Anne Bertolotti

Malfolded proteins in the endoplasmic reticulum (ER) induce cellular stress and activate c-Jun amino-terminal kinases (JNKs or SAPKs). Mammalian homologs of yeast IRE1, which activate chaperone genes in response to ER stress, also activated JNK, and IRE1 α −/− fibroblasts were impaired in JNK activation by ER stress. The cytoplasmic part of IRE1 bound TRAF2, an adaptor protein that couples plasma membrane receptors to JNK activation. Dominant-negative TRAF2 inhibited activation of JNK by IRE1. Activation of JNK by endogenous signals initiated in the ER proceeds by a pathway similar to that initiated by cell surface receptors in response to extracellular signals.

Malfolded proteins in the endoplasmic reticulum (ER) inhibit translation initiation. This response is believed to be mediated by increased phosphorylation of eukaryotic initiation factor 2α (eIF2α) and is hypothesized to reduce the work load imposed on the folding machinery during stress. Here we report that mutating the gene encoding the ER stress–activated eIF2α kinase PERK abolishes the phosphorylation of eIF2α in response to accumulation of malfolded proteins in the ER resulting in abnormally elevated protein synthesis and higher levels of ER stress. Mutant cells are markedly impaired in their ability to survive ER stress and inhibition of protein synthesis by cycloheximide treatment during ER stress ameliorates this impairment. PERK thus plays a major role in the ability of cells to adapt to ER stress.

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.

Guanabenz, a small-molecule inhibitor, protects cells from lethal accrual of misfolded proteins in the endoplasmic reticulum.

Deposition of proteins of aberrant conformation is the hallmark of many neurodegenerative diseases. Misfolding of the normally globular mutant superoxide dismutase-1 (SOD1) is a central, early, but poorly understood event in the pathogenic cascade leading to familial forms of ALS. Here we report that aggregates composed of an ALS-causing SOD1 mutant penetrate inside cells by macropinocytosis and rapidly exit the macropinocytic compartment to nucleate aggregation of the cytosolic, otherwise soluble, mutant SOD1 protein. Once initiated, mutant SOD1 aggregation is self-perpetuating. Mutant SOD1 aggregates transfer from cell to cell with remarkable efficiency, a process that does not require contacts between cells but depends on the extracellular release of aggregates. This study reveals that SOD1 aggregates, propagate in a prion-like manner in neuronal cells and sheds light on the mechanisms underlying aggregate uptake and cell-to-cell transfer.

The epithelial cells of the gastrointestinal tract are exposed to toxins and infectious agents that can adversely affect protein folding in the endoplasmic reticulum (ER) and cause ER stress. The IRE1 genes are implicated in sensing and responding to ER stress signals. We found that epithelial cells of the gastrointestinal tract express IRE1beta, a specific isoform of IRE1. BiP protein, a marker of ER stress, was elevated in the colonic mucosa of IRE1beta(-/-) mice, and, when exposed to dextran sodium sulfate (DSS) to induce inflammatory bowel disease, mutant mice developed colitis 3-5 days earlier than did wild-type or IRE1beta(+/-) mice. The inflammation marker ICAM-1 was also expressed earlier in the colonic mucosa of DSS-treated IRE1beta(-/-) mice, indicating that the mutation had its impact early in the inflammatory process, before the onset of mucosal ulceration. These findings are consistent with a model whereby perturbations in ER function, which are normally mitigated by the activity of IRE1beta, participate in the development of colitis.

Protein phosphorylation regulates virtually all biological processes. Although protein kinases are popular drug targets, targeting protein phosphatases remains a challenge. Here, we describe Sephin1 (selective inhibitor of a holophosphatase), a small molecule that safely and selectively inhibited a regulatory subunit of protein phosphatase 1 in vivo. Sephin1 selectively bound and inhibited the stress-induced PPP1R15A, but not the related and constitutive PPP1R15B, to prolong the benefit of an adaptive phospho-signaling pathway, protecting cells from otherwise lethal protein misfolding stress. In vivo, Sephin1 safely prevented the motor, morphological, and molecular defects of two otherwise unrelated protein-misfolding diseases in mice, Charcot-Marie-Tooth 1B, and amyotrophic lateral sclerosis. Thus, regulatory subunits of phosphatases are drug targets, a property exploited here to safely prevent two protein misfolding diseases.

The proteasome degrades most cellular proteins in a controlled and tightly regulated manner and thereby controls many processes, including cell cycle, transcription, signalling, trafficking and protein quality control. Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with diverse human diseases, including cancer and neurodegeneration. Target selection is an important and well-established way to control protein degradation. In addition, mounting evidence indicates that cells adjust proteasome-mediated degradation to their needs by regulating proteasome abundance through the coordinated expression of proteasome subunits and assembly chaperones. Central to the regulation of proteasome assembly is TOR complex 1 (TORC1), which is the master regulator of cell growth and stress. This Review discusses how proteasome assembly and the regulation of proteasomal degradation are integrated with cellular physiology, including the interplay between the proteasome and autophagy pathways. Understanding these mechanisms has potential implications for disease therapy, as the misregulation of proteasome function contributes to human diseases such as cancer and neurodegeneration. Protein degradation by the proteasome is crucial for the control of many cellular processes, and defects in proteasomal degradation may lead to cancer and neurodegeneration. TOR complex 1 has a key role in regulating proteasome abundance and assembly and in integrating proteasomal activity with autophagy pathways and, more generally, cell physiology.

Research Article16 September 1996free access hTAF(II)68, a novel RNA/ssDNA-binding protein with homology to the pro-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. A. Bertolotti A. Bertolotti Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author Y. Lutz Y. Lutz Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author D. J. Heard D. J. Heard Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author P. Chambon P. Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author L. Tora L. Tora Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author A. Bertolotti A. Bertolotti Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author Y. Lutz Y. Lutz Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author D. J. Heard D. J. Heard Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author P. Chambon P. Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author L. Tora L. Tora Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. Search for more papers by this author Author Information A. Bertolotti1, Y. Lutz1, D. J. Heard1, P. Chambon1 and L. Tora1 1Institut de Génétique et de Biologie Moléculaire et Cellulaire CNRS/INSERM/ULP, Illkirch, France. The EMBO Journal (1996)15:5022-5031https://doi.org/10.1002/j.1460-2075.1996.tb00882.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TFIID is the main sequence-specific DNA-binding component of the RNA polymerase II (Pol II) transcriptional machinery. It is a multiprotein complex composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s). Here we report the cloning and characterization of a novel human TBP-associated factor, hTAF(II)68. It contains a consensus RNA-binding domain (RNP-CS) and binds not only RNA, but also single stranded (ss) DNA. hTAF(II)68 shares extensive sequence similarity with TLS/FUS and EWS, two human nuclear RNA-binding pro-oncoproteins which are products of genes commonly translocated in human sarcomas. Like hTAF(II)68, TLS/FUS is also associated with a sub-population of TFIID complexes chromatographically separable from those containing hTAF(II)68. Therefore, these RNA and/or ssDNA-binding proteins may play specific roles during transcription initiation at distinct promoters. Moreover, we demonstrate that hTAF(II)68 co-purifies also with the human RNA polymerase II and can enter the preinitiation complex together with Pol II. Previous ArticleNext Article Volume 15Issue 181 September 1996In this issue RelatedDetailsLoading ...

The ubiquitin-proteasome system targets many cellular proteins for degradation and thereby controls most cellular processes. Although it is well established that proteasome inhibition is lethal, the underlying mechanism is unknown. Here, we show that proteasome inhibition results in a lethal amino acid shortage. In yeast, mammalian cells, and flies, the deleterious consequences of proteasome inhibition are rescued by amino acid supplementation. In all three systems, this rescuing effect occurs without noticeable changes in the levels of proteasome substrates. In mammalian cells, the amino acid scarcity resulting from proteasome inhibition is the signal that causes induction of both the integrated stress response and autophagy, in an unsuccessful attempt to replenish the pool of intracellular amino acids. These results reveal that cells can tolerate protein waste, but not the amino acid scarcity resulting from proteasome inhibition.

The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation.

The t(11;22) chromosomal translocation specifically linked to Ewing sarcoma and primitive neuroectodermal tumor results in a chimeric molecule fusing the amino-terminus-encoding region of the EWS gene to the carboxyl-terminal DNA-binding domain encoded by the FLI-1 gene. As the function of the protein encoded by the EWS gene remains unknown, we investigated the putative role of EWS in RNA polymerase II (Pol II) transcription by comparing its activity with that of its structural homolog, hTAFII68. We demonstrate that a portion of EWS is able to associate with the basal transcription factor TFIID, which is composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs). In vitro binding studies revealed that both EWS and hTAFII68 interact with the same TFIID subunits, suggesting that the presence of EWS and that of hTAFII68 in the same TFIID complex may be mutually exclusive. Moreover, EWS is not exclusively associated with TFIID but, similarly to hTAFII68, is also associated with the Pol II complex. The subunits of Pol II that interact with EWS and hTAFII68 have been identified, confirming the association with the polymerase. In contrast to EWS, the tumorigenic EWS–FLI-1 fusion protein is not associated with either TFIID or Pol II in Ewing cell nuclear extracts. These observations suggest that EWS and EWS–FLI-1 may play different roles in Pol II transcription.

ABSTRACT Genetic analysis of the cellular adaptation to malfolded proteins in the endoplasmic reticulum (the unfolded protein response – UPR) has revealed a novel signaling pathway initiated by activation of IRE1, an ER-resident protein kinase and endonuclease. In yeast, Ire1p activates gene expression by promoting a non-conventional splicing event that converts the mRNA encoding the Hac1p transcription factor from an inefficiently translated inactive mRNA to an actively translated one. Hac1p binds to the promoters of genes encoding chaperones and other targets of the UPR and activates them. Recently, mammalian IRE1 homologues have been identified and their response to ER stress is regulated by binding to the ER chaperone BiP. The mechanisms by which mammalian IRE1 activates gene expression have not been completely characterized and mammalian HAC1 homologues have not been identified. Surprisingly, mammalian IRE1s are able to activate both JUN N-terminal kinases and an alternative ER-stress signaling pathway mediated by the transcription factor ATF6. This indicates that the mammalian UPR is more complex than that found in yeast.

