Stephen C. Pak

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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.

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.

Extracellular serpins such as antithrombin and α1-antitrypsin are the quintessential regulators of proteolytic pathways. In contrast, the biological functions of the intracellular serpins remain obscure. We now report that the C. elegans intracellular serpin, SRP-6, exhibits a prosurvival function by blocking necrosis. Minutes after hypotonic shock, srp-6 null animals underwent a catastrophic series of events culminating in lysosomal disruption, cytoplasmic proteolysis, and death. This newly defined hypo-osmotic stress lethal (Osl) phenotype was dependent upon calpains and lysosomal cysteine peptidases, two in vitro targets of SRP-6. By protecting against both the induction of and the lethal effects from lysosomal injury, SRP-6 also blocked death induced by heat shock, oxidative stress, hypoxia, and cation channel hyperactivity. These findings suggest that multiple noxious stimuli converge upon a peptidase-driven, core stress response pathway that, in the absence of serpin regulation, triggers a lysosomal-dependent necrotic cell death routine.

Serpins compose the largest superfamily of peptidase inhibitors and are well known as regulators of hemostasis and thrombolysis. Studies using model organisms, from plants to vertebrates, now show that serpins and their unique inhibitory mechanism and conformational flexibility are exploited to control proteolysis in molecular pathways associated with cell survival, development, and host defense. In addition, an increasing number of non-inhibitory serpins are emerging as important elements within a diversity of biological systems by serving as chaperones, hormone transporters, or anti-angiogenic factors.

Decreased sequencing costs have led to an explosion of genetic and genomic data. These data have revealed thousands of candidate human disease variants. Establishing which variants cause phenotypes and diseases, however, has remained challenging. Significant progress has been made, including advances by the National Institutes of Health (NIH)-funded Undiagnosed Diseases Network (UDN). However, 6000–13,000 additional disease genes remain to be identified. The continued discovery of rare diseases and their genetic underpinnings provides benefits to affected patients, of whom there are more than 400 million worldwide, and also advances understanding the mechanisms of more common diseases. Platforms employing model organisms enable discovery of novel gene-disease relationships, help establish variant pathogenicity, and often lead to the exploration of underlying mechanisms of pathophysiology that suggest new therapies. The Model Organism Screening Center (MOSC) of the UDN is a unique resource dedicated to utilizing informatics and functional studies in model organisms, including worm (Caenorhabditis elegans), fly (Drosophila melanogaster), and zebrafish (Danio rerio), to aid in diagnosis. The MOSC has directly contributed to the diagnosis of challenging cases, including multiple patients with complex, multi-organ phenotypes. In addition, the MOSC provides a framework for how basic scientists and clinicians can collaborate to drive diagnoses. Customized experimental plans take into account patient presentations, specific genes and variant(s), and appropriateness of each model organism for analysis. The MOSC also generates bioinformatic and experimental tools and reagents for the wider scientific community. Two elements of the MOSC that have been instrumental in its success are (1) multidisciplinary teams with expertise in variant bioinformatics and in human and model organism genetics, and (2) mechanisms for ongoing communication with clinical teams. Here we provide a position statement regarding the central role of model organisms for continued discovery of disease genes, and we advocate for the continuation and expansion of MOSC-type research entities as a Model Organisms Network (MON) to be funded through grant applications submitted to the NIH, family groups focused on specific rare diseases, other philanthropic organizations, industry partnerships, and other sources of support.

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.

Inhibitory serpins are metastable proteins that undergo a substantial conformational rearrangement to covalently trap target peptidases. The serpin reactive center loop contributes a majority of the interactions that serpins make during the initial binding to target peptidases. However, structural studies on serpin-peptidase complexes reveal a broader set of contacts on the scaffold of inhibitory serpins that have substantial influence on guiding peptidase recognition. Structural and biophysical studies also reveal how aberrant serpin folding can lead to the formation of domain-swapped serpin multimers rather than the monomeric metastable state. Serpin domain swapping may therefore underlie the polymerization events characteristic of the serpinopathies. Finally, recent structural studies reveal how the serpin fold has been adapted for non-inhibitory functions such as hormone binding.

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.

Pretreatment serum squamous cell carcinoma antigen (SCCA) is a prognostic biomarker in women with cervical cancer. SCCA has not been evaluated as an early indicator of response to chemoradiation therapy (CRT). The molecular role of the two SCCA isoforms, SCCA1 (SERPINB3) and SCCA2 (SERPINB4), in cervical cancer is unknown. We hypothesised that changes in serum SCCA during definitive CRT predicts treatment response, and that SCCA1 mediates radiation resistance.Patients treated with definitive CRT for cervical squamous carcinoma with serum SCCA measured were included. SCCA immunohistochemistry was performed on tumour biopsies. Post-treatment FDG-PET/CT, recurrence, and overall survival were recorded. Radiation response of cervical tumour cell lines after SCCA1 expression or CRISPR/Cas9 knockout was evaluated by clonogenic survival assay.Persistently elevated serum SCCA during definitive CRT was an independent predictor of positive post-therapy FDG-PET/CT (P=0.043), recurrence (P=0.0046) and death (P=0.015). An SCCA1-expressing vector increased radioresistance, while SCCA knock out increased radiosensitivity of cervical tumour cell lines in vitro.Early response assessment with serum SCCA is a powerful prognostic tool. These findings suggest that escalation of therapy in patients with elevated or sustained serum SCCA and molecular targeting of SCCA1 should be considered.

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.

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

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.

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.

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 nematode Caenorhabditis elegans (C.elegans) is a multicellular organism that has become a popular model for biological and basic medical research.It has also been widely used as a model system to explore fundamental questions in multiple aspects of biology, including evolution, development, cell fate specification, stem cell regulation, tumorigenesis, and aging.The C. elegans has also been considered as an ideal model system for live animal high-throughput drug screening, as 1) their tissues are transparent at all developmental stages, 2) tissue-specific fluorescent transgenic markers to study physiological and cellular processes in vivo are well established, 3) a large number of mutant strains are available in Caenorhabditis Genetics Center (CGC) [http://www.cbs.umn.edu/research/resources/cgc], 4) whole genome sequencing has been completed, 5) they have a short lifespan (2 to 3 weeks) and a strong genetic power, and 6) the aspects of mammalian diseases can be successfully modeled in the C. elegans (O'Reilly et al., 2014).The C. elegans life cycle includes embryogenesis (~12 h), four larval stages (L1-L4; total of ~3 days) and adulthood (~10

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.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

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.

SQN-5 is a mouse serpin that is highly similar to the human serpins SCCA1 (SERPINB3) and SCCA2 (SERPINB4). Previous studies characterizing the biochemical activity of SQN-5 showed that this serpin, like SCCA2, inhibited the chymotrypsin-like enzymes mast cell chymase and cathepsin G. Using an expanded panel of papain-like cysteine proteinases, we now show that SQN-5, like SCCA1, inhibited cathepsins K, L, S, and V but not cathepsin B or H. These interactions were characterized by stoichiometries of inhibition that were nearly 1:1 and second-order rate constants of >10(4) M(-1) s(-1). Reactive site loop (RSL) cleavage analysis showed that SQN-5 employed different reactive centers to neutralize the serine and cysteine proteinases. To our knowledge, this is the first serpin that serves as a dual inhibitor of both chymotrypsin-like serine and the papain-like cysteine proteinases by employing an RSL-dependent inhibitory mechanism. The ability of serpins to inhibit both serine and/or papain-like cysteine proteinases may not be a recent event in mammalian evolution. Phylogenetic studies suggested that the SCCA and SQN genes evolved from a common ancestor approximately 250-280 million years ago. When the fact that mammals and birds diverged approximately 310 million years ago is considered, an ancestral SCCA/SQN-like serpin with dual inhibitory activity may be present in many mammalian genomes.

Headpin (SERPINB13) is a novel member of the serine proteinase inhibitor (Serpin) gene family that was originally cloned from a keratinocyte cDNA library. Western blot analysis using a headpin-specific antiserum recognized a protein with the predicted M(r) of 44kDa in lysates derived from a transformed keratinocyte cell line known to express headpin mRNA. Similarity of the reactive-site loop (RSL) domain of headpin, notably at the P1-P1(') residues, with other serpins that inhibit cysteine and serine proteinases suggests that headpin may inhibit similar proteinases. This study demonstrates that recombinant headpin indeed inhibits cathepsins K and L, but not chymotrypsin, elastase, trypsin, subtilisin A, urokinase-type plasminogen activator, plasmin, or thrombin. The second-order rate constants (k(a)) for the inhibitory reactions of rHeadpin with cathepsins K and L were 5.1+/-0.6x10(4) and 4.1+/-0.8x10(4)M(-1)s(-1), respectively. Headpin formed SDS-stable complexes with cathepsins K and L, a characteristic property of inhibitory serpins. Interactions of the RSL domain of headpin with cathepsins K and L were indicated by cleavage of headpin near the predicted P1-P1(') residues by these proteinases. These results demonstrate that the serpin headpin possesses specificity for inhibiting lysosomal cysteine proteinases.

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.

Members of the human serpin family regulate a diverse array of serine and cysteine proteinases associated with essential biological processes such as fibrinolysis, coagulation, inflammation, cell mobility, cellular differentiation, and apoptosis.Most serpins are secreted and attain physiologic concentrations in the blood and extracellular fluids.However, a subset of the serpin superfamily, the ov-serpins, also resides intracellularly.Using high throughput genomic sequence, we identified a novel member of the human ov-serpin gene family, SERPINB12.The gene mapped to the ov-serpin cluster at 18q21 and resided between SERPINB5 (maspin) and SERPINB13 (headpin).The presence of SERPINB12 in silico was confirmed by cDNA cloning.Expression studies showed that SERPINB12 was expressed in many tissues, including brain, bone marrow, lymph node, heart, lung, liver, pancreas, testis, ovary, and intestines.Based on the presence of Arg and Ser at the reactive center of the RSL, SERPINB12 appeared to be an inhibitor of trypsin-like serine proteinases.This hypothesis was confirmed because recombinant SERPINB12 inhibited human trypsin and plasmin but not thrombin, coagulation factor Xa, or urokinase-type plasminogen activator.The second-order rate constants for the inhibitory reactions were 2.5 ؎ 1.6 ؋ 10 5 and 1.6 ؎ 0.2 ؋ 10 4 M ؊1 s ؊1 , respectively.These data show that SERPINB12 encodes for a new functional member of the human ov-serpin family.

