 17 had cirrhosis, portal hypertension, or both. Nine of the 17 patients with cirrhosis or portal hypertension had a prolonged, relatively uneventful course for at least 4 years after the diagnosis of cirrhosis or portal hypertension. Two of these patients eventually underwent liver transplantation, but seven are leading relatively healthy lives for up to 23 years while carrying a diagnosis of severe alpha1-AT deficiency-associated liver disease. Patients with the prolonged stable course could be distinguished from those with a rapidly progressive course on the basis of overall life functioning but not on the basis of any other more conventional clinical or biochemical criteria.These data provide further evidence for the variable severity of liver disease associated with alpha1-AT deficiency and indicate that some patients have chronic, slowly progressing or nonprogressing cirrhosis.

Expression of alpha 1 proteinase inhibitor (alpha 1-PI) in human mononuclear phagocytes may provide a local mechanism for inactivation of serine proteases at sites of tissue injury, thereby preventing incidental damage to surrounding tissue and allowing for orderly initiation of repair. We have previously shown that serine (neutrophilic or pancreatic) elastase and lipopolysaccharide (LPS) each mediate an increase in the expression of alpha 1-PI in human peripheral blood monocytes and bronchoalveolar macrophages. In this study we demonstrate that elastase and LPS have an additive positive regulatory effect on alpha 1-PI expression. Distinct pretranslational and translational mechanisms of action for elastase and LPS, respectively, account for the additive effect. The possibility that translational regulation of alpha 1-PI by LPS involves a mechanism analogous to that of the yeast gene GCN4 during amino acid starvation and that of the human ferritin gene in response to iron is discussed.

Susceptibility to autoimmune disease is associated with null alleles at one of the two genetic loci encoding complement protein C4. These two genetic loci, C4A and C4B, are highly homologous in primary structure but encode proteins with different functional activities. Expression of C4A and C4B genes is regulated by IFN-gamma in human hepatoma cells and in murine fibroblasts transformed with the respective genes. In these cell lines, IFN-gamma has a significantly greater and longer-lasting effect on expression of C4A than that of C4B. In this study we examined synthesis and regulation of C4A and C4B in peripheral blood monocytes from normal, C4A-null, and C4B-null individuals. Synthesis of C4 in human peripheral blood monocytes decreases during time in culture. IFN-gamma mediates a concentration- and time-dependent increase in steady-state levels of C4 mRNA and a corresponding increase in synthesis of C4 in normal human monocytes. LPS decreases monocyte C4 expression and completely abrogates the effect of IFN-gamma on the expression of this gene. In contrast, LPS and IFN-gamma have a synergistic effect in upregulating expression of another class III MHC gene product, complement protein factor B. The effect of LPS on constitutive and IFN-gamma-regulated C4 synthesis is probably not mediated via release of endogenous monokines IL-1 beta, TNF-alpha, or IL-6. Synthesis of C4, and regulation of its synthesis by IFN-gamma and LPS, are similar in normal, C4A-, and C4B-null individuals. These results demonstrate the synthesis of C4 at extrahepatic sites and tissue-specific regulation of C4 gene expression.

The major histocompatibility complex-linked human complement C4 genes are highly homologous in primary structure but give rise to products which differ in complement-activating function. In order to examine the synthesis, function, and regulation of these two genes independently, cloned C4A and C4B genes were transfected into mouse fibroblast L-cells. In the stable transfected cell lines, C4A and C4B are synthesized, undergo a complex series of post-translational modifications, and each functions appropriately in activation of the classical complement pathway. A marked difference in the kinetics of complement component C1-mediated cleavage of the C4A- and C4B-alpha chains was demonstrated in the transfectants and may contribute to the differences in the intrinsic functional activity of the two C4 isotypes. In contrast to the expression of other complement genes which are affected during the hepatic acute phase response (factor B, C3), the expression of C4 was not regulated by interleukin-1 or tumor necrosis factor. Interferon-gamma, however, mediated a dose- and time-dependent increase in the expression of the C4 genes. Moreover, interferon had a significantly greater and longer-lasting effect on the synthesis of C4A than that of C4B. Differences in the expression and regulation of these two genes provide insight into the control of complement activation during inflammation.