The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1α and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.

The copper-zinc superoxide dismutase-1 (SOD1) is a highly structured protein and, a priori, one of the least likely proteins to be involved in a misfolding disease. However, more than 140, mostly missense, mutations in the SOD1 gene cause aggregation of the affected protein in familial forms of amyotrophic lateral sclerosis (ALS). The remarkable diversity of the effects of these mutations on SOD1 properties has suggested that they promote aggregation by a variety of mechanisms. Experimental assessment of surface hydrophobicity using a sensitive fluorescent-based assay, revealed that diverse ALS-causing mutations provoke SOD1 aggregation by increasing their propensity to expose hydrophobic surfaces. These findings could not be anticipated from analysis of the amino acid sequence. Our results uncover the biochemical nature of the misfolded aggregation-prone intermediate and reconcile the seemingly diverse effects of ALS-causing mutations into a unifying mechanism. Furthermore, the method we describe here will be useful for investigating and interfering with aggregation of various proteins and thereby provide insight into the molecular mechanisms underlying many neurodegenerative diseases.

Protein phosphorylation is a prevalent and ubiquitous mechanism of regulation. Kinases are popular drug targets, but identifying selective phosphatase inhibitors has been challenging. Here, we used surface plasmon resonance to design a method to enable target-based discovery of selective serine/threonine phosphatase inhibitors. The method targeted a regulatory subunit of protein phosphatase 1, PPP1R15B (R15B), a negative regulator of proteostasis. This yielded Raphin1, a selective inhibitor of R15B. In cells, Raphin1 caused a rapid and transient accumulation of its phosphorylated substrate, resulting in a transient attenuation of protein synthesis. In vitro, Raphin1 inhibits the recombinant R15B-PP1c holoenzyme, but not the closely related R15A-PP1c, by interfering with substrate recruitment. Raphin1 was orally bioavailable, crossed the blood-brain barrier, and demonstrated efficacy in a mouse model of Huntington's disease. This identifies R15B as a druggable target and provides a platform for target-based discovery of inhibitors of serine/threonine phosphatases.

The activities of human holophosphatases R15A–PP1 and R15B–PP1 on substrate eIF2α are now reconstituted in vitro, revealing that inhibitors Guanabenz and Sephin1 induce a selective conformational change in R15A and impair the recruitment of eIF2α. The reversible phosphorylation of proteins controls most cellular functions. Protein kinases have been popular drug targets, unlike phosphatases, which remain a drug discovery challenge. Guanabenz and Sephin1 are selective inhibitors of the phosphatase regulatory subunit PPP1R15A (R15A) that prolong the benefit of eIF2α phosphorylation, thereby protecting cells from proteostatic defects. In mice, Sephin1 prevents two neurodegenerative diseases, Charcot–Marie–Tooth 1B (CMT-1B) and SOD1-mediated amyotrophic lateral sclerosis (ALS). However, the molecular basis for R15A inhibition is unknown. Here we reconstituted human recombinant eIF2α holophosphatases, R15A–PP1 and R15B–PP1, whose activity depends on both the catalytic subunit PP1 (protein phosphatase 1) and either R15A or R15B. This system enabled the functional characterization of these holophosphatases and revealed that Guanabenz and Sephin1 induced a selective conformational change in R15A, detected by resistance to limited proteolysis. This altered the recruitment of eIF2α, preventing its dephosphorylation. This work demonstrates that regulatory subunits of phosphatases are valid drug targets and provides the molecular rationale to expand this concept to other phosphatases.

Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins. Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins. Polyglutamine (poly(Q)) expansion provides a toxic gain of function to nine otherwise unrelated proteins and induces progressive neurodegenerative diseases, such as Huntington disease (HD) 3The abbreviations used are: HD, Huntington disease; GFP, green fluorescent protein. 3The abbreviations used are: HD, Huntington disease; GFP, green fluorescent protein. (1Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1082) Google Scholar). A key feature of these dominantly inherited diseases is the presence of aggregated poly(Q) inclusions in affected neurons (2Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1889) Google Scholar, 3DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2284) Google Scholar). The age of onset of these dominantly inherited diseases inversely correlates with the length of the poly(Q) expansion. HD is caused by a poly(Q) expansion in a protein named Huntingtin (Htt), which leads to the aggregation of a proteolytic, amino-terminal fragment of Htt encompassing the poly(Q) repeat (2Davies S.W. Turmaine M. Cozens B.A. DiFiglia M. Sharp A.H. Ross C.A. Scherzinger E. Wanker E.E. Mangiarini L. Bates G.P. Cell. 1997; 90: 537-548Abstract Full Text Full Text PDF PubMed Scopus (1889) Google Scholar, 3DiFiglia M. Sapp E. Chase K.O. Davies S.W. Bates G.P. Vonsattel J.P. Aronin N. Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2284) Google Scholar, 4Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). However, the mechanism by which aggregated poly(Q) proteins contribute to cellular dysfunction is still a matter of debate (5Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2402) Google Scholar). Quality control pathways have evolved to protect cells from the deleterious effect of aberrant proteins, and studies in several model systems have shown that increasing Hsp70 or Hsp40 chaperones could modulate aggregation of expanded poly(Q) (6Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7841-7846Crossref PubMed Scopus (537) Google Scholar). Strikingly, however, Hsp70 or Hsp40 overexpression in Drosophila suppresses the neurotoxicity of expanded poly(Q) without removing inclusions (7Warrick J.M. Chan H.Y. Gray-Board G.L. Chai Y. Paulson H.L. Bonini N.M. Nat. Genet. 1999; 23: 425-428Crossref PubMed Scopus (722) Google Scholar, 8Cummings C.J. Sun Y. Opal P. Antalffy B. Mestril R. Orr H.T. Dillmann W.H. Zoghbi H.Y. Hum. Mol. Genet. 2001; 10: 1511-1518Crossref PubMed Scopus (422) Google Scholar), indicating that the presence of microscopically visible inclusion bodies may not necessarily determine cytotoxicity as previously proposed (9Saudou F. Finkbeiner S. Devys D. Greenberg M.E. Cell. 1998; 95: 55-66Abstract Full Text Full Text PDF PubMed Scopus (1357) Google Scholar). Yeast has been used to investigate the relationship between chaperones and poly(Q) aggregation. Although Hsp70 and Hsp40 modulate the aggregation process in yeast (6Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7841-7846Crossref PubMed Scopus (537) Google Scholar, 10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar, 11Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (295) Google Scholar, 12Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 13Gokhale K.C. Newnam G.P. Sherman M.Y. Chernoff Y.O. J. Biol. Chem. 2005; 280: 22809-22818Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), the chaperone Hsp104 is required for poly(Q) aggregation (10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar). However, except in one model (11Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (295) Google Scholar), expression of expanded poly(Q) in yeast cells is not toxic, similar to most mammalian cells (6Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7841-7846Crossref PubMed Scopus (537) Google Scholar, 10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar).Each poly(Q) disease affects a specific neuronal population and is defined by specific clinical features, whereas the affected proteins are broadly expressed (1Zoghbi H.Y. Orr H.T. Annu. Rev. Neurosci. 2000; 23: 217-247Crossref PubMed Scopus (1082) Google Scholar). The poly(Q) expansion might trigger cellular dysfunction by a similar mechanism in the different diseases and the sequence neighboring the poly(Q) stretch might confer disease specificity. In Htt, two proline repeats separated by a proline-rich region are located at the carboxyl terminus of the poly(Q) repeat and have been shown to be important for interaction with several proteins (14Liu Y.F. Deth R.C. Devys D. J. Biol. Chem. 1997; 272: 8121-8124Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 15Sittler A. Walter S. Wedemeyer N. Hasenbank R. Scherzinger E. Eickhoff H. Bates G.P. Lehrach H. Wanker E.E. Mol. Cell. 1998; 2: 427-436Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 16Faber P.W. Barnes G.T. Srinidhi J. Chen J. Gusella J.F. MacDonald M.E. Hum. Mol. Genet. 1998; 7: 1463-1474Crossref PubMed Scopus (324) Google Scholar, 17Passani L.A. Bedford M.T. Faber P.W. McGinnis K.M. Sharp A.H. Gusella J.F. Vonsattel J.P. MacDonald M.E. Hum. Mol. Genet. 2000; 9: 2175-2182Crossref PubMed Scopus (85) Google Scholar, 18Qin Z.H. Wang Y. Sapp E. Cuiffo B. Wanker E. Hayden M.R. Kegel K.B. Aronin N. DiFiglia M. J. Neurosci. 2004; 24: 269-281Crossref PubMed Scopus (146) Google Scholar, 19Zhai W. Jeong H. Cui L. Krainc D. Tjian R. Cell. 2005; 123: 1241-1253Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). A role for oligoproline on poly(Q) conformation and aggregation has recently been reported in vitro (20Bhattacharyya A. Thakur A.K. Chellgren V.M. Thiagarajan G. Williams A.D. Chellgren B.W. Creamer T.P. Wetzel R. J. Mol. Biol. 2006; 355: 524-535Crossref PubMed Scopus (204) Google Scholar). Because the link between poly(Q) aggregation and toxicity is still subjected to controversy, the effect of the proline-rich region on poly(Q) toxicity could not be anticipated. Here we have found that deleting the whole proline-rich region in Htt dramatically alters the shape of poly(Q) inclusions and unmasks toxicity in yeast cells.EXPERIMENTAL PROCEDURESYeast Strains and Manipulations—The wild-type yeast strain W303 (MAT α ade2-1 can1-100 his3-11, 15 leu2-3, 112, trp1-1, ura3-1) and isogenic hsp104 deletion strain Δhsp104 (hsp104::LEU2+) were kindly provided by S. Lindquist (Whitehead Institute for Biomedical Research, Cambridge, MA). Transformation of yeast was performed using a standard lithium acetate/polyethylene glycol method (21Gietz R.D. Woods R.A. Methods Mol. Biol. 2006; 313: 107-120PubMed Google Scholar). Yeast cells were grown in rich medium (YPD; containing 1% yeast extract, 2% bactopeptone, and 2% glucose) or in minimal glucose medium deficient for the required amino acids for plasmid selection using standard procedures (22Burke D. Dawson D. Stearns T. A Cold Spring Harbor Laboratory Course. Cold Spring Harbor Laboratory Press, New York2000Google Scholar). Growth curves were monitored in liquid-selective medium. After overnight growth, cultures were inoculated in fresh, prewarmed medium to an optical density of 0.2, and growth was followed over 12 h. Shown are mean values and S.E. as error bars of three experiments (Figs. 2A, 3C, 4C, and 5A). For the growth-plating assay, cells were grown until they reached the mid-log phase. Thereafter, cell densities were equalized, and 5 μl of cell suspension was spotted in 5-fold serial dilutions on appropriate selective media. The resulting plates were documented after 30 h of incubation at 30 °C.FIGURE 3Deletion of the proline-rich region in the amino-terminal region of mutant Htt bypasses the requirement of Hsp104 for aggregate formation. A, fluorescence microscopy of yeast cells of the indicated genotype expressing QP25–103 and Q25–103 derivatives. B, whole cell extracts of yeasts shown in A were analyzed by SDS-PAGE followed by immunoblots revealed with GFP (upper panel) or by staining with Coomassie Brilliant Blue (bottom panel). C, Q72, Q103, or empty vector does not affect Δhsp104 cell growth. OD, optical density.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Reintroduction of Hsp104 in hsp104-deleted cells restores the shape of Q47, Q72, and Q103 aggregates observed in wild-type cells. A, fluorescence microscopy of yeast cells of the indicated genotype expressing Q25–103 derivatives. B, whole cell extracts of yeasts shown in A were analyzed by SDS-PAGE followed by immunoblots revealed with GFP (upper panel) or Hsp104 antibody or by staining with Coomassie Brilliant Blue (bottom panel). C, Q72, Q103, or empty vector does not affect (Δhsp104+Hsp104) cell growth. OD, optical density. D, sucrose gradient fractionation of whole cell lysates of yeast Δhsp104 cells, overexpressing Hsp104 or not, together with Q72. The panels show immunoblots of sucrose gradient fractions revealed with anti-GFP antibody and quantification of SDS-insoluble material (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Overexpression of the chaperones Hsp104 or Ssa1 suppresses Q103 toxicity, alters inclusion shape, and aggregates sedimentation. Growth (A) and photomicrographs (B) of yeast cells expressing Q103 together with the indicated chaperones. C, fractionation of poly(Q) aggregates on sucrose gradient from extracts of yeast cells expressing QP72 and Q72 together with the indicated chaperones or empty vector.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Plasmids—QP25–103 constructs encoding the first exon of Htt fused to GFP where poly(Q) of various lengths are encoded by CAG/CAA alternate codons are described in Ref. 23Kazantsev A. Preisinger E. Dranovsky A. Goldgaber D. Housman D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11404-11409Crossref PubMed Scopus (392) Google Scholar and were PCR-subcloned into the centromeric plasmid p414GPD, which carries the glycerol 3-phosphate dehydrogenase, GPD promoter, and a TRP1 selection marker. Deletion of the prolinerich region in Htt was introduced in the QP25-QP103 constructs by PCR. 5′ and 3′ fragments flanking the proline-rich encoding sequence were generated by PCR using two sets of oligonucleotides and sewed and were as follows: Set A, 5′-GAA TTC ATG GCG ACC CTG GAA AAG C-3′ and 5′-CTC AGC CAC AGC TGG GCC CGG TTG TTG CTG TTG CTG-3′; Set B, 5′-GGCCCA GCTGTG GCTGAG-3′ and 5′-CCG CTC GAG CTT GTA CAG CTC GTC CAT G-3′. The resulting PCR products encoding Q25–103 were cloned into p414GPD. Each construct was sequenced on both strands. Hsp104 was subcloned from pFL44L Hsp104 (24Chacinska A. Szczesniak B. Kochneva-Pervukhova N.V. Kushnirov V.V. Ter-Avanesyan M.D. Boguta M. Curr. Genet. 2001; 39: 62-67Crossref PubMed Scopus (45) Google Scholar) using the NruI and XhoI site into the centromeric plasmid p416GPD.The gene encoding Ssa1 was cloned by PCR amplification on genomic DNA of the W303 strain using oligonucleotides flanked by convenient restriction sites to allow cloning into the p416GPD vector. Cloning was verified by DNA sequencing.Fluorescence Microscopy—Exponentially growing cells were used for fluorescence microscopy study, and micrographs were taken at 100× magnification.Protein Extraction, Immunoblots, and Antibodies—Yeast cells of a 20-ml exponentially growing culture were harvested by centrifugation at 3000 revolutions/min for 5 min at 4 °C. Cells were resuspended in 300 μl of spheroplasting buffer (1 m sorbitol, 10 mm MgCl2, 6.5 mg/ml zymolyase 20T (ICN Biochemicals, Inc.), 1 mm dithiothreitol, 50 mm Tris-HCl pH 7.5) and incubated for 1 h at 30°C. Spheroplasts were harvested by centrifugation at 3000 revolutions/min for 5 min at 4 °C and lysed in 1 ml of Laemmli buffer, 18 μl of which was analyzed on SDS-PAGE followed by a GFP immunoblot. Immunoblotting procedure was as described in (25Rousseau E. Dehay B. Ben-Haiem L. Trottier Y. Morange M. Bertolotti A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9648-9653Crossref PubMed Scopus (40) Google Scholar). Equal loading of whole yeast cell extracts analyzed by immunoblot was verified by Coomassie Brilliant Blue staining of 5 μl of extracts separated by SDS-PAGE. Anti-Hsp104 antibody was from Stressgen and anti-GFP antibody from BD Biosciences. Chemiluminescent images were acquired using the Chemi-smart 5000 allowing quantitative detection of chemiluminescence and signals of interest were quantified using Bio-1D software (Vilber Lourmat).Formic Acid Solubilization of Insoluble Pellets from Yeast Cells—10 ml of exponentially growing culture were harvested by centrifugation at 3000 revolutions/min for 5 min at 4 °C. Cells were resuspended in 300 μl of lysis buffer (50 mm Tris pH 7.5, 100 mm EDTA, 10 mm KCl, 1 mm dithiothreitol, 1% Triton, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 μg/ml aprotinin, and 1mm phenylmethylsulfonyl fluoride), lysed on ice for 1 h, and disrupted by 4 min of vortexing with 425–600-μm acid-washed glass beads. Cell debris were removed by centrifugation at 3000 revolutions/min for 1 min. Ultracentrifugation of 200 μl of extract at 355,000 × g at 4 °C for 1 h with a TLA120.2 rotor and an Optima Max ultracentrifuge (Beckman Coulter) was used to separate soluble and insoluble material. The resulting ultracentrifugation pellets were washed twice with phosphate-buffered saline and resuspended in 200 μl of formic acid at 37 °C for 1 h. Suspensions in formic acid were vacuum-dried and resuspended in 200 μl of Laemmli buffer. 18 μl of supernatant or solubilized pellet fraction was analyzed on SDS-PAGE followed by a GFP immunoblot.Sucrose Velocity Gradient Sedimentation—200 μl of yeast protein extracts were loaded onto a 4-ml linear 30–80% sucrose gradient and centrifuged for 15 h at 194,000 × g at 18 °C using a SW40Ti rotor (Beckman Coulter) and a L-70 Beckman ultracentrifuge. Twenty-eight fractions of 150 μl were collected in each gradient from top (fraction 1) to bottom (fraction 28) (Figs. 2C and 4D), 15 μl of which were analyzed by SDS-PAGE followed by immunoblots. Quantifications are presented as percentage of total amount of aggregated proteins loaded on the gradient.RESULTSTo study poly(Q) aggregation in yeast, we used the aminoterminal region of Htt corresponding to exon 1, which encompasses the proline-rich region and a poly(Q) stretch with either 25 (wild type), 47, 72, or 103 (mutant) glutamines fused to GFP (Fig. 1A). When Htt constructs were expressed in yeast cells, intracellular distribution of GFP fusion protein appeared as previously reported (10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar). The fluorescence in cells expressing QP25 is always diffusely distributed, whereas cells expressing QP47, -72, and -103 contain bright fluorescent foci, presumably corresponding to aggregated protein, which size augments with the length of the poly(Q) repeat (Fig. 1B). Deleting the prolinerich region in the wild-type Htt exon1, thereby generating Q25, did not alter the distribution of the protein (Fig. 1, A and B). However, when Q47, Q72, and Q103, corresponding to mutant Htt-exon1-GFP proteins lacking the proline-rich region fused to GFP (Fig. 1A), were expressed in yeast, cells contained numerous small foci (Fig. 1B). Foci formed by Q47–103 were swarming in contrast to the aggregates of QP47–103, which did not move within the cell (data not shown). Deleting the prolinerich region in Htt derivatives with expanded poly(Q) repeats increased the number of foci formed in yeast cells, although reducing their size. We next assessed the ratio of soluble and insoluble poly(Q)-containing proteins on total SDS lysates analyzed by SDS-PAGE followed by immunoblots (Fig. 1C). Although soluble proteins migrate in SDS-PAGE, SDS-insoluble aggregate poly(Q) are retained at the top of the gel (4Scherzinger E. Lurz R. Turmaine M. Mangiarini L. Hollenbach B. Hasenbank R. Bates G.P. Davies S.W. Lehrach H. Wanker E.E. Cell. 1997; 90: 549-558Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). Proteins with 25Q were completely soluble. Although QP47 formed foci visible by microscopy, insoluble poly(Q) were hardly detected in extracts of cells expressing QP47 (Fig. 1C). In contrast, insoluble Q47 proteins were readily detectable by immunoblot (Fig. 1C). Similarly, the fraction of SDS-insoluble aggregates was larger in cells expressing Q72 or Q103 than in cells expressing QP72 and QP103, respectively (Fig. 1C). This result is consistent with the fact that all of the fluorescence emanating from Q72 and Q103 is in small foci, and there is almost no trace of diffuse fluorescence (Fig. 1B). Expanded poly(Q) aggregates are sometimes difficult to transfer on immunoblot. Increasing amounts of cell lysates were analyzed by immunoblot, and both the soluble and insoluble proteins were quantified (supplemental Fig. 1). This analysis demonstrates that our experimental conditions allow a quantitative detection of soluble and insoluble poly(Q)-containing proteins. To further address whether an experimental bias might impair the conclusiveness of the experiments, proteins of cells expressing QP25, -72, -103 or Q25, -72, -103 were extracted and fractionated in soluble and insoluble fractions. The pellets, containing insoluble material, were dissolved by formic acid (26Hazeki N. Tukamoto T. Goto J. Kanazawa I. Biochem. Biophys. Res. Commun. 2000; 277: 386-393Crossref PubMed Scopus (78) Google Scholar) and subjected to immunoblot. Proteins with 25 glutamines were exclusively detected in the soluble fraction. In contrast, QP72 partitioned equally between the soluble and insoluble fraction (Fig. 1D). Deletion of the proline-rich region in QP72 reduced the solubility of the protein, as Q72 was almost exclusively detected in the solubilized pellet, in good agreement with Fig. 1C. Moreover, this analysis also indicates that QP72 and Q72 were expressed at comparable levels. There was also more Q103 in the insoluble fraction than QP103. However, the effect of the proline-rich region on the aggregation of expanded poly(Q) were more pronounced with proteins of 47 and 72 glutamines than 103. Growth of various strains was monitored both on liquid media and in spotting assays (Fig. 2, A and B). Although cells expressing different QP proteins or empty vector have a generation time of 3 h (Fig. 2, A and B, and data not shown), indicating that these expanded poly(Q)-containing proteins are not toxic in yeast as previously reported (6Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7841-7846Crossref PubMed Scopus (537) Google Scholar, 10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar), we noticed that expression of Q72 and Q103 retarded cell growth with a magnitude that increased with the length of poly(Q) (Fig. 2, A and B). Cells expressing Q72 or Q103 have a generation time of 4 or 9.6 h, respectively. This result indicated that cells expressing Q72 or Q103 have a marked growth defect. While this manuscript was under revision, the group of S. Lindquist (27Duennwald M.L. Jagadish S. Muchowski P.J. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11045-11050Crossref PubMed Scopus (230) Google Scholar) reported observations that support our conclusions. To further characterize the differences between the toxic Q72 and non-toxic QP72 proteins, total cell lysates were fractionated on sucrose gradients (28Schrodel A. de Marco A. BMC Biochem. 2005; 6: 10Crossref PubMed Scopus (110) Google Scholar). This biochemical analysis indicated that QP72 aggregates are heterogeneous and sediment in fractions ranging from 9 to 19 in contrast to the vast majority of Q72 aggregates fractionating in fractions 9–11 (Fig. 2C). Thus, the proline-rich region in Htt has a dramatic effect on the physical properties of aggregates in yeast, and its absence converts a benign protein into a toxic one.FIGURE 1Deletion of the proline-rich region in the amino-terminal region of Htt alters aggregation of expanded poly(Q) in yeast. A, schematic illustrating Htt derivatives corresponding to exon1 with or without the whole proline-rich region that is fused to GFP. B, photomicrographs of wild-type yeast cells expressing Htt derivatives of various poly(Q) lengths (25–103) containing (QP) or lacking the proline-rich region (Q) fused to GFP. C, GFP immunoblot (upper panel) and Coomassie Brilliant Blue staining (CBB, bottom panel) of whole cell lysates of yeasts shown in A. D, yeast whole cell lysates were centrifuged to separate soluble (left panel) from insoluble (right panel) material and revealed by immunoblot. SDS-insoluble pellets were solubilized using formic acid (FA) prior to immunoblot (right panel). SN, supernatant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Altering the levels of yeast chaperones modulates the extent of poly(Q) aggregation in yeast (6Muchowski P.J. Schaffar G. Sittler A. Wanker E.E. Hayer-Hartl M.K. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7841-7846Crossref PubMed Scopus (537) Google Scholar, 10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar, 11Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (295) Google Scholar, 12Schaffar G. Breuer P. Boteva R. Behrends C. Tzvetkov N. Strippel N. Sakahira H. Siegers K. Hayer-Hartl M. Hartl F.U. Mol. Cell. 2004; 15: 95-105Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 13Gokhale K.C. Newnam G.P. Sherman M.Y. Chernoff Y.O. J. Biol. Chem. 2005; 280: 22809-22818Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Among the yeast chaperones, Hsp104 has a remarkable effect, as its deletion abolishes expanded poly(Q) aggregation (10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar). Therefore, we sought to determine whether hsp104 deletion affects aggregation of expanded poly(Q) proteins lacking the proline-rich region. QP25–103 and Q25–103 proteins were expressed in hsp104 deletion strain and analyzed by fluorescent microscopy (Fig. 3A). As previously reported (10Krobitsch S. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1589-1594Crossref PubMed Scopus (446) Google Scholar), the absence of Hsp104 impedes the formation of aggregates of proteins containing up to 103 glutamines (Fig. 3A). Surprisingly, Htt derivatives with 47-, 72-, and 103Q lacking the proline-rich region formed a large and often unique focus in a substantial number of hsp104 deletion cells that were comparable in size to QP47, -72, and -103 formed in wild-type cells based on microscopic observation (Figs. 1A and 3A). Having shown in Fig. 1D and in supplemental Fig. 1 that our immunoblots can be used quantitatively to assess the level of aggregated or soluble expanded poly(Q) derivatives, Q47, Q72, and Q103 in the hsp104 deletion strain were analyzed on total protein extracts by immunoblots. No insoluble poly(Q) proteins were detected on immunoblots of total cell lysates from cells expressing QP47 and QP72, and only a trace of insoluble QP103 was formed in the hsp104 deletion strain (Fig. 3B). In contrast, traces of insoluble Q47 proteins were detected, and insoluble Q72 and Q103 were readily detected in the hsp104 deletion strain (Fig. 3B). Thus, deletion of the proline-rich region in the amino-terminal region of mutant Htt bypassed the requirement of Hsp104 for aggregate formation in a substantial number of yeast cells. Strikingly, deletion of hsp104 in cells expressing Q72 and Q103 suppressed the growth defects observed in wild-type cells expressing Q72 and Q103 (Fig. 3C). On sucrose gradients, Q72 aggregates formed in the hsp104 deletion strain exhibited a profile similar to that of QP72 expressed in the wild-type strain (Fig. 4D). Thus Q72 expressed in the hsp104 deletion strain resembled QP72 in wild-type cells, as they exhibited similar sedimentation properties and do not compromise cell growth.We next examined whether reintroducing Hsp104 affects aggregation in cells where poly(Q) aggregates were formed in the absence of this chaperone. A substantial number of cells deleted for hsp104 and expressing Q47, Q72, and Q103 contained a single bright fluorescent focus, whereas the others exhibited diffuse fluorescence, presumably corresponding to soluble proteins (Fig. 4A). Strikingly, reintroducing Hsp104 provoked the formation of numerous small swarming foci in virtually each cell expressing Q47, Q72, and Q103, whereas diffuse fluorescence disappeared (Fig. 4A). These data suggest that Hsp104 rescued the formation of aggregates whose shape and number were comparable with those formed in wild-type cells (Figs. 4A and 1B). In contrast, aggregation of QP103 is irreversibly lost upon deletion of hsp104 (Ref. 11Meriin A.B. Zhang X. He X. Newnam G.P. Chernoff Y.O. Sherman M.Y. J. Cell Biol. 2002; 157: 997-1004Crossref PubMed Scopus (295) Google Scholar and data not shown). Cell extracts were then analyzed by SDS-PAGE followed by immunoblot and revealed that reintroduction of Hsp104 in hsp104-deleted cells dramatically augments the amounts of insoluble Q47, Q72, and Q103 to levels similar to those observed in wild-type cells, while reducing the quantity of soluble Q72 and Q103 (Fig. 4B). However, Q72 and Q103 were no longer toxic in (Δhsp104+Hsp104) cells (Fig. 4C). This indicated that, despite similar aggregate shape and a similar ratio of soluble versus insoluble protein, the properties of the poly(Q)-containing protein were dramatically different in distin