High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as α1-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans. High molecular weight serpins are members of a large superfamily of structurally conserved proteins that inactivate target proteinases by a suicide substrate-like mechanism. In vertebrates, different clades of serpins distribute predominantly to either the intracellular or extracellular space. Although much is known about the function, structure, and inhibitory mechanism of circulating serpins such as α1-antitrypsin (SERPINA1) and antithrombin III (SERPINC1), relatively little is known about the function of the vertebrate intracellular (clade B) serpins. To gain a better understanding of the biology of the intracellular serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model system. A screen of the C. elegans genomic and cDNA databases revealed nine serpin genes, tandemly arrayed on chromosome V. Although the C. elegans serpins represent a unique clade (L), they share significant functional homology with members of the clade B group of intracellular serpins, since they lack typical N-terminal signal peptides and reside intracellularly. To determine whether nematode serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a dual cross-class inhibitor of the apoptosis-related serine proteinase, granzyme B, and the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of temporal and spatial expression indicated that SRP-2 was present during early embryonic development and highly expressed in the intestine and hypoderm of larval and adult worms. Transgenic animals engineered to overexpress SRP-2 were slow growing and/or arrested at the first, second, or third larval stages. These data suggest that perturbations of serpin-proteinase balance are critical for correct postembryonic development in C. elegans. Serpins are a unique class of proteinase inhibitors that irreversibly neutralize target proteinases by a mechanism involving the conformational distortion of the proteinase. Serpins have been identified in animals, plants, insects, and certain viruses (1Silverman G.A. Bird P.I. Carrell R.W. Coughlin P.B. Gettins P.G. Irving J.I. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar, 2Gettins P.G. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (983) Google Scholar). More recently, serpins have been detected in prokaryotes (3Irving J.A. Steenbakkers P.J. Lesk A.M. Op den Camp H.J. Pike R.N. Whisstock J.C. Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (106) Google Scholar). A search of genome data bases provides evidence for >500 serpins that are grouped into 17 clades (plus >10 unclassified orphans) based upon phylogenetic relationships (4Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (507) Google Scholar). In humans, ∼35 serpins have been identified. These serpins are distributed among nine clades (A-I), and most are secreted and function in the circulation or extracellular spaces. These serpins regulate proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and extracellular matrix remodeling. In contrast, serpins belonging to clade B reside predominantly intracellularly and have been implicated in regulating apoptosis, tumor progression, and metastasis (5Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (154) Google Scholar). However, their biological functions in terms of an intact organism have not been well defined. To date, no naturally occurring mutations with an identifiable phenotype have been identified in humans or rodents. Moreover, targeted mutagenesis studies in mice have revealed no overt phenotype (serpin b2) (6Dougherty K.M. Pearson J.M. Yang A.Y. Westrick R.J. Baker M.S. Ginsburg D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 686-691Crossref PubMed Scopus (99) Google Scholar) or appear to be embryonic lethal (serpin b5) (7Zhang M. Hendrix M.J.C. Maspin. Landes Bioscience, Georgetown, TX2002: 96-116Google Scholar). Thus, we sought to develop a more tractable model to study intracellular serpin activity within the context of a whole organism. Data base analysis of the Caenorhabditis elegans genome reveals the presence of several intracellular serpins (8Whisstock J.C. Irving J.A. Bottomley S.P. Pike R.N. Lesk A.M. Proteins. 1999; 36: 31-41Crossref PubMed Scopus (19) Google Scholar, 9Zang X. Maizels R.M. Trends Biochem. Sci. 2001; 26: 191-197Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The presence of these genes and the ease of genetic manipulations in this organism make it possible to conduct a comprehensive analysis of serpin biochemistry within the context of a whole animal. As a first step, we identified and cloned nine serpin genes. Sequence analysis suggested that all of the nine are transcribed, but only five are likely to encode for functional proteinase inhibitors. Initial biochemical characterization of one family member, SRP-2, demonstrated that it functions as a dual, cross-class inhibitor of both serine and cysteine proteinases. Analysis of the SRP-2::GFP 1The abbreviations used are: GFP, green fluorescent protein; RSL, reactive site loop; cat, cathepsin; Suc-, succinyl-; pNA, para-nitroanilide; VLK-pNA, d-Val-Leu-Lys-pNA; EGR-pNA, H-Glu-Gly-Arg-pNA; Z-, benzyloxycarbonyl-; SI, stoichiometry of inhibition; MALDI, matrix-associated laser desorption ionization; MS, mass spectroscopy; UTR, untranslated region; GST, glutathione S-transferase; FMK, fluoromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AFC, 7-amino-4-trifluoromethyl coumarin. 1The abbreviations used are: GFP, green fluorescent protein; RSL, reactive site loop; cat, cathepsin; Suc-, succinyl-; pNA, para-nitroanilide; VLK-pNA, d-Val-Leu-Lys-pNA; EGR-pNA, H-Glu-Gly-Arg-pNA; Z-, benzyloxycarbonyl-; SI, stoichiometry of inhibition; MALDI, matrix-associated laser desorption ionization; MS, mass spectroscopy; UTR, untranslated region; GST, glutathione S-transferase; FMK, fluoromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AFC, 7-amino-4-trifluoromethyl coumarin. expression pattern indicates that SRP-2 is expressed in numerous cell types throughout embryonic and postembryonic development. To determine whether SRP-2 played a role in C. elegans development, null mutants and transgenic animals overexpressing SRP-2 were generated. Whereas null mutants showed no overt developmental phenotype, animals overexpressing SRP-2 displayed severe developmental abnormalities characterized by slow growth, defective molting, and early (L1/L2) larval arrest/death. Although the mode of SRP-2 action is currently unknown, we hypothesize that SRP-2 plays a role in regulating proteinase activity during development and that an imbalance in the serpin/proteinase equilibrium has deleterious consequences during C. elegans development. cDNA Isolation and DNA Sequencing—First strand cDNA was prepared from total C. elegans RNA using Superscript™ II RNase H- reverse transcriptase (Invitrogen). The full-length srp-2 cDNA containing the putative open reading frame and the 5′/3′-UTR sequence was amplified using splice leader (SL1) sense (5′-TATATACTCGAGGGTTTAATTACCCAAGTTTG-3′) and srp-2, 3′UTR-specific antisense (5′-TATATACTCGAGCTTTATTTCTTCCAAAAAATATTCCGATC-3′) primers. An XhoI restriction site was included in the primers (underlined) to simplify cloning. The resulting PCR fragment was subcloned into pBluescript®II KS (Stratagene, La Jolla, CA) and sequenced using the automated DNA sequencing facility of the Mental Retardation Research Center, Children's Hospital (Boston, MA). Sequences were analyzed using MacVector 7.1 (Accelrys Inc., Princeton, NJ), ClustalW 1.8, and BLAST programs (Wormbase (available on the World Wide Web at www.wormbase.org/) and the National Center for Biotechnology Information (available on the World Wide Web at www.ncbi.nlm.nih.gov/). Construction of Glutathione S-Transferase (GST) Fusion Proteins—A 1.1-kb cDNA fragment containing the complete coding sequence of SRP-2 was generated by PCR from the pBluescript®II KS plasmid containing the full-length srp-2 cDNA. The sense (5′-ATATGAATTCCCGACAATGTCCGATAACGC-3′) and antisense (5′-TATATACTCGAGTCAAGCATGAACCCCAAGG-3′) primers were designed to facilitate in-frame insertion into the bacterial expression vector, pGEX-6P-1 (Amersham Biosciences). The PCR product was digested with the restriction endonucleases EcoRI and XhoI (sites underlined in primers), gel-purified, and ligated into the corresponding sites of the pGEX6P-1 vector. Recombinant clones were verified for intact srp-2 sequence and in-frame insertion by DNA sequencing. Purification of GST-SRP-2 Fusion Protein—The GST-SRP-2 fusion protein was batch-purified using glutathione-Sepharose 4B beads (Amersham Biosciences) using a modification of a procedure previously described (10Schick C. Kamachi Y. Bartuski A.J. Cataltepe S. Schechter N.M. Pemberton P.A. Silverman G.A. J. Biol. Chem. 1997; 272: 1849-1855Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). In brief, 10 ng of pGEX-SRP-2 plasmid was transformed into the competent Escherichia coli strain, BL21, and cultured overnight at 37 °C on LB plates supplemented with 100 μg/ml ampicillin. Colonies were collected using 5 ml of LB broth and inoculated directly into a flask containing 1 liter of LB plus 100 μg/ml ampicillin. The bacteria were grown in a 37 °C shaker to an A600 of 0.5. The flask was cooled on ice for 3 min and recombinant protein expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 500 μm. To reduce formation of inclusion bodies, induction was carried out at 25 °C for 4 h. Cells were harvested by centrifugation and stored at -80 °C. The frozen cell pellet was lysed by incubating in 60 ml of freshly made prep buffer (100 mm NaCl, 100 mm Tris-HCl, pH 8.0, 50 mm EDTA, 2% Triton X-100) supplemented with 1.5 mg/ml lysozyme and a Complete™ protease inhibitor mixture tablet (Roche Applied Science). The lysate was cleared by centrifugation at 12,000 × g for 30 min and transferred to a fresh tube containing 2 ml of 50% glutathione-Sepharose 4B. To facilitate the binding of the recombinant protein, the tube was gently shaken for 30 min at 4 °C. Beads were collected by centrifugation at 500 × g and washed twice in prep buffer and twice in PBST (10 mm phosphate buffer, 27 mm KCl, 137 mm NaCl, 0.1% Tween 20, pH 7.4). The GST-SRP-2 protein was eluted from the beads using three 1-ml washes of glutathione elution buffer (10 mm reduced glutathione, 50 mm Tris-HCl, pH 8.0). Protein concentration in the eluate was determined by Bradford analysis (Protein Assay Kit II; Bio-Rad), and protein purity was checked by SDS-PAGE analysis. Thermal Stability Assay—Aliquots of recombinant GST-SRP-2 protein (final concentration 0.5 mg/ml) were incubated for 7 min at temperatures ranging from 25 to 75 °C. The solution was placed on ice for 1 min and centrifuged (12,000 × g for 10 min at 4 °C), and the supernatant was analyzed by SDS-PAGE (see below). Enzymes, Inhibitors, and Substrates—Human cathepsin B (catB), catG, catL, and catS; human chymotrypsin; human neutrophil elastase; human kallikrein; human pancreatic trypsin; and human plasmin were purchased from Athens Research and Technology Inc. (Athens, GA). catK and -V were prepared as described (11Bromme D. Li Z. Barnes M. Mehler E. Biochemistry. 1999; 38: 2377-2385Crossref PubMed Scopus (199) Google Scholar, 12Bromme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). Human granzyme B was purchased from Enzyme Systems Products (Livermore, CA). Rat granzyme B was a generous gift from Dr. Charles Craik (University of California, San Francisco, CA). Endoproteinase Glu-C (V8) was purchased from Worthington. Papain was purchased from Roche Applied Science. Mast cell chymase was a generous gift from Dr. Norman Schechter (University of Pennsylvania). Subtilisin A and urokinase-type plasminogen activator were purchased from Sigma. Recombinant human caspases-3, -8, and -9 were purchased from A. G. Scientific, Inc. (San Diego, CA). Inhibitors (Z-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK), Z-Ile-Glu-Thr-Asp-FMK (Z-IETD-FMK), Z-Leu-Glu-His-Asp-FMK (Z-LEHD-FMK), and Z-Val-Ala-Asp-FMK (Z-VAD-FMK)) were purchased from Enzyme Systems Products. Diisopropylfluorophosphate and acetyl-Ile-Glu-Thr-Asp-aldehyde were purchased from Calbiochem. Phenylmethanesulfonyl fluoride and trans-epoxysuccinyl-l-leucylamido(4-guanidino)-butane (E64) were purchased from Sigma. Ecotin was a generous gift from Dr. Charles Craik. Enzyme substrates were purchased from Sigma (succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA), methoxy-Suc-Ala-Ala-Pro-Val-pNA, and d-Val-Leu-Lys-pNA (VLK-pNA)), Bachem Bioscience, Inc. (King of Prussia, PA) (acetyl-Ile-Glu-Pro-Asp-pNA (Ac-IEPD-pNA), H-Glu-Gly-Arg-pNA (EGR-pNA), and succinyl-Ala-Ala-Pro-Glu-pNA (Suc-AAPE-pNA)), Molecular Probes, Inc. (Eugene, OR) ((Z-Phe-Arg)2-R110) (Z-FR)2-R110)), and Enzyme Systems Products (acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC), Ac-Ile-Glu-Pro-Asp-AFC (Ac-IEPD-AFC), Ac-Ile-Glu-Thr-Asp-AFC (Ac-IETD-AFC), and Ac-Leu-Glu-His-Asp-AFC (Ac-LEHD-AFC)). Phosphate-buffered saline (10 mm phosphate buffer, 27 mm KCl, 137 mm NaCl, pH 7.4) was used in enzymatic reactions with trypsin, plasmin, human neutrophil elastase, chymotrypsin, kallikrein, subtilisin A, urokinase-type plasminogen activator, and mast cell chymase. Cathepsin reaction buffer (50 mm sodium acetate, pH 5.5, 4 mm dithiothreitol, 1 mm EDTA) was used with papain and catB, -K, -L, -S, and -V. Unique reaction buffers were used with catG (50 mm HEPES, 150 mm NaCl, 5% N,N-dimethylformamide, pH 7.4), endoproteinase-Glu-C (V8) (25 mm ammonium carbonate (pH 7.8) or 50 mm Tris adjusted to pH 7.8 using H3PO4), caspases (20 mm PIPES, 100 mm NaCl, 10 mm dithiothreitol, 1 mm EDTA, 0.1% CHAPS, 10% sucrose, pH 7.4), and granzyme B (50 mm Tris, pH 7.4, 100 mm NaCl, 0.01% Tween 20). Determination of Enzyme and Inhibitor Concentrations—The specific activity of granzyme B was determined as follows. Trypsin was active site-titrated using 4-methylumbelliferyl p-guanidinobenzoate (Sigma) (13Jameson G.W. Roberts D.V. Adams R.W. Kyle W.S. Elmore D.T. Biochem. J. 1973; 131: 107-117Crossref PubMed Scopus (287) Google Scholar). Assuming 1:1 stoichiometry, the concentration of ecotin was standardized against the active site-titrated trypsin. Granzyme B was standardized against the active site-titrated ecotin, assuming 1:1 stoichiometry, using Ac-IEPD-pNA or Ac-IEPD-AFC as the substrate. Activities of the cysteine proteinases (catK, -L, -S, and -V) were determined by active site titration using E64 as previously described (14Salvesen G. Nagase H. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. IRL Press, Oxford1989: 83-104Google Scholar). The concentration of recombinant SRP-2 was determined by Bradford analysis (Bio-Rad Protein Assay Kit II), thermal stability (see above), and amino acid composition analysis by postcolumn ninhydrin dectection on a Beckman 6300 amino acid analyzer (Beckman Instruments). Assays for Inhibition—Proteinase inhibitory activity of SRP-2 was determined by mixing enzyme and inhibitor in the appropriate buffer, incubating for 15 min at 25 °C, and measuring residual enzyme activity as previously described (10Schick C. Kamachi Y. Bartuski A.J. Cataltepe S. Schechter N.M. Pemberton P.A. Silverman G.A. J. Biol. Chem. 1997; 272: 1849-1855Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Residual enzyme activity was determined by measuring substrate hydrolysis over time (velocity) using either a THERMOmax (in the case of -pNA substrates) or fmax (in the case of -AFC and -R110 substrates) microplate readers (Molecular Devices, Sunnyvale, CA). For the UV-visible substrate, -pNA, a wavelength of 405 nm was used. For the fluorescent substrates, -AFC and -R110, the excitation/emission spectra were 390 nm/510 nm and 485 nm/538 nm, respectively. The concentrations of enzyme, inhibitor and substrate are listed in Table II and the buffers are listed above. Percentage enzyme inhibition = 100 × (1 - (velocity of inhibited enzyme reaction/velocity of uninhibited enzyme reaction)).Table IIInhibitory profile of SRP-2Proteinase (final concentration)SRP-2Ratio ([I]/[E])InhibitionSubstrate (final concentration)nm%Human granzyme B (50 nm)50010100Ac-IEPD-AFC (5 μm)Rat granzyme B (25 nm)50020100Ac-IEPD-pNA (1 mm)Cathepsin B (100 nm)80080(Z-FR)2-R110 (5 μm)Cathepsin G (50 nm)3006100Suc-AAPF-pNA (1 mm)Cathepsin K (25 nm)4001696(Z-FR)2-R110 (5 μm)Cathepsin L (25 nm)4001699(Z-FR)2-R110 (5 μm)Cathepsin S (50 nm)2004100(Z-FR)2-R110 (5 μm)Cathepsin V (20 nm)20010100(Z-FR)2-R110 (5 μm)Chymotrypsin (50 nm)35070Suc-AAPF-pNA (1 mm)Endoproteinase Glu-C (V8) (30 nm)300100Suc-AAPE-pNA (1 mm)Human neutrophil elastase (50 nm)35070MeO-Succ-AAPV-pNA (0.5 mm)Kallikrein (50 nm)30060(Z-FR)2-R110 (5 μm)Mast cell chymase (50 nm)400855Suc-AAPF-pNA (1 mm)Papain (50 nm)300650(Z-FR)2-R110 (5 μm)Plasmin (50 nm)500100VLK-pNA (0.1 mm)Subtilisin A (25 nm)3001284Suc-AAPF-pNA (1 mm)Trypsin (50 nm)35070EGR-pNA (0.5 mm)Urokinase-type plasminogen activator (50 nm)500100EGR-pNA (0.1 mm) Open table in a new tab Binding Stoichiometries—Assays for binding between SRP-2 and human granzyme B (25 nm) was performed in a volume of 100 μl in 96-well microtiter plates (EIA/RIA plates, Costar, Cambridge, MA). The inhibitor, SRP-2 (concentration range 0-50 nm), was incubated with the enzyme, granzyme B, for 15 min at 25 °C. The substrate, Ac-IEPD-AFC, was added to a final concentration of 10 μm. The velocity of substrate hydrolysis was measured using the fmax microplate reader. The partitioning ratio of the inhibitor-enzyme association was determined by plotting the fractional activity (velocity of the inhibited enzyme reaction/velocity of the uninhibited enzyme reaction) versus the initial ratio of the inhibitor to enzyme ([I]0/[E]0) (14Salvesen G. Nagase H. Beynon R.J. Bond J.S. Proteolytic Enzymes: A Practical Approach. IRL Press, Oxford1989: 83-104Google Scholar). The x-intercept (i.e. the stoichiometry of inhibition (SI)) was determined by linear regression analysis. Enzyme Kinetics—The interaction of SRP-2 with human granzyme B was determined by the progress curve method (15Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). Under these pseudo-first order conditions, a constant amount of enzyme (25 nm) was mixed with different concentrations of inhibitor (0-800 nm) and substrate (final concentration 100 μm). The rate of product formation was measured using the fmax microplate reader. Since the inhibition of human granzyme B is assumed to be irreversible over the course of the reaction, product formation is described as shown below, where the amount of product formation (P) proceeds at an initial velocity (vz) and is inhibited over time (t) at the following rate (kobs). P=vz/kobs×(1-e-kobst)(Eq. 1) For each combination of enzyme and inhibitor, a kobs was calculated by nonlinear regression of the data using Equation 1. A second order rate constant (k′) was determined by plotting a series of kobs versus the respective inhibitor concentration and measuring the slope of the line (k′ = Δkobs/Δ[I]). Since the inhibitor is in competition with substrate and the rate depends on the SI, the second order rate constant (k′) was corrected for the substrate concentration, SI of the enzyme and inhibitor, and the Km of the enzyme for the substrate in order to calculate the ka as shown. ka=k'×(1+[S]/Km)×SI(Eq. 2) The Km of human granzyme B for Ac-IEPD-AFC in granzyme B assay buffer (50 mm Tris, pH 7.4, 100 mm NaCl, 0.01% Tween 20) was 585 μm. Complex Formation—SRP-2-granzyme B complexes were formed by incubating 9 μg of GST-SRP-2 protein with 0.5 μg of human granzyme B in granzyme B assay buffer for 15 min at 25 °C. The mixture components were separated by SDS-PAGE and higher molecular weight protein bands were visualized following staining with Coomassie Brilliant Blue R-250. SRP-2-catV complexes were formed by incubating 1 μg of GST-SRP-2 with 50 ng of catV in cathepsin assay buffer for 3 min at 25 °C. Following separation by SDS-PAGE, complex bands were visualized by immunoblotting (see below). SDS-PAGE and Immunoblottng—Proteins were mixed with 2× gel loading buffer (4% SDS, 20% glycerol, 120 mm Tris-HCl, pH 6.8, 0.01% bromphenol blue, 2% β-mercaptoethanol), heated to 95 °C for 5 min and separated by SDS-PAGE according to the method of Laemmli (16Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206631) Google Scholar). The running buffer was 25 mm Tris-base, 250 mm glycine, and 0.1% SDS, pH 8.3. Protein bands were visualized after staining in a solution containing 0.25% Coomassie Brilliant Blue R-250, 45% methanol, 10% acetic acid. For immunoblot detection, proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4 °C onto reinforced nitrocellulose (NitroPure, Osmonics Inc., Westborough, MA). The transfer buffer was 25 mm Tris-base, pH 8.0, and 190 mm glycine, pH 8.3. The immunodetection of SRP-2 was performed using a polyclonal rabbit anti-SRP-2 antibody diluted to 1:2000 as the primary antibody and a horseradish peroxidase-linked antibody (Amersham Biosciences) diluted to 1:2000 as a secondary antibody. The immunoblot was visualized using the ECL detection kit (Amersham Biosciences). Matrix-associated Laser Desorption Ionization Mass Spectroscopy (MALDI-MS)—Human granzyme B (1 μg) was mixed with SRP-2 (25 μg) in granzyme B assay buffer (20 μl) for 15 min at 25 °C. For catV, 125 ng of enzyme was mixed with 6 μg of GST-SRP-2 in cathepsin assay buffer (20 μl) for 3 min at 25 °C. The mixture components were separated by MALDI-MS at the Wistar Protein Microchemistry Facility (Philadelphia, PA). Worm Strain and Culture Conditions—The wild-type worm strain used in this study was C. elegans Bristol (N2). Worms were routinely cultured at 25 °C on standard nematode growth medium agar plates (17Lewis J.A. Fleming J.T. Methods Cell Biol. 1995; 48: 3-29Crossref PubMed Scopus (500) Google Scholar) seeded with E. coli strain OP50 (Caenorhabditis Genetics Center (CGC), available on the World Wide Web at biosci.umn.edu/CGC/CGChomepage.htm). Generation of the srp-2 promoter::gfp and srp-2::gfp Expression Constructs—A translational srp-2 promoter::gfp fusion gene was made by ligating a 6.3-kb genomic fragment, containing the first three exons of the srp-2 gene and 5.5 kb of the upstream promoter sequence in frame into the GFP expression vector pPD95.81. For the srp-2 overexpression construct, the 5.5-kb promoter region, the full-length (1.6-kb) srp-2 gene (minus the stop codon), and the 0.85-kb gfp fragment was cloned into the canonical expression vector pPD49.26. The gfp gene used in these experiments contained five engineered introns and the mutation S65C for enhanced expression and fluorescence, respectively. The plasmids pPD95.81 and pPD49.26 were a kind gift from Dr. Andrew Fire (Carnegie Institution of Washington, Baltimore, MD). Transgenic Animals Expressing srp-2 Promoter::gfp and srp-2::gfp Fusion Genes—To obtain germ line expression of the srp-2 promoter::gfp and srp-2::gfp fusion genes, appropriate plasmid DNA (100 ng/μl) was microinjected into the gonads of young adult hermaphrodites as previously described (18Mello C. Fire A. Methods Cell Biol. 1995; 48: 451-482Crossref PubMed Scopus (1151) Google Scholar). Most injected DNA exist as extrachromosomal arrays that are lost frequently during meiosis and mitosis. Thus, we also generated strains where the transgene was stably integrated into the C. elegans genome. Approximately 100 L4 larvae of a transgenic line carrying the extrachromosomal array of srp-2 promoter::gfp or srp-2::gfp were irradiated with γ rays from a 137Cs source (3500 rads). GFP-positive worms were transferred, in pairs, to 25 plates and allowed to grow for ∼10-12 days. Worms were harvested with 5 ml of M9 buffer (22 mm KH2PO4, 42 mm Na2HPO4, 86 mm NaCl, 1 mm MgSO4), and a small aliquot (50 μl) was transferred to 25 new plates with food. Following 4-5 days of growth, 150 GFP-positive worms (six from each plate) were cloned onto individual plates. Stable integrants were identified, 3 days later, by the presence of 100% GFP-positive worms on the plate. To remove background mutations, worms were backcrossed six times using the wild-type N2 males. Identification of Novel Serpin Genes in C. elegans—The availability of a complete sequence of the C. elegans genome prompted us and others to consider whether serpin genes were present in this organism. By mining genome data bases, Whisstock and colleagues (8Whisstock J.C. Irving J.A. Bottomley S.P. Pike R.N. Lesk A.M. Proteins. 1999; 36: 31-41Crossref PubMed Scopus (19) Google Scholar) identified seven amino acid sequences bearing significant sequence homology to the archetypical serpin, α1-antitrypsin. In a similar report, Zang and Maizels (9Zang X. Maizels R.M. Trends Biochem. Sci. 2001; 26: 191-197Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) confirmed electronically the presence of the seven serpin genes and provided evidence for an additional serpin sequence, bringing the total number to 8. These reports, however, were based on data obtained in silico, and no attempt was made to verify gene expression by cDNA cloning. To determine whether these serpin genes are expressed, we designed oligonucleotide primers specific to the eight serpin genes and performed reverse transcription-PCR on C. elegans total RNA. Appropriately sized (∼1.2-kb) cDNA fragments were amplified and subcloned into the plasmid, pBluescript (pBS). Sequence analysis confirmed amplification of full-length cDNAs corresponding to the GENEFINDER-predicted genes, C05E4.1, C05E4.3, C03G6.18, C03G6.19, F20D6.4, F09C6.5, and F09C6.4. However, only a partial cDNA corresponding to F20D6.3 was isolated. To determine whether additional serpin genes were present in the C. elegans genome, newer versions of the C. elegans WORMPEP (available on the World Wide Web at www.sanger.ac.uk/Projects/C_elegans/wormpep/) and genomic data bases were rescreened using the BLAST algorithm (available on the World Wide Web at www.sanger.ac.uk/cgi-bin/nph-Blast_Server.html) and the human SERPINB4 as the query sequence. In addition to the eight serpins previously reported, we identified an additional serpin sequence, Y32G9A.4, expanding the total number of C. elegans serpins to nine. Furthermore, a screen of Yuji Kohrara's EST data base (available on the World Wide Web at www.ddbj.nig.ac.jp/c-elegans/html/CE_INDEX.html) identified yet another previously unidentified sequence, yk411c9, mapping to the YAC, Y32G9A. The yk411c9 sequence was identical to the nucleotide sequence of serpin, Y32G9A.4, except for a single base pair change, converting a Met → Val, in the P1 position of the RSL. Although this change appears to be conservative, it could alter the proteinase-inhibitory profile of the serpin. Further scrutiny is required to determine if this single nucleotide difference represents a simple cloning artifact, a sequencing error, a polymorphism, or the presence of two genes. The proximal hinge region and the distal “serpin signature motif” of the RSL in the C terminus of a se