The second component of complement (C2), is a class III major histocompatibility complex gene product and a glycoprotein in the classical complement activating system. Synthesis in the human hepatoma-derived cell line HepG2 results in three intracellular forms: an 84-kDa form secreted in 1-2 h; 79-kDa and 70-kDa forms that remain cell-associated for intervals up to 12 h. All three forms are C2 polypeptides as demonstrated by inhibition of immunoprecipitation with unlabeled C2 and the presence of common major peptide fragments following chymotryptic digestion. The cell-associated forms of C2 are not products of proteolysis as demonstrated by experiments with multiple proteinase inhibitors and by observations of the kinetics of synthesis. Inhibition of core glycosylation by tunicamycin and deglycosylation by acid hydrolysis indicate that the three intracellular C2 polypeptides are glycosylated to a similar extent. Although the 84-kDa form of C2 is susceptible to C1s cleavage, the two cell-associated forms are not. Cell-free biosynthesis by mRNA from HepG2 or human liver results in three primary translation products corresponding to the three unglycosylated forms of C2. These results indicate that HepG2 cells synthesize C2 protein in both secreted and cell-associated forms and that each form is derived from a separate primary translation product.

In a1-antitrypsin (AT) deficiency, a point mutation renders a hepatic secretory glycoprotein prone to misfolding and polymerization.The mutant protein accumulates in the endoplasmic reticulum of liver cells and causes hepatic fibrosis and hepatocellular carcinoma by a gain-of-function mechanism.Genetic and/or environmental modifiers determine whether an affected homozygote is susceptible to hepatic fibrosis/carcinoma.Two types of proteostasis mechanisms for such modifiers have been postulated: variation in the function of intracellular degradative mechanisms and/or variation in the signal transduction pathways that are activated to protect the cell from protein mislocalization and/or aggregation.In recent studies we found that carbamazepine, a drug that has been used safely as an anticonvulsant and mood stabilizer, reduces the hepatic load of mutant AT and hepatic fibrosis in a mouse model by enhancing autophagic disposal of this mutant protein.These results provide evidence that pharmacological manipulation of endogenous proteostasis mechanisms is an appealing strategy for chemoprophylaxis in disorders involving gain-of-function mechanisms.

Autophagy plays an essential role in cell survival/death and functioning. Modulation of autophagy has been recognized as a promising therapeutic strategy against diseases/disorders associated with uncontrolled growth or accumulation of biomolecular aggregates, organelles, or cells including those caused by cancer, aging, neurodegeneration, and liver diseases such as α1-antitrypsin deficiency. Numerous pharmacological agents that enhance or suppress autophagy have been discovered. However, their molecular mechanisms of action are far from clear. Here, we collected a set of 225 autophagy modulators and carried out a comprehensive quantitative systems pharmacology (QSP) analysis of their targets using both existing databases and predictions made by our machine learning algorithm. Autophagy modulators include several highly promiscuous drugs (e.g., artenimol and olanzapine acting as activators, fostamatinib as an inhibitor, or melatonin as a dual-modulator) as well as selected drugs that uniquely target specific proteins (~30% of modulators). They are mediated by three layers of regulation: (i) pathways involving core autophagy-related (ATG) proteins such as mTOR, AKT, and AMPK; (ii) upstream signaling events that regulate the activity of ATG pathways such as calcium-, cAMP-, and MAPK-signaling pathways; and (iii) transcription factors regulating the expression of ATG proteins such as TFEB, TFE3, HIF-1, FoxO, and NF-κB. Our results suggest that PKA serves as a linker, bridging various signal transduction events and autophagy. These new insights contribute to a better assessment of the mechanism of action of autophagy modulators as well as their side effects, development of novel polypharmacological strategies, and identification of drug repurposing opportunities.

AbstractAlpha-1-antitrypsin (α1-AT) deficiency is a well known cause of emphysema in adults. A subgroup of deficient individuals develops liver injury during infancy and childhood. In fact, it is the most common genetic cause of liver disease in children. Although lung injury is due to the decrease in α1 -AT function in the lung, allowing uninhibited elasto-lytic destruction of its connective tissue integrity, liver injury is probably due to retention of the mutant α1-AT molecule in the endoplasmic reticulum (ER) of liver cells. Recent studies have shown that the mutant α1-AT molecule polymerizes in the ER by a novel loop-sheet insertion mechanism. Other recent studies show that the subgroup of deficient individuals is susceptible to liver injury by virtue of unlinked genetic traits and/or environmental factors which interfere with degradation of the mutant α1-AT molecules within the ER.Key Words: alpha-1-antitrypsinemphysemaendoplasmic reticulum degradationliver disease

The serpin-enzyme complex (SEC) receptor was originally identified using a synthetic peptide (peptide 105Y) based on the sequence of a candidate receptorbinding domain of ␣1-antitrypsin (1-AT) and was subsequently shown to be a receptor on the surface of hepatocytes, monocytes, and neutrophils for recognition of ␣1-AT-elastase and several other serpin-enzyme complexes (Perlmutter, D.