The proteasome is essential for the selective degradation of most cellular proteins. To survive overwhelming demands on the proteasome arising during environmental stresses, cells increase proteasome abundance. Proteasome assembly is known to be complex. How stressed cells overcome this vital challenge is unknown. In an unbiased suppressor screen aimed at rescuing the defects of a yeast Rpt6 thermosensitive proteasome mutant, we identified a protein, hereafter named Adc17, as it functions as an ATPase dedicated chaperone. Adc17 interacts with the amino terminus of Rpt6 to assist formation of the Rpt6-Rpt3 ATPase pair, an early step in proteasome assembly. Adc17 is important for cell fitness, and its absence aggravates proteasome defects. The abundance of Adc17 increases upon proteasome stresses, and its function is crucial to maintain homeostatic proteasome levels. Thus, cells have mechanisms to adjust proteasome assembly when demands increase, and Adc17 is a critical effector of this process.

Cells and organisms have evolved numerous mechanisms to cope with and to adapt to unexpected challenges and harsh conditions. Proteins are essential to perform the vast majority of cellular and organismal functions. To maintain a healthy proteome, cells rely on a network of factors and pathways collectively known as protein quality control (PQC) systems, which not only ensure that newly synthesized proteins reach a functional conformation but also are essential for surveillance, prevention, and rescue of protein defects. The main players of PQC systems are chaperones and protein degradation systems: the ubiquitin-proteasome system and autophagy. Here we provide an integrated overview of the diverse PQC systems in eukaryotic cells in health and diseases, with an emphasis on the key regulatory aspects and their cross talks. We also highlight how PQC regulation may be exploited for potential therapeutic benefit.

Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases.

Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.