Abstract Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant ( k ass ) for inhibitory complex formation (1.9×10 4 m /s and 5.8×10 4 m /s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus . The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus -derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.

The classical form of α1-antitrypsin deficiency (ATD) is characterized by intracellular accumulation of the misfolded variant α1-antitrypsin Z (ATZ) and severe liver disease in some of the affected individuals. In this study, we investigated the possibility of discovering novel therapeutic agents that would reduce ATZ accumulation by interrogating a C. elegans model of ATD with high-content genome-wide RNAi screening and computational systems pharmacology strategies. The RNAi screening was utilized to identify genes that modify the intracellular accumulation of ATZ and a novel computational pipeline was developed to make high confidence predictions on repurposable drugs. This approach identified glibenclamide (GLB), a sulfonylurea drug that has been used broadly in clinical medicine as an oral hypoglycemic agent. Here we show that GLB promotes autophagic degradation of misfolded ATZ in mammalian cell line models of ATD. Furthermore, an analog of GLB reduces hepatic ATZ accumulation and hepatic fibrosis in a mouse model in vivo without affecting blood glucose or insulin levels. These results provide support for a drug discovery strategy using simple organisms as human disease models combined with genetic and computational screening methods. They also show that GLB and/or at least one of its analogs can be immediately tested to arrest the progression of human ATD liver disease.