The outcome of the hepatic portoenterostomy (Kasai) procedure for biliary atresia is improved when it is performed before 90 days of age. However, it is not known whether intervention before 30 days is better than intervention between 30 and 90 days.The authors reviewed the records of all patients seen by the Pediatric Gastroenterology Service at St. Louis Children's Hospital from 1984-1999 to ascertain the outcome of patients who underwent Kasai procedure before or after 30 days of age.Of 92 patients with biliary atresia treated at St. Louis Children's Hospital over 15 years, 9 underwent the Kasai procedure before 30 days of age. Liver transplantation was necessary in 77.8% of these patients at a mean age of 11.0 +/- 4.26 months, as compared with 53.4% at 32.14 +/- 7.14 months for the remainder of the patients who underwent the procedure after 30 days of age.Although these data suggest that outcomes are worse for patients who undergo the procedure before 30 days of age, they may reflect a difference in the pathogenesis of biliary atresia that brings it to clinical attention earlier and may provide further evidence that biliary atresia is a phenotype for a number of distinct underlying disease processes.

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alpha 1-Antitrypsin (alpha 1-AT) deficiency is the most common genetic cause of liver disease in children and genetic disease for which children undergo liver transplantation. It also causes cirrhosis and hepatocellular carcinoma in adults. Studies by Sveger in Sweden have shown that only a subgroup of the population with homozygous PiZZ alpha 1-AT deficiency develop clinically significant liver injury. Other studies have shown that the mutant alpha 1-AT Z molecule undergoes polymerization in the endoplasmic reticulum and that a subpopulation of alpha 1-AT-deficient individuals may be susceptible to liver injury because they also have a trait that reduces the efficiency by which the mutant alpha 1-AT Z molecule is degraded in the endoplasmic reticulum.

We have shown that bile acid efflux and ecto-ATPase activities are two distinct properties of a single rat liver hepatocyte canalicular membrane protein (Sippel, C. J., Suchy, F. J., Ananthanarayanan, M., and Perlmutter, D. H. (1993) J. Biol. Chem. 268, 2083-2091). Bile acid efflux in COS cells transfected with this rat hepatocyte canalicular bile acid transport/ectoATPase cDNA is stimulated by ATP and inhibited by nonhydrolyzable ATP analogs. In this study, we depleted transfected COS cells of ATP to examine whether bile acid efflux mediated by this transporter was dependent on ATP or just stimulated by ATP. We also used mutagenesis of an ATPase consensus sequence in the ectoplasmic domain to examine the relationship of ATPase activity to bile acid efflux mediated by the same polypeptide. The results indicate that bile acid transport is abrogated by ATP depletion and reconstituted by exogenous ATP in a concentration-dependent and saturable manner. Introduction of mutations at amino acids Gly97 and Arg98 in the ATPase consensus sequence abrogated ATPase activity but did not affect synthesis or cell surface delivery of the transporter and did not affect its bile acid transport activity. Taken together, the data indicate that bile acid efflux mediated by the rat hepatocyte canalicular bile acid transport/ecto-ATPase protein is dependent on ATP but not on its own ATPase activity. The data, therefore, imply that 1) ATP affects its bile acid transport activity through an entirely distinct mechanism; and 2) if there is any functional relationship between the ecto-ATPase and bile acid transport properties, it is mediated indirectly through regulation of net ATP concentrations in the canalicular space by the ecto-ATPase.

A fraction of intestinal alkaline phosphatase (IAP) is secreted into blood. To study this process, enzyme secretion was examined in a fetal (IRD-98) and a differentiated (Caco-2) intestinal cell line. Tissue-unspecific alkaline phosphatase (AP) activity in the IRD-98 cells increased 20-fold after addition of 1.5 mM sodium butyrate and 40 mM NaCl, but no AP activity was secreted into the medium. In contrast, newly synthesized IAP in Caco-2 cells was secreted into the medium. AP secretion increased with time and was inhibited by monensin. Medium AP was still partially bound to membranes as assessed by Triton X-114 phase separation and could be released by the addition of serum. Analysis by sodium dodecyl sulfate polyacrylamide gels and by isoelectric focussing showed that secreted AP gave a pattern similar to that of the AP released from membranes by phospholipase D treatment. When Caco-2 cells were grown on filters, AP activity was found in both basolateral (75%) and luminal (25%) media. These data demonstrate that the secretion of a particulate AP with extracellular release from the membrane can account for the appearance of the intestinal isozyme in both the serum and the lumen.