Organisms have evolved mechanisms to cope with and adapt to unexpected challenges and harsh conditions. Unfolded or misfolded proteins represent a threat for cells and organisms, and the deposition of misfolded proteins is a defining feature of many age-related human diseases, including the increasingly prevalent neurodegenerative diseases. These protein misfolding diseases are devastating and currently cannot be cured, but are hopefully not incurable. In fact, the aggregation-prone and potentially harmful proteins at the origins of protein misfolding diseases are expressed throughout life, whereas the diseases are late onset. This reveals that cells and organisms are normally resilient to disease-causing proteins and survive the threat of misfolded proteins up to a point. This Commentary will outline the limits of the cellular resilience to protein misfolding, and discuss the possibility of pushing these limits to help cells and organisms to survive the threat of misfolding proteins and to avoid protein quality control catastrophes.

Reversible phosphorylation of proteins is a post-translational modification that regulates all aspect of life through the antagonistic action of kinases and phosphatases. Protein kinases are well characterized, but protein phosphatases have been relatively neglected. Protein phosphatase 1 (PP1) catalyzes the dephosphorylation of a major fraction of phospho-serines and phospho-threonines in cells and thereby controls a broad range of cellular processes. In this review, I will discuss how phosphatases were discovered, how the view that they were unselective emerged and how recent findings have revealed their exquisite selectivity. Unlike kinases, PP1 phosphatases are obligatory heteromers composed of a catalytic subunit bound to one (or two) non-catalytic subunit(s). Based on an in-depth study of two holophosphatases, I propose the following: selective dephosphorylation depends on the assembly of two components, the catalytic subunit and the non-catalytic subunit, which serves as a high-affinity substrate receptor. Because functional complementation of the two modules is required to produce a selective holophosphatase, one can consider that they are split enzymes. The non-catalytic subunit was often referred to as a regulatory subunit, but it is, in fact, an essential component of the holoenzyme. In this model, a phosphatase and its array of mostly orphan substrate receptors constitute the split protein phosphatase system. The set of potentially generalizable principles outlined in this review may facilitate the study of these poorly understood enzymes and the identification of their physiological substrates.

In Ewing tumor, the (11;22) chromosomal translocation produces a chimeric molecule composed of the amino-terminal domain of EWS fused to the carboxyl-terminal DNA-binding domain of FLI-1. Previously, we have identified a novel protein TAFII68, which is highly similar to EWS and another closely related protein TLS (also called FUS). We demonstrate that the N-terminus of TAFII68 efficiently stimulates transcription when fused to two different DNA binding domains and that overexpression of TAFII68-FLI-1 chimeras in NIH3T3 cells leads to oncogenic transformation. We have also investigated the molecular mechanisms which could account for the transcriptional activation and the oncogenic transformation potential of the N-termini of TAFII68 and EWS. Thus, we have tested whether the artificial recruitment of components of the preinitiation complex (PIC) or a histone acetyltransferase (HAT) could bypass the requirement for the activation domains of either EWS or TAFII68. Recruitment of individual components of the transcription machinery or the GCN5 HAT is not sufficient to promote activation from FLI-1 responsive genes either in transfection experiments or in oncogenic transformation assays. These results suggest that the TAFII68 or EWS activation domains enhance a step after PIC formation in the transcriptional activation process.

Selective and reversible phosphorylation is one of the most common post‐translational modifications of proteins. Although kinase inhibitors are popular in drug development programmes, selective pharmacological manipulation of phosphatase activity has been challenging to achieve. We review recent advances in the development of selective inhibitors of dephosphorylation events and discuss the potential applications of small‐molecule phosphatase inhibitors.

Caroline Sibilla and Anne Bertolotti MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom Correspondence: aberto{at}mrc-lmb.cam.ac.uk

The deposition of proteins of abnormal conformation is one of the major hallmarks of the common neurodegenerative diseases including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, frontotemporal dementia, and prion diseases. Protein quality control systems have evolved to protect cells and organisms against the harmful consequences of abnormally folded proteins that are constantly produced in small amounts. Mutations in rare inherited forms of neurodegenerative diseases have provided compelling evidence that failure of protein quality control systems can drive neurodegeneration. With extensive knowledge of these systems, and the notion that protein quality control may decline with age, many laboratories are now focussing on manipulating these evolutionarily optimized defence mechanisms to reduce the protein misfolding burden for therapeutic benefit.

Aggregation of misfolded proteins is a characteristic of several neurodegenerative diseases. The huntingtin amino-terminal fragment with extended polyglutamine repeat forms aggregates closely associated with chaperones both in the cytoplasm and the nucleus. Because each cellular compartment contains distinct chaperones and because the molecular mechanisms controlling polyglutamine aggregation are largely unknown, we decided to investigate the influence of different cellular environments on the aggregation of this pathological protein. Here, we show that aggregation of a protein containing a polyglutamine stretch of pathological length is abolished when its expression is targeted to the endoplasmic reticulum. Once retrogradely transported outside the endoplasmic reticulum, the aggregation-prone polyglutamine-containing protein recovers its ability to aggregate. When expressed in the mitochondria, a protein containing 73 glutamines is entirely soluble, whereas the nucleocytosolic equivalent has an extremely high tendency to aggregate. Our data imply that polyglutamine aggregation is a property restricted to the nucleocytosolic compartment and suggest the existence of compartment-specific cofactors promoting or preventing aggregation of pathological proteins.

Deposition of misfolded proteins with a polyglutamine expansion is a hallmark of Huntington disease and other neurodegenerative disorders. Impairment of the proteolytic function of the proteasome has been reported to be both a cause and a consequence of polyglutamine accumulation. Here we found that the proteasomal chaperones that unfold proteins to be degraded by the proteasome but also have non-proteolytic functions co-localized with huntingtin inclusions both in primary neurons and in Huntington disease patients and formed a complex independently of the proteolytic particle. Overexpression of Rpt4 or Rpt6 facilitated aggregation of mutant huntingtin and ataxin-3 without affecting proteasomal degradation. Conversely, reducing Rpt6 or Rpt4 levels decreased the number of inclusions in primary neurons, indicating that endogenous Rpt4 and Rpt6 facilitate inclusion formation. In vitro reconstitution experiments revealed that purified 19S particles promote mutant huntingtin aggregation. When fused to the ornithine decarboxylase destabilizing sequence, proteins with expanded polyglutamine were efficiently degraded and did not aggregate. We propose that aggregation of proteins with expanded polyglutamine is not a consequence of a proteolytic failure of the 20S proteasome. Rather, aggregation is elicited by chaperone subunits of the 19S particle independently of proteolysis.