Abstract Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans ( C. elegans ) null for a cysteine protease inhibitor, srp-6 , undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3 , respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans , this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

The squamous cell carcinoma antigen (SCCA) serves as a serologic marker for advanced squamous cell carcinomas (SCC) of the uterine cervix, lung, esophagus, head and neck and vulva. Elevations in serum levels of SCCA following treatment for SCC correlate with tumor relapse or metastasis. Recent molecular studies show that SCCA is transcribed by two nearly identical genes (SCCA1 and SCCA2) that encode for members of the high molecular weight serine proteinase inhibitor (serpin) family. Despite a high degree of similarity in their amino acid sequences, SCCA1 and SCCA2 have distinct biochemical properties: SCCA1 is an inhibitor of papain like cysteine proteinases, such as cathepsins (cat) L, S and K, whereas SCCA2 inhibits chymotrypsin-like serine proteinases, catG and mast cell chymase. In this paper, we report the generation and characterization of anti-SCCA1 and anti-SCCA2 specific monoclonal antibodies (MAbs). Using these MAbs, we developed an enzyme-linked immunoassay (ELISA) that discriminated between SCCA1 and SCCA2 without any cross-reaction. This assay measured both the native and complexed forms of SCCA1 and SCCA2. The sensitivity of detection of SCCA1 and SCCA2 assays were 0.17 ngml(-1) and 0.19 ngml(-1), respectively. Mean inter- and intra-assay coefficients of variation were 12.1% and 9.9% for SCCA1 assay and 12% and 8.8% for SCCA2 assay, respectively. Recovery and parallellism studies indicated that SCCA1 and SCCA2 were detected in the plasma and amniotic fluids without any major interference by the biologic fluid components. This assay provides a simple and accurate procedure for the quantitation of total SCCA1 and SCCA2.

Headpin is a novel serine proteinase inhibitor (serpin) that is down-regulated in squamous cell carcinoma of the oral cavity and in squamous cell carcinoma cell lines of the head and neck. Using a panel of 18q21.3 YAC clones, we mapped and cloned the HEADPIN gene. The gene spans 10 kb and is composed of eight exons and seven introns. The genomic structure is identical with some other ovalbumin serpins (ov-serpins) in terms of the numbers, position and phasing of the intron/exon boundaries. HEADPIN was mapped within the serpin cluster in 18q21.3 between MASPIN and SCCA2 as follows: cen-MASPIN-HEADPIN-SCCA2-SCCA1-tel. The transcription start site was determined and the promoter activity of the 5'-flanking region was analyzed. Luciferase promoter assays in HaCaT cells showed that the -432 to -144 nucleotide region has functional promoter activity. The activity of the promoter/enhancer was not observed in head and neck cancer cell lines TU167 and UMSCC1 which lack headpin expression. These data suggest that the differential expression of headpin in normal and carcinoma-derived cells is regulated at the transcriptional level. Understanding the genomic organization and transcriptional regulation of the ov-serpins clustered within 18q21. 3 provides a critical framework for assessing their potential role in cancer.

SERPINB11, the last of 13 human clade B serpins to be described, gave rise to seven different isoforms. One cDNA contained a premature termination codon, two contained splice variants, and four contained full-length open reading frames punctuated by eight single nucleotide polymorphisms (SNPs). The SNPs encoded amino acid variants located within the serpin scaffold but not the reactive site loop (RSL). Although the mouse orthologue, Serpinb11, could inhibit trypsin-like peptidases, SERPINB11 showed no inhibitory activity. To determine whether the human RSL targeted a different class of peptidases or the serpin scaffold was unable to support inhibitory activity, we synthesized chimeric human and mouse proteins, in which the RSLs had been swapped. The human RSL served as a trypsin inhibitor when supported by mouse scaffold sequences. Conversely, the mouse RSL on the human scaffold showed no inhibitory activity. These findings suggested that variant residues in the SERPINB11 scaffold impaired serpin function. SDS-PAGE analysis supported this notion as RSL-cleaved SERPINB11 was unable to undergo the stressed-to-relaxed transition typical of inhibitory type serpins. Mutagenesis studies supported this hypothesis, since the reversion of amino acid sequences in helices D and I to those conserved in other clade B serpins partially restored the ability of SERPINB11 to form covalent complexes with trypsin. Taken together, these findings suggested that SERPINB11 SNPs encoded amino acids in the scaffold that impaired RSL mobility, and HapMap data showed that the majority of genomes in different human populations harbored these noninhibitory SERPINB11 alleles. Like several other serpin superfamily members, SERPINB11 has lost inhibitory activity and may have evolved a noninhibitory function. SERPINB11, the last of 13 human clade B serpins to be described, gave rise to seven different isoforms. One cDNA contained a premature termination codon, two contained splice variants, and four contained full-length open reading frames punctuated by eight single nucleotide polymorphisms (SNPs). The SNPs encoded amino acid variants located within the serpin scaffold but not the reactive site loop (RSL). Although the mouse orthologue, Serpinb11, could inhibit trypsin-like peptidases, SERPINB11 showed no inhibitory activity. To determine whether the human RSL targeted a different class of peptidases or the serpin scaffold was unable to support inhibitory activity, we synthesized chimeric human and mouse proteins, in which the RSLs had been swapped. The human RSL served as a trypsin inhibitor when supported by mouse scaffold sequences. Conversely, the mouse RSL on the human scaffold showed no inhibitory activity. These findings suggested that variant residues in the SERPINB11 scaffold impaired serpin function. SDS-PAGE analysis supported this notion as RSL-cleaved SERPINB11 was unable to undergo the stressed-to-relaxed transition typical of inhibitory type serpins. Mutagenesis studies supported this hypothesis, since the reversion of amino acid sequences in helices D and I to those conserved in other clade B serpins partially restored the ability of SERPINB11 to form covalent complexes with trypsin. Taken together, these findings suggested that SERPINB11 SNPs encoded amino acids in the scaffold that impaired RSL mobility, and HapMap data showed that the majority of genomes in different human populations harbored these noninhibitory SERPINB11 alleles. Like several other serpin superfamily members, SERPINB11 has lost inhibitory activity and may have evolved a noninhibitory function. Serpins are a superfamily of serine and cysteine peptidase inhibitors that contain ∼1500 family members and are found in all domains of life (Eukarya, Eubacteria, and Archaea) as well as many Poxviridae (reviewed in Refs. 1Gettins P.G. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar, 2Law R.H. Zhang Q. McGowan S. Buckle A.M. Silverman G.A. Wong W. Rosado C.J. Langendorf C.G. Pike R.N. Bird P.I. Whisstock J.C. Genome Biol. 2006; 7: 216-226Crossref PubMed Scopus (487) Google Scholar, 3Silverman G.A. Bird P.I. Carrell R.W. Coughlin P.B. Gettins P.G. Irving J.I. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1064) Google Scholar, 4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar). Unlike canonical inhibitors, inhibitory serpins employ a unique suicide substrate-like inhibitory mechanism to neutralize their target peptidases. The surface-exposed reactive site loop (RSL) 4The abbreviations used are: RSLreactive site loopSIstoichiometry of inhibitionSNPsingle nucleotide polymorphisms4B and s5Bstrand 4B and 5B, respectivelyhA, hB, hD, and hIhelix A, B, D, E, and I, respectivelycontiggroup of overlapping clonesGSTglutathione S-transferasepNAp-nitroanilidePBSphosphate-buffered salineMALDI-MSmatrix-assisted laser desorption ionization mass spectroscopyserpinserine peptidase inhibitor4-NA4-nitroanalide serves as a pseudosubstrate and binds to the active site of the peptidase. Upon cleavage of the RSL, the metastable serpin molecule undergoes a major conformational rearrangement and traps the covalently attached peptidase in a distorted, inactive form (5Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (948) Google Scholar). A small proportion of serpins are noninhibitory and aid in diverse functions, such as hormone transport (e.g. SERPINA7/thyroxine binding globulin) and protein folding (e.g. SERPINH1/HSP47) (6Dafforn T.R. Della M. Miller A.D. J. Biol. Chem. 2001; 276: 40319-49310Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 7Zhou A. Wei Z. Read R.J. Carrell R.W. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13321-13326Crossref PubMed Scopus (89) Google Scholar). reactive site loop stoichiometry of inhibition single nucleotide polymorphism strand 4B and 5B, respectively helix A, B, D, E, and I, respectively group of overlapping clones glutathione S-transferase p-nitroanilide phosphate-buffered saline matrix-assisted laser desorption ionization mass spectroscopy serine peptidase inhibitor 4-nitroanalide Serpins are classified into 17 clades based on phylogenetic relationships (8Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (512) Google Scholar). So far, 36 human serpins from nine clades (A–I) have been identified (2Law R.H. Zhang Q. McGowan S. Buckle A.M. Silverman G.A. Wong W. Rosado C.J. Langendorf C.G. Pike R.N. Bird P.I. Whisstock J.C. Genome Biol. 2006; 7: 216-226Crossref PubMed Scopus (487) Google Scholar). The majority of serpins are plasma proteins that serve as critical regulators of important physiological processes, such as blood coagulation, fibrinolysis, and inflammation. In contrast, clade B serpins exist predominantly, but not exclusively, as intracellular proteins with a cytoplasmic or nucleocytoplasmic distribution (9Uemura Y. Pak S.C. Luke C. Cataltepe S. Tsu C. Schick C. Kamachi Y. Pomeroy S.L. Perlmutter D.H. Silverman G.A. Int. J. Cancer. 2000; 89: 368-377Crossref PubMed Scopus (67) Google Scholar, 10Bird C.H. Blink E.J. Hirst C.E. Buzza M.S. Steele P.M. Sun J. Jans D.A. Bird P.I. Mol. Cell. Biol. 2001; 21: 5396-5407Crossref PubMed Scopus (101) Google Scholar). Other distinct features of clade B serpins include the absence of both cleavable N-terminal signal peptides and terminal extensions (N- or C-) relative to the prototypical serpin, α1-antitrypsin (SERPINA1) (reviewed in Refs. 4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar and 11Remold-O'Donnell E. FEBS Lett. 1993; 315: 105-108Crossref PubMed Scopus (214) Google Scholar). Although the functions of many clade B serpins are unknown, inhibitory members appear to protect cells from endogenous (e.g. lysosomal or granule enzymes) or exogenous (e.g. microbial or inflammatory cell) peptidase-mediated injury (4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar, 12Bird P.I. Silverman G.A. Cooper D.N. Nature Encyclopedia of the Human Genome. 4. Nature Publishing Group, London, U.K.2003: 462-468Google Scholar). To date, a single intracellular serpin, SERPINB5, does not function as an inhibitor, is unable to undergo the stressed to relaxed (S-R) transition (13Law R.H. Irving J.A. Buckle A.M. Ruzyla K. Buzza M. Bashtannyk-Puhalovich T.A. Beddoe T.C. Nguyen K. Worrall D.M. Bottomley S.P. Bird P.I. Rossjohn J. Whisstock J.C. J. Biol. Chem. 2005; 280: 22356-22364Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 14Pemberton P.A. Wong D.T. Gibson H.L. Kiefer M.C. Fitzpatrick P.A. Sager R. Barr P.J. J. Biol. Chem. 1995; 270: 15832-15837Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), and performs a crucial role in preventing the development of invasive breast and prostrate cancers (15Luo J.L. Tan W. Ricono J.M. Korchynskyi O. Zhang M. Gonias S.L. Cheresh D.A. Karin M. Nature. 2007; 446: 690-694Crossref PubMed Scopus (364) Google Scholar, 16Zou Z. Anisowicz A. Hendrix M.J. Thor A. Neveu M. Sheng S. Rafidi K. Seftor E. Sager R. Science. 1994; 263: 526-529Crossref PubMed Scopus (829) Google Scholar). Thirteen clade B serpin genes reside in the human genome (4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar). Three map to 6p25, and 10 map to 18q21.3 (4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar). The syntenic regions in the mouse genome are 13A3.2 and 1D1, respectively. The clade B cluster at 13A3.2 is greatly expanded (n = 15) relative to that of the human locus at 6p25 (17Kaiserman D. Knaggs S. Scarff K.L. Gillard A. Mirza G. Cadman M. McKeone R. Denny P. Cooley J. Benarafa C. Remold-O'Donnell E. Ragoussis J. Bird P.I. Genomics. 2002; 79: 349-362Crossref PubMed Scopus (54) Google Scholar). Except for the SCCA locus, which contains SERPINB3 and -B4 in humans and Serpinb3a, -b, -c, and -d in mice, the remaining eight clade B serpins mapping to human 18q21 and mouse 1D1 show 1:1 orthology and are highly conserved in terms of gene structure, gene order, and amino acid sequence (18Askew D.J. Askew Y.S. Kato Y. Turner R.F. Dewar K. Lehoczky J. Silverman G.A. Genomics. 2004; 84: 176-184Crossref PubMed Scopus (31) Google Scholar). These correlations suggest that the functional activity of the clade B serpins has been retained throughout mammalian evolution and that knowledge gained about any one of these genes should help define the overall activity of each orthologous pair. Amino acid analysis of the human and mouse RSLs confirms that these orthologous pairs are likely to demonstrate identical inhibitory profiles (18Askew D.J. Askew Y.S. Kato Y. Turner R.F. Dewar K. Lehoczky J. Silverman G.A. Genomics. 2004; 84: 176-184Crossref PubMed Scopus (31) Google Scholar). Of this set of eight orthologous clade B serpins, only human SERPINB11 and mouse Serpinb11 have yet to be characterized. The goal of this study was to use recombinant human and mouse proteins to define the biochemical activity of this pair of clade B serpins. Although Serpinb11 inhibited trypsin-like peptidases, single nucleotide polymorphisms (SNPs) in the human gene generated several different amino acid variants within the serpin scaffold that precluded proper inhibitory function in vitro. Based on current haplotype data, the majority of SERPINB11 alleles encoded proteins that lack detectable inhibitory activity. Identification of SERPINB11 and Its Mouse Orthologue—SERPINB11 (per the nomenclature guidelines, human genes (italicized) and gene products (not italicized) are uppercase, and mouse gene and gene products are lowercase after the first letter) was identified by electronic hybridization using the BLAST2 algorithm. Serpinb11 was identified using the SERPINB11 cDNA sequence and BLAST programs to query the mouse genomic and EST data bases as reported (18Askew D.J. Askew Y.S. Kato Y. Turner R.F. Dewar K. Lehoczky J. Silverman G.A. Genomics. 2004; 84: 176-184Crossref PubMed Scopus (31) Google Scholar). cDNA Isolation and DNA Sequencing—The SERPINB11 cDNA was amplified from prostate and lung first-strand cDNAs (Clontech) using specific primers (forward 1, 5′-ATGGGTTCTCTCAGCACAGCTAAC-3′; reverse 1, 5′-GAGGTGTGAACAGCTTTTTGG-3′) as described (19Cataltepe S. Gornstein E.R. Schick C. Kamachi Y. Chatson K. Fries J. Silverman G.A. Upton M.P. J. Histochem. Cytochem. 2000; 48: 113-122Crossref PubMed Scopus (134) Google Scholar). A heminested PCR was performed using the cDNA templates from first-round PCR with forward 1 and reverse 2 primers (reverse 2, 5′-CATCTGCAACTCTGAGCCTTG-3′). The resulting PCR fragments were ligated into pBluescript (Stratagene, La Jolla, CA) or pCR®2.1-TOPO® (Invitrogen) and sequenced. Sequences were analyzed using MacVector 6.0 (Accelrys Inc., Princeton, NJ). Serpinb11 was amplified and cloned from a mouse lung cDNA library (Clontech) as described (18Askew D.J. Askew Y.S. Kato Y. Turner R.F. Dewar K. Lehoczky J. Silverman G.A. Genomics. 2004; 84: 176-184Crossref PubMed Scopus (31) Google Scholar, 20Askew D.J. Askew Y.S. Kato Y. Luke C.J. Pak S.C. Bromme D. Silverman G.A. Genomics. 2004; 84: 166-175Crossref PubMed Scopus (19) Google Scholar). Site-directed Mutagenesis—SERPINB11 cDNA sequences were mutated using the QuikChange kit per the manufacturer's instructions (Stratagene). All mutations were confirmed by DNA sequencing, as described (21Luke C. Schick C. Tsu C. Whisstock J.C. Irving J.A. Bromme D. Juliano L. Shi G.P. Chapman H.A. Silverman G.A. Biochemistry. 2000; 39: 7081-7091Crossref PubMed Scopus (49) Google Scholar). Production of Recombinant Proteins—SERPINB11 coding sequences were excised from pBluescript or pCR®2.1-TOPO® by BamHI/XhoI digestion. The Serpinb11 coding sequence was excised from pBluescript by EcoRI/XhoI digestion. The cDNAs were subcloned into the bacterial expression vector pGEX-6P-1 (Amersham Biosciences). GST fusion proteins were batch-purified using glutathione-Sepharose 4B beads (Amersham Biosciences) as described (22Schick C. Kamachi Y. Bartuski A.J. Cataltepe S. Schechter N.M. Pemberton P.A. Silverman G.A. J. Biol. Chem. 1997; 272: 1849-1855Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). RSL Swap Mutations—The RSL of Serpinb11 was flanked by PstI sites located 5′ in the proximal hinge region and 3′ in the pGEX-6P-1 backbone. To generate a PstI site in the identical position within the proximal hinge region of SERPINB11d, an A1014T transversion was introduced by site-directed mutagenesis. To facilitate the RSL swap, a second PstI site located in the proximal portion of SERPINB11d (site not present in the mouse sequence) was eliminated also by a site-directed transversion (A153T). The PstI RSL fragments of SERPINB11d and Serpinb11 were isolated after restriction endonuclease digestion and agarose gel electrophoresis. The fragments were ligated into PstI-digested Serpinb11-pGEX-6P-1 and SERPNB11d-pGEX-6P-1, respectively. Correct insertions of the human and mouse RSLs into the mouse and human serpin scaffolds, respectively, were confirmed by DNA sequence analysis. Enzymes, Substrates, and Buffers—Human plasmin, human trypsin, human cathepsin G and cathepsin L, human chymotrypsin, human kallikrein, human neutrophil elastase, and subtilisin A were purchased from Athens Research and Technology, Inc. (Athens, GA). Cathepsins K and V were prepared as described (23Bromme D. Okamoto K. Wang B.B. Biroc S. J. Biol. Chem. 1996; 271: 2126-2132Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar, 24Bromme D. Li Z. Barnes M. Mehler E. Biochemistry. 1999; 38: 2377-2385Crossref PubMed Scopus (201) Google Scholar). Urokinase-type plasminogen activator and subtilisin A were purchased from Sigma. Thrombin was purchased from Calbiochem-Novabiochem. γ-Tryptase and β1-tryptase were generously provided by Dr. Richard Stevens (Brigham and Women's Hospital, Harvard Medical School, Boston, MA). Clostripain was obtained from Sigma and was stored at 1 unit/ml in 0.1 m calcium acetate. Enzyme substrates were purchased from Sigma (succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (succinyl-AAPF-pNA), methoxy-succinyl-Ala-Ala-Pro-Val-pNA, d-Val-Leu-Lys-pNA (VLK-pNA), and N-(p-tosyl)-Gly-Pro-Lys 4-NA); Bachem Bioscience, Inc. (King of Prussia, PA) (H-Glu-Gly-Arg-pNA, succinyl-Ala-Ala-Pro-Arg-para-nitroanilide (succinyl-AAPR-pNA), and H-d-Leu-Thr-Arg-pNA); and Molecular Probes, Inc. (Eugene, OR) ((benzyloxycarbonyl-Pro-Arg)2-R110) and (benzyloxycarbonyl-Phe-Arg)2-R110)). PBS (137 mm NaCl, 27 mm KCl, 10 mm phosphate buffer, pH 7.4) was used in enzymatic reactions with cathepsin G, chymotrypsin, human neutrophil elastase, plasmin, thrombin, trypsin, subtilisin A, urokinase-type plasminogen activator, γ-tryptase, and β1-tryptase. Cathepsin reaction buffer (50 mm sodium acetate, pH 5.5, 4 mm dithiothreitol, 1 mm EDTA) was used with cathepsins K, L, and V. Determination of Protein Concentrations—Trypsin and plasmin were active site-titrated using 4-methylumbelliferyl-para-guanidinobenzoate (Sigma) as described (25Askew Y.S. Pak S.C. Luke C.J. Askew D.J. Cataltepe S. Mills D.R. Kato H. Lehoczky J. Dewar K. Birren B. Silverman G.A. J. Biol. Chem. 2001; 276: 49320-49330Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The concentrations of recombinant serpins were determined by using the Bio-Rad protein assay kit II (Bio-Rad). Screening Assays for Enzyme Inhibition—The inhibitory activities of Serpinb11 and SERPINB11 were determined initially by mixing the inhibitor with various peptidases in appropriate buffer, incubating for 30 min at 25 °C, and measuring residual enzyme activity as described (22Schick C. Kamachi Y. Bartuski A.J. Cataltepe S. Schechter N.M. Pemberton P.A. Silverman G.A. J. Biol. Chem. 1997; 272: 1849-1855Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Residual enzyme activity was determined by adding the appropriate substrate and measuring hydrolysis over time (velocity) using either a THERMOmax (in the case of UV-visible substrates) or fmax (in the case of fluorescent substrates) microplate reader (Molecular Devices, Sunnyvale, CA). All screening assays were performed at an inhibitor/enzyme ratio of >5 as listed in Table 1.TABLE 1Comparison of human SERPINB11 amino acid variants-with other speciesSerpinGenBank™ accessionStructural motif and amino acid position in SERPINB11 (equivalent position in SERPINA1)hC 51 (73)hD 90 (99)hD 91 (100)hF 148 (156)s3A 181 (187)s3A 188 (194)hH 267 (271)s6A 293 (295)hI 303 (305)SERPINB11Human genomicAC069356EStopFTTWTTSHuman genomicaCelera genomic sequence.EAW63158?Stop?TAWTIPHuman cDNA (-B11a)AF419953AEFMIRTIPHuman cDNA (-B11b)AF419954AEFTTRTIPHuman cDNA (-B11c)AF419955AEFMTRTIPHuman cDNANM_080475AEFMTRTIPHuman cDNABC069596AEFMTRTIPHuman cDNA (-B11d)bThe designation of conservative (italic type) and nonconservative (bold face type) amino acid variants in SERPNB11d are relative the consensus motif.AY792323AESTAWAIPHuman cDNA (-B11e)AY792324AEFTIPHuman cDNA (-B11f)AY792325AStopHuman cDNABC130372AStopFTAWTTSHuman cDNABC130370AStopFTAWTTSHuman cDNADR006192AStopFHuman cDNA (-B11g)AY792326ATIPMouse genomicNM025867ALISAWMVSMouse cDNAAY367772ALISAWMVSMouse cDNABC010313ALISAWMVSMouse cDNAAK009855.1ALISAWMVSMouse cDNAAK009003.1ALISAWMVSChimp genomicXM523958ALFAWTISDog genomicXP541073ALFTAWTISRat genomicXP222495ALVSAWMVSSERPINB1P30740ALNTAWKERSERPINB2P05120ELSKAWKESSERPINB3P29508ALLKAWKETSERPINB4P48594ALLKAWKETSERPINB5P36952AVTQAWSKNSERPINB6P35237ALLHAWKENSERPINB7NP003775LVFNAWNKASERPINB8P50452ALLHAWKERSERPINB9P50453ALLHAWKEHSERPINB10P48595ALIDAWTDSSERPINB12NP536722ALIEAWKDDSERPINB13NP036529AFIKMWKDAConsensusALVLIFAWKTMIVEDSTNa Celera genomic sequence.b The designation of conservative (italic type) and nonconservative (bold face type) amino acid variants in SERPNB11d are relative the consensus motif. Open table in a new tab Stoichiometry of Inhibition (SI)—Assays were performed in a final volume of 100 μl in a 96-well microtiter plate (Costar, Cambridge, MA). Varying amounts of inhibitor were incubated with enzyme for 30 min at 25 °C. Substrate, at a final concentration of 1 mm VLK-pNA for plasmin and 0.2 mm H-Glu-Gly-Arg-pNA for trypsin, was added to each well, and the velocity of substrate hydrolysis was determined by measuring the A405 using the THERMOmax microplate reader. Enzyme Kinetics—The apparent second order rate constants for the interaction of trypsin or plasmin with serpin were determined by the progress curve method under pseudo-first order conditions (26Morrison J.F. Walsh C.T. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). A constant amount of enzyme (2.0 nm trypsin or 10 nm plasmin) and substrate (final concentration 2.0 mm AAPR-pNA or 0.5 mm VLK-pNA, respectively) were mixed with different concentrations of inhibitor (0–1000 nm), and product formation was measured over time. Since the inhibition of trypsin or plasmin is assumed to be irreversible, the course of the reaction can be fit via nonlinear regression, where product formation (P) proceeds at an initial velocity (vz) and is inhibited over time (t) at a rate (kobs). P=(vz/kobs)×[1−e(−kobst)]Eq. 1 For each combination of enzyme and inhibitor, a kobs was calculated by nonlinear regression analysis using Equation 1. A second order rate constant (k′) was determined by plotting a series of kobs versus the respective inhibitor concentration and measuring the slope of the line (k′=Δkobs/Δ[I]). Since the inhibitor was in competition with the substrate, the second order rate constant (k′) was corrected (ka) for the substrate concentration and the Km of the enzyme for the substrate. ka=k′×(1+[S]/Km)Eq. 2 The Km of human trypsin for AAPR-pNA in PBS was 50 μm, and that of plasmin for VLK-pNA in PBS was 230 μm. All kinetic studies were repeated 2–4 times. SDS-PAGE and Immunoblotting—Proteins were mixed with 2–4× gel loading buffer (4% SDS, 20% glycerol, 120 mm Tris-HCl, pH 6.8, 0.01% bromphenol blue, 2% β-mercaptoethanol), heated to 95 °C for 3–5 min, and separated by SDS-PAGE (10% acrylamide; 19% T, 1% C) according to the method of Laemmli (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar). The running buffer (pH 8.3) was 25 mm Tris-base, 250 mm glycine, and 0.1% SDS. Protein bands were visualized after staining in a solution of 0.25% Coomassie Brilliant Blue R-250, 45% methanol, and 10% acetic acid. For immunodetection, proteins separated by SDS-PAGE were electroblotted at 100 V for 1 h at 4 °C onto reinforced nitrocellulose (NitroPure, Osmonics Inc., Westborough, MA) as described (22Schick C. Kamachi Y. Bartuski A.J. Cataltepe S. Schechter N.M. Pemberton P.A. Silverman G.A. J. Biol. Chem. 1997; 272: 1849-1855Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The transfer buffer was 25 mm Tris-base, pH 8.0, and 190 mm glycine, pH 8.3. Recombinant serpin was detected using a monoclonal goat anti-GST antibody (Amersham Biosciences) diluted 1:1000 as the primary antibody and a horseradish peroxidase-linked anti-goat antibody (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) diluted 1:10,000 as the secondary antibody. Immunoblots were visualized using the ECL detection kit (Amersham Biosciences). Complex Formation—Recombinant serpin (20 ng/μl PBS, pH 7.4) in the presence or absence of trypsin (20:1 molar ratio) was incubated at 25 °C. At 1, 5, and 20 min, 20-μl aliquots were removed, mixed with 4× Laemmli loading buffer, boiled for 3 min, and analyzed by SDS-PAGE and immunoblotting. Partial Digestion of Serpin Proteins—Clostripain was diluted to 0.15 units/ml in 3.3 mm dithiothreitol and 0.5× PBS, pH 7.4, and activated by incubation for 2 h at 25 °C. For partial digestion, 5 μg of recombinant serpin in 51 μl of PBS, pH 7.4, was incubated 20 min at 25 °C with 51 μl of activated clostripain in 10 mm dithiothreitol. The amount of clostripain in each reaction varied with the serpin and was adjusted to cleave ∼50% of the RSL substrates: 0.00024 units for Serpinb3b, 0.00006 units for Serpinb11 and Serpinb11-hRSL, and 0.00003 units for SERPINB11d and SERPINB11d-(E90L,S91F,P303S). Digestion was terminated by the addition of EDTA pH 8.0 to a final concentration of 25 mm. Thermostability Assays—Assays were performed as described (21Luke C. Schick C. Tsu C. Whisstock J.C. Irving J.A. Bromme D. Juliano L. Shi G.P. Chapman H.A. Silverman G.A. Biochemistry. 2000; 39: 7081-7091Crossref PubMed Scopus (49) Google Scholar). Briefly, aliquots containing 1 μg of undigested or partially digested recombinant serpin were incubated at temperatures ranging from 37 to 95 °C, transferred to ice for 10 min, and then centrifuged at 12,000 × g for 10 min at 4 °C. Supernatants were analyzed by SDS-PAGE and immunoblotting. Matrix-assisted Laser Desorption Ionization Mass Spectroscopy (MALDI-MS)—Human trypsin (0.2 μg) or plasmin (0.6 μg) was mixed with Serpinb11 (4.4 μg) in 20 μl of PBS. Following either a 3- or 30-min incubation at 25 °C, the protein mixtures were frozen and stored at −80 °C. The mixture components were separated by MALDI-MS at the Wistar Protein Microchemistry Facility (Philadelphia, PA). Tissue Expression Survey—First-strand cDNA samples from human adult tissues (multiple tissue cDNA panels; Clontech) were assayed by PCR for SERPINB11 transcripts using specific primers (forward 2 (5′-AAGCAGCTGAATTCGGGGACG-3′) and reverse 2 (see above)) that amplified a 490-bp product (19Cataltepe S. Gornstein E.R. Schick C. Kamachi Y. Chatson K. Fries J. Silverman G.A. Upton M.P. J. Histochem. Cytochem. 2000; 48: 113-122Crossref PubMed Scopus (134) Google Scholar). To control for template integrity, cDNA samples were assayed for a 616-bp β-actin fragment (forward primer, 5′-CCTCGCCTTTGCCGATCC-3′; reverse primer, 5′-GGATCTTCATGAGGTAGTCAGTC-3′). Similarly, first-strand cDNA samples from adult mouse tissues and mouse embryos were screened for Serpinb11 transcripts by PCR using specific primers (forward 1, 5′-GGGTAAAAGTGCAGTTGTGAATATG-3′; reverse 1, 5′-GGAGAAGCAAACTTGCCAGC-3′) (18Askew D.J. Askew Y.S. Kato Y. Turner R.F. Dewar K. Lehoczky J. Silverman G.A. Genomics. 2004; 84: 176-184Crossref PubMed Scopus (31) Google Scholar, 20Askew D.J. Askew Y.S. Kato Y. Luke C.J. Pak S.C. Bromme D. Silverman G.A. Genomics. 2004; 84: 166-175Crossref PubMed Scopus (19) Google Scholar). These primers amplified a 554-bp product. As a control for template integrity, glyceraldehyde-3-phosphate dehydrogenase primers (Clontech) were used to amplify a 983-bp product. Statistical Analysis—The Student's t test was used to compare the differences between the means of second order rate constants. Identification of a Novel Human Clade B Serpin and Its Mouse Orthologue—To identify novel clade B genomic sequences, we used the BLAST2 algorithm and exon 8 of SERPINB4 to query the human genomic DNA sequence deposited in GenBank™ (25Askew Y.S. Pak S.C. Luke C.J. Askew D.J. Cataltepe S. Mills D.R. Kato H. Lehoczky J. Dewar K. Birren B. Silverman G.A. J. Biol. Chem. 2001; 276: 49320-49330Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This exon was chosen, since it contained the highly conserved hinge region of the RSL (28Hopkins P.C. Carrell R.W. Stone S.R. Biochemistry. 1993; 32: 7650-7657Crossref PubMed Scopus (169) Google Scholar) and the PROSITE serpin signature motif. A significant match was detected in the DNA sequence from the human 18q21 contig clone RP11-69807 (GenBank™ accession number AP001583). Using a combination of gene prediction programs, a novel serpin gene was identified that had the typical features of clade B serpins, including eight exons and the absence of an N-terminal hydrophobic signal sequence (4Silverman G.A. Whisstock J.C. Askew D.J. Pak S.C. Luke C. Cataltepe S. Irving J.A. Bird P.I. Cell. Mol. Life Sci. 2004; 61: 301-325Crossref PubMed Scopus (155) Google Scholar). The gene was designated SERPINB11 by the HUGO Gene Nomenclature Committee. The genomic sequence of SERPINB11, however, contained an early termination codon in exon 4. Since this mutation would lead to either a truncated and dysfunctional peptide of 89 residues or an mRNA targeted for decay (29Maquat L.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 89-99Crossref PubMed Scopus (951) Google Scholar, 30Maquat L.E. J. Cell Sci. 2005; 118: 1773-1776Crossref PubMed Scopus (223) Google Scholar), we sought to determine whether the reference SERPINB11 in GenBank™ was a pseudogene or a simple sequence variant with a nonsense mutation. We designed oligonucleotide primers encompassing the putative start and stop codons of SERPINB11, and used these reagents to amplify DNA fragments (full-length open reading frame ∼1200 bp) from both lung and prostate first-strand cDNAs. DNA fragments were subcloned into pBluescript or pCR®2.1-TOPO® and analyzed by DNA sequencing. Seven different transcripts were identified; four contained full-length coding sequences that could be distinguished by different combinations of eight different SNPs (designated SERPINB11a, -b, -c, and -d; Fig. 1, A and B), two were splice variants (designated SERPINB11e and -g; Fig. 1B), and one contained a nonse