Article1 March 2018free access Source DataTransparent process Prion-like protein aggregates exploit the RHO GTPase to cofilin-1 signaling pathway to enter cells Zhen Zhong MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Laura Grasso MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Caroline Sibilla MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Tim J Stevens MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nicholas Barry MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Anne Bertolotti Corresponding Author [email protected] orcid.org/0000-0002-9185-0558 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Zhen Zhong MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Laura Grasso MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Caroline Sibilla MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Tim J Stevens MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Nicholas Barry MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Anne Bertolotti Corresponding Author [email protected] orcid.org/0000-0002-9185-0558 MRC Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Author Information Zhen Zhong1, Laura Grasso1,‡, Caroline Sibilla1,‡, Tim J Stevens1, Nicholas Barry1 and Anne Bertolotti *,1 1MRC Laboratory of Molecular Biology, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1223 267054; E-mail: [email protected] EMBO J (2018)37:e97822https://doi.org/10.15252/embj.201797822 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein aggregation is a hallmark of diverse neurodegenerative diseases. Multiple lines of evidence have revealed that protein aggregates can penetrate inside cells and spread like prions. How such aggregates enter cells remains elusive. Through a focused siRNA screen targeting genes involved in membrane trafficking, we discovered that mutant SOD1 aggregates, like viruses, exploit cofilin-1 to remodel cortical actin and enter cells. Upstream of cofilin-1, signalling from the RHO GTPase and the ROCK1 and LIMK1 kinases controls cofilin-1 activity to remodel actin and modulate aggregate entry. In the spinal cord of symptomatic SOD1G93A transgenic mice, cofilin-1 phosphorylation is increased and actin dynamics altered. Importantly, the RHO to cofilin-1 signalling pathway also modulates entry of tau and α-synuclein aggregates. Our results identify a common host cell signalling pathway that diverse protein aggregates exploit to remodel actin and enter cells. Synopsis Protein aggregates associated with neurodegenerative diseases can penetrate cells and spread like prions. Results from an RNAi screen show that pathological protein aggregates exploit signaling and cytoskeleton-remodeling pathways also used by viruses to enter host cells. Mutant SOD1 protein aggregates use cofilin-1 to remodel actin and invade host cells. Signaling through RHO GTPase, ROCK1 and LIMK1 to cofilin-1 remodels actin to facilitate aggregate entry. Cofilin-1 activity is altered in the spinal cord of SOD1G93A transgenic mice. Tau and α-synuclein aggregates also exploit RHO GTPase signaling to cofilin-1 to enter cells. Introduction The deposition of proteins of abnormal conformation is at the origin of a broad range of human age-related diseases including the devastating neurodegenerative diseases Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), amyotrophic lateral sclerosis (ALS), dementia and prion diseases (Soto, 2003). Prion diseases were traditionally thought to be unique because the misfolded protein in this group of diseases, PrP, is infectious (Prusiner, 1998). A prion is a proteinaceous infectious particle which can propagate its misfolded and pathogenic conformation by corrupting the normally soluble host protein. In the recent years, an explosion of studies has challenged our views on neurodegenerative diseases. In fact, the diverse protein aggregates that characterize neurodegenerative diseases share the defining properties of prions and can spread their abnormal conformation like prions, where seeds made of protein aggregates can induce aggregation of the soluble counterpart in a host (Brundin et al, 2010; Frost & Diamond, 2010; Jucker & Walker, 2011; Münch & Bertolotti, 2012; Prusiner, 2012). Therefore, knowledge on the extremely rare prion diseases has opened up new avenues for the understanding of more common neurodegenerative diseases such as AD or ALS. Amongst the diverse neurodegenerative diseases, ALS is an attractive model to study the propagation of protein aggregates because its defining clinical features are reminiscent of prion diseases. Like prion diseases, ALS is a devastating and rapidly progressive neurodegenerative disease. ALS manifests by muscle weakness, which always begins at a focal point, but the body region involved is highly variable between individuals. The symptoms then spread contiguously to adjacent regions by an orderly process (Gowers, 1892; Ravits & La Spada, 2009). The spreading of SOD1 aggregates from cell to cell by a prion-like mechanism, which has not only been observed in cells (Grad et al, 2011, 2014; Münch et al, 2011) but also in mice (Ayers et al, 2014, 2015; Thomas et al, 2017), could explain the defining pathological hallmarks of ALS. With this model, the site of disease corresponds to the misfolding of the disease-causing protein and the spreading of the symptoms results from the spreading of the SOD1 prion. Numerous studies have demonstrated the propagation of diverse disease-related protein aggregates in animal models (Sibilla & Bertolotti, 2017). However, the mechanisms underlying these properties are largely unknown. Cellular models recapitulating the propagation of protein aggregates are essential to conduct mechanistic studies. In most cases, the protein aggregates associated with neurodegenerative diseases are intracellular and replicate inside cells. Therefore, amongst the diverse steps of the prion cycle, the entry of protein aggregates into their host is a particularly important one because it is a first step in a pathological cascade, and so far, this process is ill-defined. We have previously shown that SOD1 aggregates enter cells by macropinocytosis (Münch et al, 2011), a route later found to be also exploited by tau aggregates (Holmes et al, 2013). Here, we aimed at identifying cellular factors involved in aggregate entry. Knowing that aggregates enter cells by macropinocytosis, a poorly defined endocytic pathway, we designed a targeted siRNA screen using a membrane trafficking siRNA library to identify host cell factors involved in aggregate entry, with the hope to provide some mechanistic insights on this elusive prion-like cycle. Our work identifies a critical cellular factor, cofilin-1 required to remodel cortical actin, enabling entry of diverse protein aggregates. Upstream of cofilin-1 and controlling aggregate entry is the RHO-ROCK1-LIMK1 signalling pathway. These findings reveal host cellular factors critical for aggregate entry. Remarkably, cofilin-1 is also exploited by some viruses to enter cells, thereby highlighting an analogy between entry of protein aggregates and viruses. Results An siRNA screen identifies modifiers of mutant SOD1 aggregate uptake We previously found that SOD1 aggregates enter cells by macropinocytosis (Münch et al, 2011), a finding confirmed by others (Zeineddine et al, 2015) and relevant to diverse disease-causing aggregates (Holmes et al, 2013). However, beyond the knowledge that aggregates transit through vesicles to enter cells, the cellular factors involved in this process are unknown. To shed light on this process, we conducted a screen with a membrane trafficking siRNA library. We confirmed that, in our cellular system, aggregates penetrated inside cells using high-resolution confocal microscopy (Movie EV1). Aggregate uptake was quantified by high-throughput and automated image analysis in human 293T cells using high-content microscopy, following reverse transfection with pools of four siRNAs per gene prior to Dylight-650-labelled mutant SOD1 aggregates inoculation (Figs 1A and EV1A and B and Materials and Methods). The technical reproducibility of four independent screens allowed us to combine the different dataset, and hits were ranked according to the combined Z-score of all experimental repeats (Figs EV2A and B, and 1B). The top hit of the screen was PICALM (Fig EV2B), a clathrin-coated-pit adaptor protein homologous to AP180, which we previously reported (Münch et al, 2011). Knockdown of PICALM increased SOD1 aggregate uptake indirectly by the following mechanism: it decreases clathrin-mediated endocytosis, to which cells compensate by increasing other endocytic routes, in turn resulting in increasing SOD1 aggregate uptake (Münch et al, 2011). The presence of PICALM in the screen provided confidence that the siRNA screen identified relevant hits. This also confirms our previous findings showing that clathrin-mediated endocytosis does not mediate SOD1 aggregate entry (Münch et al, 2011). Figure 1. An unbiased high-content screen identifies modifiers of aggregate entry Scheme representing the experimental workflow. Combined Z-scores for each siRNA knockdown or mock transfection, presented as a scatter plot with the x-axis indicating the screening well. Z-score points representing hits above 2.0 are coloured in orange and below −2 in blue. Horizontal lines illustrate the Z-score threshold (± 3.8) used for the secondary screen, selecting 15 of the most significant candidate genes. Flow cytometry analysis of 293T cells 16 h after inoculation with 0.8 μM Dylight-650-SOD1H46R aggregates. Cells were transfected with the indicated siRNA or control (CTRL) siRNA 3 days before their inoculation with aggregates. Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample (n = 2–5). Flow cytometry analysis of SK-N-AS cells 1 h after inoculation with 0.8 μM Dylight-650-SOD1H46R aggregates and 3 days after transfection with the indicated siRNA. Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample (n = 3–10). Data information: Data are means ± SEM. *P ≤ 0.05, **P ≤ 0.01. ***P ≤ 0.001. ****P ≤ 0.0001. Results were analysed by ordinary one-way ANOVA followed by multiple comparisons. Source data are available online for this figure. Source Data for Figure 1 [embj201797822-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Representative images of the siRNA screen Representative primary image samples extracted from the siRNA screen with the Nikon high-content microscope (20× 0.75 NA objective). Upper row: Subregion taken from a three-channel image of 293T cells treated for 16 h with Dylight-650-labelled SOD1 aggregates. The sample was co-stained with H33342 (blue) to reveal nuclei and CellMask (green) to reveal plasma membrane. Lower row: results of image segmentation using the Nikon NIS-Elements general analysis. Aggregates within cells are identified as green dots. CellMask reveals the outline of the plasma membrane artificially coloured as purple boundary. Yellow dots are rejected spots, being either aggregates outside of cells or containing saturated pixels. Red circle lines delineate the cell nuclei. Scale bar: 50 μm. High-content images of 293T cells 3 days after transfection with control (CTRL) siRNA, PCALM siRNA or RAB5C siRNA and stained with H33342 (blue) to reveal nuclei, CellMask green plasma membrane stain (green). Cells were fixed and imaged after 16 h flowing inoculation with Dylight-650-labelled SOD1 aggregates (red). Scale bar: 50 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Overview of siRNA screen procedure Upper panel: Colour matrix representing the combined Z-score (protein aggregates per cell normalized to control) for each siRNA knockdown reconstructed at original well-plate grid locations. Summary of siRNA screening results for the top 15 genes. Hits are ranked according to the absolute value of the combined Z-scores (screens 1–4), normalized from average of experimental replicates (screen 1, screen 2, screen 3 and screen 4). P-value is the estimated probability of obtaining a value as combined Z-scores; the cell count corresponds to the mean of cell count Z-scores from four independent biological experiments. Overview of the validation strategy of siRNA screen. Number of individual siRNA (out of 4) per hit modifying SOD1 aggregate entry in secondary screen. Download figure Download PowerPoint We next performed a secondary validation screen for the top 15 genes (Z-score > 3.8 or < −3.8) using stringent validation criteria previously used in an siRNA screen for HIV restriction factors (Liu et al, 2011) (Fig EV2C and D). 9 out of 15 genes passed the secondary screen. The hits were next tested in an orthogonal experimental set-up, measuring aggregate uptake by flow cytometry, as described (Münch et al, 2011). First, we confirmed that the method enabled the detection of internalized aggregates (Fig EV3), as previously reported (Münch et al, 2011). This second validation yielded five genes: cofilin-1, RHO-associated protein kinase 1 (ROCK1), the Ras-related proteins RAB5C, RAB10 and sorting nexin-1 (SNX1) (Figs EV2C and D, and 1C). We also confirmed that individual siRNA against CFL1, ROCK1, RAB5C, RAB10 as well as SNX1 efficiently reduced their respective mRNA levels (Fig EV4). Having performed the primary screen and validation in 293T cells, we next tested the different modifiers in a neuronal cell line. The relevance of the five validated modifiers of SOD1 aggregate uptake was confirmed in the neuronal cell line SK-N-AS (Figs 1D and EV5). We next performed additional controls to assess the selectivity of the hits identified. RAB5C is one of three isoforms of RAB5 and the siRNA screen selectively identified RAB5C as a modifier of SOD1 aggregate entry. We confirmed that this effect was indeed specific because knockdown of RAB5A or RAB5B did not affect SOD1 aggregate uptake (Fig EV6). These results validate the screening method and identify modifiers of SOD1 aggregate uptake. Click here to expand this figure. Figure EV3. Confocal images showing SOD1 aggregates are inside cells after trypsinization and prior to FACS analysisSK-N-AS cells were trypsinized and fixed after 2 h following inoculation with Dylight-650-labelled SOD1 aggregates. Images were acquired with the Zeiss 710 confocal microscope (Carl Zeiss Ltd.) by 63× objective focusing on nuclei layer, to ensure that the images enable the visualization of the cellular content. Scale bar: 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. siRNA knockdown of the five modifiers isolated in the screenmRNA levels measured by quantitative RT–PCR 2 days after transfection with the indicated siRNA. Data are means ± SEM (n ≥ 2). Ordinary one-way ANOVA followed by multiple comparisons. ****P ≤ 0.0001. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Modifiers of SOD1 aggregate entryFlow cytometry analysis of SK-N-AS cells 16 h after inoculation with 0.8 μM Dylight-650-SOD1H46R aggregates and 3 days after transfection with the indicated siRNA. Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample. Data are means ± SEM (n = 3). Ordinary one-way ANOVA followed by multiple comparisons. ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV6. SOD1 aggregate uptake following RAB5 knockdownFlow cytometry analysis of SK-N-AS cells 1 h after inoculation with 0.8 μM Dylight-650-SOD1H46R aggregates and 3 days after transfection with the indicated siRNA. Data are means ± SEM (n ≥ 2). Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample and analysed by ordinary one-way ANOVA followed by multiple comparisons. **P ≤ 0.01. Source data are available online for this figure. Download figure Download PowerPoint Signalling through RHO, ROCK1, LIMK1 to cofilin-1 regulates SOD1 aggregate entry into cells Remarkably, two of the top five hits, ROCK1 and cofilin-1, belonged to the same pathway. This provided strong evidence to suggest that the ROCK1-cofilin-1 pathway was important for aggregate uptake and to examine this pathway in depth. Cofilin-1 is an actin depolymerization factor, a crucial regulator of actin dynamics (Lappalainen & Drubin, 1997) whose activity is restricted by phosphorylation by the LIM-kinase 1(LIMK1) (Mizuno et al, 1998; Maekawa et al, 1999). LIMK1 is itself regulated by the kinases ROCK1 and RHO (Maekawa et al, 1999) (Fig 2A). Indeed, knockdown of ROCK1 decreased cofilin-1 phosphorylation (Fig 2B and C). Likewise, the RHO inhibitor CT04 as well as the ROCK1 inhibitor Y27632 also decreased cofilin-1 phosphorylation (Fig 2D–G). Conversely, overexpression of LIMK1 increased cofilin-1 phosphorylation (Fig 2H and I). Having found that cofilin-1 siRNA decreased aggregate uptake whilst ROCK1 siRNA increased aggregate uptake (Fig 1C and D), we next tested whether the diverse manipulations of the RHO to cofilin-1 signalling pathway described above affected aggregate uptake. We found that both the RHO inhibitor CT04 and the ROCK inhibitor Y27632 increased mutant SOD1 aggregate uptake (Fig 2J and K). Conversely, overexpression of LIMK1 decreased aggregate uptake (Fig 2L). These findings were validated in primary neurons where both RHO inhibition and ROCK inhibition increased mutant SOD1 aggregate uptake (Fig 2M). These results provide multiple independent lines of evidence establishing that a decrease in RHO signalling increases cofilin-1 activity to increase aggregate entry. Figure 2. Signalling from RHO to cofilin-1 controls aggregate entry into cells A. Cartoon depicting the RHO-ROCK1, LIMK1 signalling pathway to cofilin-1 (CFL1). B. Immunoblots of the indicated proteins in lysates of SK-N-AS cells 3 days after transfection with ROCK1 siRNA or control siRNA. C. Quantifications of triplicates of (B). The graph depicts levels of phospho-cofilin-1 (pCFL1) relative to total CFL1 in cells with ROCK1 siRNA knockdown compared with control siRNA treatment. Data are means ± SEM (n = 3). Unpaired t-test. Two-tailed **P = 0.0043. D. Immunoblots of the indicated proteins in lysates of SK-N-AS cells treated with CT04 or vehicle (CTRL). E. Quantifications of replicates of (D). Data are means ± SEM (n = 3). Unpaired t-test. Two-tailed ***P = 0.0003. F, G. Same as (D, E) with Y27632. Data are means ± SEM (n = 4). Unpaired t-test. Two-tailed **P = 0.0081. H. Immunoblots of the indicated proteins in lysates of SK-N-AS cells 2 days after transfection with pTGSH-LIMK1 or empty vector. I. Quantifications of replicates of (H). Data are means ± SEM (n = 4). Unpaired t-test. Two-tailed **P = 0.0021. J, K. Flow cytometry analysis of SK-N-AS cells 1 h after inoculation with 0.8 μM Dylight-650-SOD1H46R aggregates and following a 15 h treatment with 1 μg/ml CT04 (J) or 10 μM Y27632 (K) or vehicle (CTRL). Data are means ± SEM (n ≥ 3). Unpaired t-test. Two-tailed P-values are 0.0001 and 0.0097 for treatment with CT04 and Y27632, respectively. L. Flow cytometry analysis of aggregate uptake in SK-N-AS cells transfected with pTGSH-LIMK1 or empty vector as a control 2 days before inoculation with labelled SOD1 aggregates. Data are means ± SEM (n = 3). Unpaired t-test. Two-tailed **P = 0.0064. M. Flow cytometry analysis of aggregate uptake 1 h after inoculation of aggregates in primary neuron in the presence of 1 μg/ml CT04 or 10 μM Y27632 or vehicle (CTRL) added 1 h before aggregates. Data are means ± SEM (n = 3). Unpaired t-test. Two-tailed P-values are 0.0009 and 0.0254 for treatment with CT04 and Y27632, respectively. Data information: Representative results are shown in (B, D, F, H). Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample on a LSRFortessaTM (BD Biosciences) in (J–M). Source data are available online for this figure. Source Data for Figure 2 [embj201797822-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Remodelling of the cortical actin barrier through cofilin-1 is required for SOD1 aggregate entry To gain insights into the underlying mechanism by which cofilin-1 facilitates aggregate entry, we next examined actin dynamics, knowing that cofilin-1 is an actin depolymerizing factor. As expected (Mizuno, 2013), knockdown of cofilin-1 increased cortical actin, as revealed by phalloidin staining (Fig 3A) a finding confirmed by biochemical fractionation of F-actin and G-actin (Fig 3B and C). ROCK1 knockdown had the opposite effect on the F/G-actin ratio (Fig 3D and E) and aggregate uptake (Fig 1C and D). Both the RHO inhibitor CT04 and the ROCK inhibitor Y27632, which increased mutant SOD1 aggregate uptake (Fig 2J and K) and decreased cofilin-1 phosphorylation (Fig 2D–G), decreased F/G-actin ratio (Fig 3F and G). Jasplakinolide (Holzinger, 2009), which promotes actin polymerization, also reduced SOD1 aggregate uptake (Fig 3H and I). These results suggest that cortical actin forms a restriction barrier to aggregate entry and inhibition of RHO signalling promotes reorganization of cortical actin to facilitate aggregate entry (Fig 3J). Figure 3. Cofilin-1 controls cortical actin, a barrier to SOD1 aggregate entry A. Confocal images of 293T cells 3 days after transfection with cofilin-1 (CFL1) siRNA or control siRNA and stained with H33342 (blue) to reveal nuclei, anti-CFL1 antibody (green) and phalloidin (red) to reveal actin filaments. Scale bar: 10 μm. B. Fractionation of cell lysates followed by immunoblots to reveal F-actin, G-actin. SK-N-AS cells were transfected with control or CFL1 siRNA 3 days before the analysis. C. Quantifications of replicates of (B). The graph depicts levels of F-actin/G-actin ratio in cells transfected with or without CFL1 siRNA. Data are means ± SEM (n = 4). Unpaired t-test. Two-tailed **P = 0.0068. D, E. As in (B, C) except that cells were transfected with control or ROCK1 siRNA. Data are means ± SEM (n = 2). Unpaired t-test. Two-tailed **P = 0.0046. F. Fractionation of SK-N-AS cell lysates followed by immunoblots to reveal F-actin, G-actin. Before lysis, cells were treated for 16 h with 1 μg/ml CT04 or 10 μM Y27632. G. Quantification of replicated experiments as in (F). Data are means ± SEM (n = 4). Unpaired t-test. Two-tailed P-values are 0.0083 and 0.0043 for treatment with CT04 and Y27632, respectively. H. Immunoblots of F-actin, G-actin and total actin in fractionated lysates from SK-N-AS cells after a treatment with vehicle (DMSO) or 25 nM Jasplakinolide for 16 h. I. Flow cytometry analysis of aggregate entry, 1 h after inoculation of SK-N-AS cells with 0.8 μM Dylight-650-SOD1H46R aggregates following a treatment with vehicle (DMSO) or 25 nM Jasplakinolide for 16 h. Fluorescence intensity was measured by flow cytometry on 10,000 cells per sample (n = 2). Data are means ± SEM. Unpaired t-test. Two-tailed *P = 0.0155. J. Cartoon depicting the regulation of acting remodelling by the RHO to CFL1 pathway. Data information: Representative results are shown. (C, E, G, I) *P ≤ 0.05, **P ≤ 0.01. Source data are available online for this figure. Source Data for Figure 3 [embj201797822-sup-0005-SDataFig3.zip] Download figure Download PowerPoint To further investigate how alteration of cofilin-1 regulates aggregate entry, we monitored cofilin-1 activity following aggregate inoculation. Because cofilin-1 is regulated by phosphorylation (Mizuno et al, 1998; Maekawa et al, 1999), we examined the levels of phospho-cofilin-1 by immunoblots at different time after aggregate inoculation. The total cofilin-1 protein levels did not measurably vary following aggregate inoculation (Fig 4A). However, phosphorylation of cofilin-1 decreased between 10 and 60 min following aggregate inoculation (Fig 4A and B). Interestingly, following this initial decrease, phospho-cofilin-1 levels then increased 1 h after aggregate inoculation. This indicates that aggregates elicited a transient decrease in cofilin-1 phosphorylation, further supporting the notion that aggregates exploit cofilin-1 to enter cells and remodel actin. The increased cofilin-1 phosphorylation following an initial decrease indicates the existence of a negative feedback response where cells react to the initial alteration by increasing cofilin-1 phosphorylation. Indeed, cofilin-1 dephosphorylation leads to actin disassembly, which in turn increases cofilin-1 phosphorylation (Nagata-Ohashi et al, 2004; Soosairajah et al, 2005; Liu et al, 2015). Importantly, the changes in cofilin-1 phosphorylation were not observed when cells were inoculated with dextran (Mr ∼10,000) instead of SOD1 aggregates (Fig 4C and D), establishing the selective nature of the changes induced by SOD1 aggregates (Fig 4A and B). Bi-phasic changes in cofilin-1 phosphorylation as observed here (Fig 4A and B) is also a hallmark of HIV-1 infection (Yoder et al, 2008). The role of cofilin-1 in aggregate entry identified here reveals a mechanistic analogy between SOD1 aggregate entry and the entry of some viruses, which also exploits cofilin-1 to remodel actin and enter cells (Yoder et al, 2008; Xiang et al, 2012; Zheng et al, 2013). Figure 4. SOD1 aggregates transiently alter cofilin-1 phosphorylation in cells Immunoblots of the indicated proteins in lysates of SK-N-AS cells inoculated with SOD1 aggregates for the indicated time. Quantifications of triplicates of (A). The graph depicts the level of pCFL1 relative to total CFL1 during the SOD1 aggregates inoculation in SK-N-AS cells. SK-N-AS cells inoculated with dextran (Mr 10,000) for the indicated time were lysed and analysed by immunoblots with the indicated antibodies. Quantifications of triplicates of (C). The graph depicts the level of pCFL1 relative to total CFL1 during the dextran (Mr 10,000) inoculation in SK-N-AS cells. Data information: Representative results are shown. (B, D) Ordinary one-way ANOVA followed by multiple comparisons (n = 3). n.s.: P > 0.05, ***P ≤ 0.001. ****P ≤ 0.0001. Source data are available online for this figure. Source Data for Figure 4 [embj201797822-sup-0006-SDataFig4.zip] Download figure Download PowerPoint Cofilin-1 phosphorylation increases over time in SOD1G93A transgenic mice To examine the possible pathological relevance of our findings, we monitored cofilin-1 activity in transgenic mice expressing the human ALS-causing mutant SOD1G93A (Gurney et al, 1994). The transgenic SOD1G93A mice develop a motor neuron disease that closely resembles human ALS with progressive deposition of mutant SOD1 (Johnston et al, 2000; Wang et al, 2009) and progressive motor neuron loss leading to motor deficits. As previous noted in different transgenic SOD1 lines (Johnston et al, 2000; Wang et al, 2009), insoluble SOD1G93A accumulated in the spinal cord of SOD1G93A transgenic mice, as the disease progressed (Fig 5A). Coincidentally, phosphorylation of cofilin-1 increased over time in the SOD1G93A transgenic mice, whilst the total levels of cofilin-1 were not measurably altered (Fig 5A and B). This is analogous to what has been observed in cells, where an increase in cofilin-1 phosphorylation follows an initial transient decrease (Fig 4A and B). Whilst the bi-phasic nature of the changes in cofilin-1 phosphorylation cannot be captured in a mouse model, the alteration of cofilin-1 activity in vivo demonstrates the pathophysiological importance of cofilin-1 in this disease model. The changes in cofilin-1 phosphorylation observed in the spinal cord of SOD1 transgenic mice were associated with an increased F/G-actin ratio (Fig 5C and D). These alterations were specific to the spinal cord, the affected tissue in the SOD1G93A mice because no such changes were observed in the brains of the transgenic mice (Fig 5E and F). These results argue that cofilin-1 signalling is altered in SOD1G93A mice. Figure 5. SOD1 aggregates alter cofilin-1 phosphorylation in SOD1G93A transgenic mice Immunoblots of the indicated proteins in spinal cord lysates of SOD1G93A transgenic mice or wild-type mice from 4 to 20 weeks of age. Quantifications of the pCFL1/CFL1 ratio in immunoblots such as the ones shown in (A). The graph depicts levels of pCFL1 relative to total CFL1 in lumbar region of the spinal cord of SOD1G93A compared with wild type. Data are means ± SEM (n = 4). Unpaired t-test. Two-tailed P-values are 0.2359, 0.0363 and 0.0376 in each age group separately. Actin fractionation followed