Recent breakthrough discoveries have shown that committed cell fates can be reprogrammed by genetic, chemical and environmental manipulations. The germline of the nematode Caenorhabditis elegans provides a tractable system for studying cell fate reprogramming within the context of a whole organism. To explore the possibility of using C. elegans in high-throughput screens (HTS), we developed a high-throughput workflow for testing compounds that modulate cell fate reprogramming. We utilized puf-8; lip-1 mutants that have enhanced MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling. Wild-type C. elegans hermaphrodites produce both sperm and oocytes, and are thus self-fertile. However, puf-8; lip-1 mutants produce only sperm and are sterile. Notably, compounds that pharmacologically down-regulate MPK-1 (an ERK homolog)/MAP kinase (MAPK) signaling are able to reprogram germ cell fate and restore fertility to these animals. puf-8; lip-1 mutants provide numerous challenges for HTS. First, they are sterile as homozygotes and must be maintained as heterozygotes using a balancer chromosome. Second, homozygous animals for experimentation must be physically separated from the rest of the population. Third, a high quality, high-content assay has not been developed to measure compound effects on germ cell fate reprogramming. Here we describe a semi-automated high-throughput workflow that enables effective sorting of homozygous puf-8; lip-1 mutants into 384-well plates using the COPAS™ BIOSORT. In addition, we have developed an image-based assay for rapidly measuring germ cell reprogramming by measuring the number of viable progeny in wells. The methods presented in this report enable the use of puf-8; lip-1 mutants in HTS campaigns for chemical modulators of germ cell reprogramming within the context of a whole organism.

In the classic form of α1-antitrypsin deficiency (ATD), the misfolded α1-antitrypsin Z (ATZ) variant accumulates in the endoplasmic reticulum (ER) of liver cells. A gain-of-function proteotoxic mechanism is responsible for chronic liver disease in a subgroup of homozygotes. Proteostatic response pathways, including conventional endoplasmic reticulum-associated degradation and autophagy, have been proposed as the mechanisms that allow cellular adaptation and presumably protection from the liver disease phenotype. Recent studies have concluded that a distinct lysosomal pathway called endoplasmic reticulum-to-lysosome completely supplants the role of the conventional macroautophagy pathway in degradation of ATZ. Here, we used several state-of-the-art approaches to characterize the proteostatic responses more fully in cellular systems that model ATD.We used CRISPR-mediated genome editing coupled to a cell selection step by fluorescence-activated cell sorter to perform screening for proteostasis genes that regulate ATZ accumulation and combined that with selective genome editing in 2 other model systems.Endoplasmic reticulum-associated degradation genes are key early regulators and multiple autophagy genes, from classic as well as from ER-to-lysosome and other newly described ER-phagy pathways, participate in degradation of ATZ in a manner that is temporally regulated and evolves as ATZ accumulation persists. Time-dependent changes in gene expression are accompanied by specific ultrastructural changes including dilation of the ER, formation of globular inclusions, budding of autophagic vesicles, and alterations in the overall shape and component parts of mitochondria.Macroautophagy is a critical component of the proteostasis response to cellular ATZ accumulation and it becomes more important over time as ATZ synthesis continues unabated. Multiple subtypes of macroautophagy and nonautophagic lysosomal degradative pathways are needed to respond to the high concentrations of misfolded protein that characterizes ATD and these pathways are attractive candidates for genetic variants that predispose to the hepatic phenotype.

In the classical form of α1-antitrypsin deficiency a misfolded variant α1-antitrypsin Z accumulates in the endoplasmic reticulum of liver cells and causes liver cell injury by gain-of-function proteotoxicity in a sub-group of affected homozygotes but relatively little is known about putative modifiers. Here we carried out genomic sequencing in a uniquely affected family with an index case of liver failure and 2 homozygous siblings with minimal or no liver disease. Their sequences were comparedto sequences in well-characterized cohorts of homozygotes with or without liver disease and then candidate sequence variants were tested for changes in kinetics of ATZ degradation in iPS-derived hepatocyte-like cells derived from the affected siblings themselves.Specific variants in autophagy genes MTMR12 and FAM134A could each accelerate the degradation of ATZ in cells from the index patient but both MTMR12 and FAM134A variants were needed to slow degradation of ATZ in cells from a protected sib, indicating that inheritance of both variants is needed to mediate the pathogenic effects of hepatic proteotoxicity at the cellular level. Analysis of homozygote cohorts showed that multiple patient-specific variants in proteostasis genes are likely to explain liver disease susceptibility at the population level.These results validate the concept that genetic variation in autophagy function can determine susceptibility to liver disease in ATD and provide evidence that polygenic mechanisms and multiple patient-specific variants are likely needed for proteotoxic pathology.

The clade B/intracellular serpins protect cells from peptidase-mediated injury by forming covalent complexes with their targets. SERPINB12 is expressed in most tissues, especially at cellular interfaces with the external environment. This wide tissue distribution pattern is similar to that of granzyme A (GZMA). Because SERPINB12 inhibits trypsin-like serine peptidases, we determined whether it might also neutralize GZMA. SERPINB12 formed a covalent complex with GZMA and inhibited the enzyme with typical serpin slow-binding kinetics. SERPINB12 also inhibited Hepsin. SERPINB12 may function as an endogenous inhibitor of these peptidases.

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

AbstractIntroduction: Many human diseases result from a failure of a single protein to achieve the correct folding and tertiary conformation. These so-called 'conformational diseases' involve diverse proteins and distinctive cellular pathologies. They all engage the proteostasis network (PN), to varying degrees in an attempt to mange cellular stress and restore protein homeostasis. The insulin/insulin-like growth factor signaling (IIS) pathway is a master regulator of cellular stress response, which is implicated in regulating components of the PN.Areas covered: This review focuses on novel approaches to target conformational diseases. The authors discuss the evidence supporting the involvement of the IIS pathway in modulating the PN and regulating proteostasis in Caenorhabditis elegans. Furthermore, they review previous PN and IIS drug screens and explore the possibility of using C. elegans for whole organism-based drug discovery for modulators of IIS-proteostasis pathways.Expert opinion: An alternative approach to develop individualized therapy for each conformational disease is to modulate the global PN. The involvement of the IIS pathway in regulating longevity and response to a variety of stresses is well documented. Increasing data now provide evidence for the close association between the IIS and the PN pathways. The authors believe that high-throughput screening campaigns, which target the C. elegans IIS pathway, may identify drugs that are efficacious in treating numerous conformational diseases.Keywords:: Caenorhabditis elegansconformational diseasedrug discoveryinsulin signalingproteostasis network NotesThis box summarizes key points contained in the article.

The clade B serpins occupy a unique niche among a larger superfamily by predominantly regulating intracellular proteolysis. In humans, there are 13 family members that map to serpin gene clusters at either 6p25 or 18q21. While most of these serpins display a unique inhibitory profile and appear to be well conserved in mammals, the clade B loci of several species show evidence of relatively recent genomic amplification events. However, it is not clear whether these serpin gene amplification events yield paralogs with functional redundancy or, through selective pressure, inhibitors with more diverse biochemical activities. A recent comparative genomic analysis of the mouse clade B cluster at 1D found nearly complete conservation of gene number, order, and orientation relative to those of 18q21 in humans. The only exception was the squamous cell carcinoma antigen (SCCA) locus. The human SCCA locus contains two genes, SERPINB3 (SCCA1) and SERPINB4 (SCCA2), whereas the mouse locus contains four serpins and three pseudogenes. At least two of these genes encoded functional, dual cross-class proteinase inhibitors. Mouse Serpinb3a was shown previously to inhibit both chymotrypsin-like serine and papain-like cysteine proteinases. We now report that mouse Serpinb3b extends the inhibitory repertoire of the mouse SCCA locus to include a second cross-class inhibitor with activity against both papain-like cysteine and trypsin-like serine proteinases. These findings confirmed that the genomic expansion of the clade B serpins in the mouse was associated with a functional diversification of inhibitory activity.

α1-antitrypsin deficiency (ATD) predisposes patients to both loss-of-function (emphysema) and gain-of-function (liver cirrhosis) phenotypes depending on the type of mutation. Although the Z mutation (ATZ) is the most prevalent cause of ATD, >120 mutant alleles have been identified. In general, these mutations are classified as deficient (<20% normal plasma levels) or null (<1% normal levels) alleles. The deficient alleles, like ATZ, misfold in the ER where they accumulate as toxic monomers, oligomers and aggregates. Thus, deficient alleles may predispose to both gain- and loss-of-function phenotypes. Null variants, if translated, typically yield truncated proteins that are efficiently degraded after being transiently retained in the ER. Clinically, null alleles are only associated with the loss-of-function phenotype. We recently developed a C. elegans model of ATD in order to further elucidate the mechanisms of proteotoxicity (gain-of-function phenotype) induced by the aggregation-prone deficient allele, ATZ. The goal of this study was to use this C. elegans model to determine whether different types of deficient and null alleles, which differentially affect polymerization and secretion rates, correlated to any extent with proteotoxicity. Animals expressing the deficient alleles, Mmalton, Siiyama and S (ATS), showed overall toxicity comparable to that observed in patients. Interestingly, Siiyama expressing animals had smaller intracellular inclusions than ATZ yet appeared to have a greater negative effect on animal fitness. Surprisingly, the null mutants, although efficiently degraded, showed a relatively mild gain-of-function proteotoxic phenotype. However, since null variant proteins are degraded differently and do not appear to accumulate, their mechanism of proteotoxicity is likely to be different to that of polymerizing, deficient mutants. Taken together, these studies showed that C. elegans is an inexpensive tool to assess the proteotoxicity of different AT variants using a transgenic approach.

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C. This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI = 1, o/e = 0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the proband's phenotypes.

Members of the intracellular serpin family may help regulate apoptosis, tumor progression, and metastasis. However, their in vivo functions in the context of a whole organism have not been easily defined. To better understand the biology of these serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model organism. Previous in silico analysis suggested that the C. elegans genome harbors nine serpin-like sequences bearing significant similarities to the human clade B intracellular serpins. However, only five genes appear to encode full-length serpins with intact reactive site loops. To determine if this was the case, we have cloned and expressed a putative inhibitory-type C. elegans serpin, srp-3. Analysis of SRP-3 inhibitory activity indicated that SRP-3 was a potent inhibitor of the serine peptidases, chymotrypsin and cathepsin G. Spatial and temporal expression studies using GFP and LacZ promoter fusions indicated that SRP-3 was expressed primarily in the anterior body wall muscles, suggesting that it may play a role in muscle cell homeostasis. Combined with previous studies showing that SRP-2 is an inhibitor of the serine peptidase, granzyme B, and lysosomal cysteine peptidases, these data suggested that C. elegans expressed at least two inhibitory-type serpins with nonoverlapping expression and inhibitory profiles. Moreover, the profiles of these clade L serpins in C. elegans share significant similarities with the profiles of clade B intracellular serpin members in higher vertebrates. This degree of conservation suggests that C. elegans should prove to be a valuable resource in the study of metazoan intracellular serpin function.

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules mediate the inhibition of gene expression. RNAi in C. elegans can be achieved by simply feeding animals with bacteria expressing dsRNA against the gene of interest. This "feeding" method has made it possible to conduct genome-wide RNAi experiments for the systematic knockdown and subsequent investigation of almost every single gene in the genome. Historically, these genome-scale RNAi screens have been labor and time intensive. However, recent advances in automated, high-throughput methodologies have allowed the development of more rapid and efficient screening protocols. In this report, we describe a fast and efficient, liquid-based method for genome-wide RNAi screening.

Delineating the phylogenetic relationships among members of a protein family can provide a high degree of insight into the evolution of domain structure and function relationships. To identify an early metazoan member of the high molecular weight serine proteinase inhibitor (serpin) superfamily, we initiated a cDNA library screen of the cnidarian, Cyanea capillata. We identified one serpin cDNA encoding for a full-length serpin, jellypin. Phylogenetic analysis using the deduced amino acid sequence showed that jellypin was most similar to the platyhelminthe Echinococcus multiocularis serpin and the clade P serpins, suggesting that this serpin evolved ∼1000 million years ago (MYA). Modeling of jellypin showed that it contained all the functional elements of an inhibitory serpin. In vitro biochemical analysis confirmed that jellypin was an inhibitor of the S1 clan SA family of serine proteinases. Analysis of the interactions between the human serine proteinases, chymotrypsin, cathepsin G, and elastase, showed that jellypin inhibited these enzymes in the classical serpin manner, forming a SDS stable enzyme/inhibitor complex. These data suggest that the coevolution of serpin structure and inhibitory function date back to at least early metazoan evolution, ∼1000 MYA.

Neurobeachin (NBEA) was initially identified as a candidate gene for autism. Recently, variants in NBEA have been associated with neurodevelopmental delay and childhood epilepsy. Here, we report on a novel NBEA missense variant (c.5899G > A, p.Gly1967Arg) in the Domain of Unknown Function 1088 (DUF1088) identified in a child enrolled in the Undiagnosed Diseases Network (UDN), who presented with neurodevelopmental delay and seizures. Modeling of this variant in the Caenorhabditis elegans NBEA ortholog, sel-2, indicated that the variant was damaging to in vivo function as evidenced by altered cell fate determination and trafficking of potassium channels in neurons. The variant effect was indistinguishable from that of the reference null mutation suggesting that the variant is a strong hypomorph or a complete loss-of-function. Our experimental data provide strong support for the molecular diagnosis and pathogenicity of the NBEA p.Gly1967Arg variant and the importance of the DUF1088 for NBEA function.

Hepatocytes are metabolically active cells of the liver that play an important role in the biosynthesis of proteins including α1-antitrypsin. Mutations in the α1-antitrypsin gene can lead to protein misfolding, polymerization/aggregation and retention of protein within the endoplasmic reticulum of hepatocytes. The intracellular accumulation of α1-antitrypsin aggregates can lead to liver disease and increased likelihood of developing hepatocellular carcinomas. Of note, only ∼10% of individuals with α1-antitrypsin-deficiency develop severe liver disease suggesting that there are other genetic and/or environmental factors that determine disease outcome. The nematode, Caenorhabditis elegans, is a powerful genetic model organism to study molecular aspects of human disease. In this review, we discuss the functional similarities between the intestinal cells of C. elegans and human hepatocytes and how a C. elegans model of α1-antitrypsin-deficiency can be used as a tool for identifying genetic modifiers and small molecule drugs.

Familial encephalopathy with neuroserpin inclusions bodies (FENIB) is a serpinopathy that induces a rare form of presenile dementia. Neuroserpin contains a classical signal peptide and like all extracellular serine proteinase inhibitors (serpins) is secreted via the endoplasmic reticulum (ER)-Golgi pathway. The disease phenotype is due to gain-of-function missense mutations that cause neuroserpin to misfold and aggregate within the ER. In a previous study, nematodes expressing a homologous mutation in the endogenous Caenorhabditis elegans serpin, srp-2, were reported to model the ER proteotoxicity induced by an allele of mutant neuroserpin. Our results suggest that SRP-2 lacks a classical N-terminal signal peptide and is a member of the intracellular serpin family. Using confocal imaging and an ER colocalization marker, we confirmed that GFP-tagged wild-type SRP-2 localized to the cytosol and not the ER. Similarly, the aggregation-prone SRP-2 mutant formed intracellular inclusions that localized to the cytosol. Interestingly, wild-type SRP-2, targeted to the ER by fusion to a cleavable N-terminal signal peptide, failed to be secreted and accumulated within the ER lumen. This ER retention phenotype is typical of other obligate intracellular serpins forced to translocate across the ER membrane. Neuroserpin is a secreted protein that inhibits trypsin-like proteinase. SRP-2 is a cytosolic serpin that inhibits lysosomal cysteine peptidases. We concluded that SRP-2 is neither an ortholog nor a functional homolog of neuroserpin. Furthermore, animals expressing an aggregation-prone mutation in SRP-2 do not model the ER proteotoxicity associated with FENIB.

Abstract Aging is a common risk factor in neurodegenerative disorders and the ability to investigate aging of neurons in an isogenic background would facilitate discovering the interplay between neuronal aging and onset of 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), a primary neuronal subtype affected in Huntington’s disease (HD), identified pathways associated with RCAN1, a negative regulator of calcineurin. Notably, RCAN1 undergoes age-dependent increase at the protein level detected in reprogrammed MSNs as well as in human postmortem striatum. In patient-derived MSNs of adult-onset HD (HD-MSNs), counteracting RCAN1 by gene knockdown (KD) rescued HD-MSNs from degeneration. The protective effect of RCAN1 KD was associated with enhanced chromatin accessibility of genes involved in longevity and autophagy, mediated through enhanced calcineurin activity, which in turn dephosphorylates and promotes nuclear localization of TFEB transcription factor. Furthermore, we reveal that G2-115 compound, an analog of glibenclamide with autophagy-enhancing activities, reduces the RCAN1-Calcineurin interaction, phenocopying the effect of RCAN1 KD. Our results demonstrate that RCAN1 is a potential genetic or pharmacological target whose reduction-of-function increases neuronal resilience to neurodegeneration in HD through chromatin reconfiguration.

Kinesin motor proteins transport intracellular cargo, including mRNA, proteins, and organelles. Pathogenic variants in kinesin-related genes have been implicated in neurodevelopmental disorders and skeletal dysplasias. We identified de novo, heterozygous variants in KIF5B, encoding a kinesin-1 subunit, in four individuals with osteogenesis imperfecta. The variants cluster within the highly conserved kinesin motor domain and are predicted to interfere with nucleotide binding, although the mechanistic consequences on cell signaling and function are unknown.To understand the in vivo genetic mechanism of KIF5B variants, we modeled the p.Thr87Ile variant that was found in two patients in the C. elegans ortholog, unc-116, at the corresponding position (Thr90Ile) by CRISPR/Cas9 editing and performed functional analysis. Next, we studied the cellular and molecular consequences of the recurrent p.Thr87Ile variant by microscopy, RNA and protein analysis in NIH3T3 cells, primary human fibroblasts and bone biopsy.C. elegans heterozygous for the unc-116 Thr90Ile variant displayed abnormal body length and motility phenotypes that were suppressed by additional copies of the wild type allele, consistent with a dominant negative mechanism. Time-lapse imaging of GFP-tagged mitochondria showed defective mitochondria transport in unc-116 Thr90Ile neurons providing strong evidence for disrupted kinesin motor function. Microscopy studies in human cells showed dilated endoplasmic reticulum, multiple intracellular vacuoles, and abnormal distribution of the Golgi complex, supporting an intracellular trafficking defect. RNA sequencing, proteomic analysis, and bone immunohistochemistry demonstrated down regulation of the mTOR signaling pathway that was partially rescued with leucine supplementation in patient cells.We report dominant negative variants in the KIF5B kinesin motor domain in individuals with osteogenesis imperfecta. This study expands the spectrum of kinesin-related disorders and identifies dysregulated signaling targets for KIF5B in skeletal development.

Protein misfolding, polymerization, and/or aggregation are hallmarks of serpinopathies and many other human genetic disorders including Alzheimer's, Huntington's, and Parkinson's disease. While higher organism models have helped shape our understanding of these diseases, simpler model systems, like Caenorhabditis elegans, offer great versatility for elucidating complex genetic mechanisms underlying these diseases. Moreover, recent advances in automated high-throughput methodologies have promoted C. elegans as a useful tool for drug discovery. In this chapter, we describe how one could model serpinopathies in C. elegans and how one could exploit this model to identify small molecule compounds that can be developed into effective therapeutic drugs.

Background & Aims Insulin signaling is known to regulate essential proteostasis mechanisms. Methods The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. Results We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator–activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. Conclusions These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies. Insulin signaling is known to regulate essential proteostasis mechanisms. The analyses here examined effects of insulin signaling in the PiZ mouse model of α1-antitrypsin deficiency in which hepatocellular accumulation and proteotoxicity of the misfolded α1-antitrypsin Z variant (ATZ) causes liver fibrosis and cancer. We first studied the effects of breeding PiZ mice to liver-insulin-receptor knockout (LIRKO) mice (with hepatocyte-specific insulin-receptor gene disruption). The results showed decreased hepatic ATZ accumulation and liver fibrosis in PiZ x LIRKO vs PiZ mice, with reversal of those effects when we bred PiZ x LIRKO mice onto a FOXO1-deficient background. Increased intracellular degradation of ATZ mediated by autophagy was identified as the likely mechanism for diminished hepatic proteotoxicity in PiZ x LIRKO mice and the converse was responsible for enhanced toxicity in PiZ x LIRKO x FOXO1-KO animals. Transcriptomic studies showed major effects on oxidative phosphorylation and autophagy genes, and significant induction of peroxisome proliferator–activated-receptor-γ-coactivator-1α (PGC1α) expression in PiZ-LIRKO mice. Because PGC1α plays a key role in oxidative phosphorylation, we further investigated its effects on ATZ proteostasis in our ATZ-expressing mammalian cell model. The results showed PGC1α overexpression or activation enhances autophagic ATZ degradation. These data implicate suppression of autophagic ATZ degradation by down-regulation of PGC1α as one mechanism by which insulin signaling exacerbates hepatic proteotoxicity in PiZ mice, and identify PGC1α as a novel target for development of new human α1-antitrypsin deficiency liver disease therapies.

Definitive chemoradiation therapy (CRT) is the standard of care for cervical cancer but recurrence after CRT is common. More complete understanding of the molecular mechanisms underlying resistance to CRT is necessary for the development of novel therapies to improve patient outcomes. Elevated serum squamous cell carcinoma antigen (SCCA) is strongly prognostic for recurrence and death after CRT for cervical cancer; however, changes in expression of SERPINB3 (the gene for SCCA) after exposure to CRT, and the molecular function of SCCA in cervical cancer pathogenesis are not well understood. The purpose of this study is to evaluate changes in SERPINB3 expression in cervical tumors resistant to CRT and to test the hypothesis that SCCA protects cervical tumor cells from radiation. With patient consent, cervical tumor specimens from 21 women undergoing definitive CRT at our institution were collected prior to the start of treatment and after 3 weeks of CRT (mid-treatment), providing matched pairs. SERPINB3 expression was determined using whole transcriptome RNA sequencing and reads per kilobase per million (RPKM) normalization method. Exogenous SERPINB3 was overexpressed with an adenoviral vector in cervical tumor cell lines with low endogenous expression. CRISPR-Cas9 technology was used to knock out (KO) SERPINB3 in cervical tumor cell lines with high endogenous SCCA. Cultured cells were treated with increasing doses of ionizing radiation and clonogenic survival was determined. Gene expression analysis of paired patient tumor specimens revealed that SERPINB3 mRNA transcript remained elevated or increased in 7 (33%) patients [pre-treatment: median 253 RPKM (range 119-468), mid-treatment: median = 343 RPKM (range 132-808)). SERPINB3 transcript decreased or remained low in 14 (66%) patients [pre-treatment: median = 208 RPKM (range 6-772), mid-treatment: median = 36 RPKM (range 6-121)]. Absolute recurrence rate was 57% in patients with maintained or elevated tumor SERPINB3 expression compared to 21% for patients with low or decreased expression. Stable overexpression of SERPINB3 conferred greater clonogenic survival after RT compared to parent cell lines. Conversely, CRISPR-Cas9 directed KO of SERPINB3 resulted in increased radiation sensitivity. SCCA is an established serum biomarker for poor prognosis in cervical cancer. These data provide evidence that SCCA expression is commonly maintained during CRT in tumors from patients who experience recurrence, and that SCCA mediates cervical tumor cell response to radiation in vitro. Further studies are ongoing to dissect the molecular mechanism of this phenotype, and could lead to development of novel therapies for increasing the effectiveness of CRT in cervical cancer.

VOLUME 285 (2010) PAGES 24299–24305 PAGE 24303: In the right-hand column (line 18), the sentence should read as follows: An example of the latter situation occurred with the α1-antitrypsin paralog (Serpina1b) knock-out, which only partly removes elastase inhibitory activity in the circulation, suggesting that Serpina1a also contributes to elastase control in the mouse (44). Ref. 14 in the supplemental data should read as follows: Benarafa, C., Priebe, G. P., and Remold-O'Donnell, E. (2007) J. Exp Med. 204, 1901–1909. Download .pdf (.16 MB) Help with pdf files

To investigate the role of insulin degrading enzymes in heterologous insulin gene expression, Chinese hamster ovary (CHO) cells were transfected with a mammalian expression vector carrying the cDNA for the human pre-proinsulin under the control of the RSV promoter. Stable transfectants were isolated and characterised in regard to insulin transcription, translation, processing and secretion. Levels of insulin expressed by these cells were extremely low, peaking at around 5–10 ng/106 cells/24 hours. Analysis of the secreted product indicated inefficient processing with less than 10% of the total product being fully processed. [125I]-Insulin degradation assays revealed insulin degrading activity in the cytosol and in the conditioned medium of CHO cells. In both cases the insulin degrading activity was significantly inhibited by the addition of bacitracin, a peptide antibiotic and an inhibitor of insulin degrading enzyme (IDE). No noticeable effects were seen with the addition of a general protease inhibitor, PMSF. When transfected CHO cultures were supplemented with small quantities of bacitracin insulin expressionimproved considerably. These results combined support the hypothesis that IDEs are directly involved in inhibiting overexpression of recombinant human insulin in transfected CHO cells.

Abstract The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy (CRT). Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3 -deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces cell death morphologically consistent with necrosis, with biochemical and cellular features of lysoptosis. Moreover, rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. These data suggest targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Abstract Autophagy and apoptosis regulate cell survival and death, and are implicated in the pathogenesis of many diseases. The same type of stress signals can induce either process, but it is unclear how cells ‘assess’ cellular damage and make a ‘life’ or ‘death’ decision by activating autophagy or apoptosis. A computational model of coupled apoptosis and autophagy is built here to study the systems-level dynamics of the underlying signaling network. The model explains the differential dynamics of autophagy and apoptosis in response to various experimental 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 intensity of stress. The model reveals that this dynamic cell fate decision is conferred by a core regulatory network involving cytoplasmic Ca 2+ 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. The model identified Ca 2+ /calmodulin-dependent kinase kinase β (CaMKKβ) as a determinant of the opposite roles of cytoplasmic Ca 2+ in autophagy regulation. The results also demonstrated that the model could contribute to the development of pharmacological strategies modulate cell fate decisions.

Frameshifting-stimulating pseudoknots, referred to as pseudoknots unless specified otherwise, exhibit classic H-type pseudoknot-folds with two stacked stems (S1 and S2) and two loops (L1 and L2) (Fig. 1A). Structural elements responsible for the stability and the integrity of the pse udoknots include quasi-continuous base-stacking in S1 and S2 and an extensive S1-L2 triplex interaction.Structural diversity among pseudoknots appears to be one of major culprits for various vi ral systems to achieve different -1RFS efficiencies optimized to their own needs. The pseu-doknots in BWYV (beet western yellow virus) and SRV-1 (simian retrovirus-1) provide an interesting instance for the structural diversity. BWYV and other luteoviral pseudoknots, which mediate P1-P2 production through -1RFS, are relatively small and tightly folded with a characteristically very short S2 (3 base pairs) (Fig. 1A). These luteoviral pseudoknots produce considerable degrees of -1RFS and show a strong sequence preference in both L1 and L2 for efficient -1RFS.

ABSTRACT We describe a proband evaluated through the Undiagnosed Diseases Network (UDN) who presented with microcephaly, developmental delay, and refractory epilepsy with a de novo p.Ala47Thr missense variant in the protein phosphatase gene, PPP5C . This gene has not previously been associated with a Mendelian disease, and based on the population database, gnomAD, the gene has a low tolerance for loss-of-function variants (pLI=1, o/e=0.07). We functionally evaluated the PPP5C variant in C. elegans by knocking the variant into the orthologous gene, pph-5, at the corresponding residue, Ala48Thr. We employed assays in three different biological processes where pph-5 was known to function through opposing the activity of genes, mec-15 and sep-1. We demonstrated that, in contrast to control animals, the pph-5 Ala48Thr variant suppresses the neurite growth phenotype and the GABA signaling defects of mec-15 mutants, and the embryonic lethality of sep-1 mutants. The Ala48Thr variant did not display dominance and behaved similarly to the reference pph-5 null, indicating that the variant is likely a strong hypomorph or complete loss-of-function. We conclude that pph-5 Ala48Thr is damaging in C. elegans. By extension in the proband, PPP5C p.Ala47Thr is likely damaging, the de novo dominant presentation is consistent with haplo-insufficiency, and the PPP5C variant is likely responsible for one or more of the proband’s phenotypes.

Abstract Mutations in the autophagy gene EPG5 are responsible for Vici syndrome, a multisystem human genetic disease with a combined immunodeficiency component. However, the mechanism(s) by which EPG5 regulates immune signaling are incompletely understood. Previously, we found that Epg5−/− mice are resistant to influenza and exhibit hyperinflammation in the lungs including elevated IL1B/IL-1β and TNF/TNFa. Here, we show that the disruption of Epg5 results in protection against multiple enteric viruses including norovirus and rotavirus. RNA sequencing revealed that IFN-λ responses are highly upregulated in the intestines of Epg5−/− mice. Further, mice lacking Epg5 exhibit substantial alterations of the intestinal microbiota. Surprisingly, germ-free Epg5−/− mice showed persistent inflammation of both the intestine and lung, suggesting a microbiota independent mechanism. Epg5−/−Ifnlr1−/− mice, which lack the receptor for IFN-λ, regained susceptibility to viral infection but maintained microbial dysbiosis, indicating that IFN-λ signaling is the primary mediator of resistance to enteric viruses but is dispensable for microbial dysbiosis in Epg5−/− mice. Intriguingly, epg-5-mutant Caenorhabditis elegans animals are also resistant to Orsay virus, which is an intestinal cell-tropic virus and the only known virus that naturally infects C. elegans. This study unveils an important role for autophagy gene Epg5 in protection of mammalian hosts by modulating intestinal IFN-λ responses, which is unexpectedly independent of the microbiota, and raises the possibility of a conserved viral resistance mechanism that is shared between C. elegans and mammals.

You have accessJournal of UrologyBladder Cancer: Invasive II (MP32)1 Apr 2019MP32-19 ADJUVANT CHEMOTHERAPY VERSUS OBSERVATION AFTER RADICAL CYSTECTOMY FOR PATIENTS WITH NODE-POSITIVE BLADDER CANCER Sahyun Pak*, Teak Jun Shin, Hwiwoo Kim, Donghyun Lee, Dalsan You, In Gab Jeong, Cheryn Song, Jae-Lyun Lee, Bumsik Hong, Hyuk Hong, Kim Choung-Soo, and Hanjong Ahn Sahyun Pak*Sahyun Pak* More articles by this author , Teak Jun ShinTeak Jun Shin More articles by this author , Hwiwoo KimHwiwoo Kim More articles by this author , Donghyun LeeDonghyun Lee More articles by this author , Dalsan YouDalsan You More articles by this author , In Gab JeongIn Gab Jeong More articles by this author , Cheryn SongCheryn Song More articles by this author , Jae-Lyun LeeJae-Lyun Lee More articles by this author , Bumsik HongBumsik Hong More articles by this author , Hyuk HongHyuk Hong More articles by this author , Kim Choung-SooKim Choung-Soo More articles by this author , and Hanjong AhnHanjong Ahn More articles by this author View All Author Informationhttps://doi.org/10.1097/01.JU.0000555876.53407.76AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVES: This study aimed to compare adjuvant chemotherapy (AC) versus observation after radical cystectomy in patients with pathologically node-positive bladder cancer (pN+). We also attempted to identify pN+ patients most likely to benefit from AC after RC. METHODS: Outcomes were reviewed in patients with pTanyN1-3M0 bladder cancer treated with RC between January 1995 and June 2017. Patients who underwent RC alone were assigned to the RC group and those who received AC were assigned to the AC group. Baseline characteristics between two groups were controlled with inverse probability of treatment weighting (IPTW)-adjusted analyses. Variables prognostic for survival were assessed by stratified time-varying covariate Cox model. RESULTS: Of 281 enrolled patients, 122 (43.4%) underwent RC alone and 159 (56.6%) received AC. The 3-year IPTW-adjusted rates of metastasis-free survival was similar between AC and RC groups (35.8% vs. 31.2%, p=0.471), whereas IPTW-adjusted overall survival rate was higher in the AC than RC group (46.4% vs. 33.7%, p=0.024). In the time-varying covariate Cox model, AC was an independent predictor of overall survival (HR=0.48; 95% CI, 0.34–0.67; P< 0.0001). When subdivided by tertiles of lymph node density (LND), the 3-year overall survival rates were similar between the AC and RC alone in patients with LND <9% (58.7% vs. 51.7%, p=0.878), whereas 3-year overall survival rates of AC group were higher than RC group in patients with LND 9-25% (53.4% vs. 23.7%, p=0.003) and LND ≥25% (27.4% vs. 16.1%, p=0.032). The numbers needed to treat to prevent one death at 3 years were 3 and 9 in patients with LND 9-25% and ≥25%, respectively. CONCLUSIONS: AC after RC was associated with improved overall survival in patients with node-positive bladder cancer. Patients with intermediate nodal burden may benefit most from AC, whereas the effect of AC was not apparent in low nodal burden disease. Randomized studies are needed to confirm the results of the present study. Source of Funding: None Seoul, Korea, Republic of© 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 201Issue Supplement 4April 2019Page: e451-e451 Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sahyun Pak* More articles by this author Teak Jun Shin More articles by this author Hwiwoo Kim More articles by this author Donghyun Lee More articles by this author Dalsan You More articles by this author In Gab Jeong More articles by this author Cheryn Song More articles by this author Jae-Lyun Lee More articles by this author Bumsik Hong More articles by this author Hyuk Hong More articles by this author Kim Choung-Soo More articles by this author Hanjong Ahn More articles by this author Expand All Advertisement PDF downloadLoading ...

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