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital response to many forms of stress, including protein-misfolding stress in the endoplasmic reticulum (ER stress). It is believed to cause a general reduction in protein synthesis while enabling translation of few transcripts. Such a reduction of protein synthesis comes with the threat of depleting essential proteins, a risk thought to be mitigated by its transient nature. Here, we find that translation attenuation is not uniform, with cytosolic and mitochondrial ribosomal subunits being prominently downregulated. Translation attenuation of these targets persists after translation recovery. Surprisingly, this occurs without a measurable decrease in ribosomal proteins. Explaining this conundrum, translation attenuation preferentially targets long-lived proteins, a finding not only demonstrated by ribosomal proteins but also observed at a global level. This shows that protein stability buffers the cost of translational attenuation, establishing an evolutionary principle of cellular robustness.

Proteins destined for secretion, membrane insertion,and retention within the lumen of the exocytic compartment are synthesized on ribosomes bound to the endoplasmic reticulum (ER). The nascent peptide is translocated across the ER membrane and encounters theluminal environment, where it undergoes specific posttranslational modifications and folding reactions. Theflux of such client proteins through the ER varies considerably between different cell types and is influenced substantially by physiological conditions. For example, theload on the ER of a pancreatic acinar cell that secreteslarge quantities of digestive enzymes will be muchgreater than that placed on a maturing erythroblast whoseribosomes are active mainly in the synthesis of cytoplasmic proteins such as globins...

The deposition of misfolded proteins is the hallmark of the late-onset, rapidly progressive and devastating neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. These diseases are caused by a gain of toxic properties associated with the propensity of otherwise soluble proteins to misfold. What governs the deposition of the disease-causing proteins in aged neurons is unclear, but recent evidence suggests that once misfolded, the diverse proteins associated with the neurodegenerative diseases can induce aggregation of their soluble counterpart, thereby sharing one of the defining properties of prions. In addition to the seeded polymerization, prions have the ability to replicate their aberrant conformation indefinitely and are transmissible. Are these properties also shared by diverse misfolded proteins?

Comment on: Münch C, et al. Proc Natl Acad Sci USA 2011; 108:3548-53.

Article25 April 2022Open Access Transparent process Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response Aleksandra P Pitera Aleksandra P Pitera orcid.org/0000-0001-7004-1615 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Conceptualization, Formal analysis, Validation, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Maria Szaruga Maria Szaruga orcid.org/0000-0003-1673-1855 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Formal analysis, Validation, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Sew-Yeu Peak-Chew Sew-Yeu Peak-Chew orcid.org/0000-0002-7602-6384 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: ​Investigation, Writing - review & editing Search for more papers by this author Steven W Wingett Steven W Wingett orcid.org/0000-0002-2343-0711 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Software, Formal analysis, Writing - review & editing Search for more papers by this author Anne Bertolotti Corresponding Author Anne Bertolotti [email protected] orcid.org/0000-0002-9185-0558 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Aleksandra P Pitera Aleksandra P Pitera orcid.org/0000-0001-7004-1615 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Conceptualization, Formal analysis, Validation, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Maria Szaruga Maria Szaruga orcid.org/0000-0003-1673-1855 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Formal analysis, Validation, ​Investigation, Visualization, Writing - review & editing Search for more papers by this author Sew-Yeu Peak-Chew Sew-Yeu Peak-Chew orcid.org/0000-0002-7602-6384 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: ​Investigation, Writing - review & editing Search for more papers by this author Steven W Wingett Steven W Wingett orcid.org/0000-0002-2343-0711 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Software, Formal analysis, Writing - review & editing Search for more papers by this author Anne Bertolotti Corresponding Author Anne Bertolotti [email protected] orcid.org/0000-0002-9185-0558 MRC Laboratory of Molecular Biology, Cambridge, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Aleksandra P Pitera1, Maria Szaruga1, Sew-Yeu Peak-Chew1, Steven W Wingett1 and Anne Bertolotti *,1 1MRC Laboratory of Molecular Biology, Cambridge, UK *Corresponding author. Tel: +44 1223 267054; E-mail: [email protected] The EMBO Journal (2022)41:e109985https://doi.org/10.15252/embj.2021109985 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic. SYNOPSIS Treatment with Halofuginone (HF), an inhibitor of the glutamyl-prolyl-tRNA synthetase, activates the integrated stress response (ISR) leading to both gene expression reprogramming and translational attenuation. However, the decrease of translation induced by HF is atypical as it occurs independently of GCN2 and eIF2α phosphorylation. ISR-related gene expression changes induced by HF depend on GCN2 and eIF2α phosphorylation. HF-induced translation attenuation is independent of GCN2 and eIF2α phosphorylation. HF induces translation elongation defect. Proline-dependent cancer cell lines are more sensitive to HF-induced cytotoxicity. Introduction Halofuginone (HF), a derivative of the natural product febrifugine extracted from the hydrangea Dichroa febrifuga, has been used for centuries in Chinese medicine to treat malaria (Pines & Spector, 2015). Halofuginone was synthesized to alleviate the toxicity of febrifugine and has been widely utilized in veterinary practice for more than two decades to treat parasites in poultry and cattle (Daugschies et al, 1998). HF also exhibits a wide range of experimental and clinical activities, including antifibrotic properties, inhibition of angiogenesis and metastasis (Pines & Spector, 2015). HF binds and inhibits the EPRS with low nanomolar potency and as a result induces the ISR by activating GCN2 (Sundrud et al, 2009; Keller et al, 2012). However, recent data revealed some non-ISR activities associated with anti-inflammatory properties (Kim et al, 2020). The ISR is a vital homeostatic pathway elicited by phosphorylation of the translation initiation factor eIF2α on serine 51 to allow cells to adapt and survive changes in their environment. ISR signaling triggers a coordinated response consisting of attenuation of translation initiation and reprogramming gene expression (Sonenberg & Hinnebusch, 2009; Wek, 2018). In humans, this response is orchestrated by four eIF2α kinases that sense different signals and two phosphatases. The kinase GCN2 is activated by amino acid shortage, PKR by double-stranded RNA during viral infections, HRI by heme deficiency and PERK (or PEK) by accumulation of misfolded proteins in the endoplasmic reticulum (Sonenberg & Hinnebusch, 2009; Wek, 2018). Two eIF2α phosphatases reverse the activity of the four eIF2α kinases. They are split enzymes composed of a common catalytic subunit, protein phosphatase 1 (PP1), bound to one of two specific substrate receptors: stress-inducible PPP1R15A (R15A), or the constitutive PPP1R15B (R15B) (Bertolotti, 2018). Unlike PP1 in isolation, the holoenzymes R15A-PP1 and R15B-PP1 are selective for their substrate (Harding et al, 2009; Carrara et al, 2017; Bertolotti, 2018). The antagonistic actions of the four eIF2α kinases and the two phosphatases tune the phosphorylation levels of eIF2α to the cellular needs and conditions. Because of its central role in controlling cell and organismal survival, the ISR has emerged as a prime target for new pharmacological manipulations (Costa-Mattioli & Walter, 2020; Luh & Bertolotti, 2020), particularly for approaches aimed at restoring proteostasis (Balch et al, 2008). Compounds that either prolong (Guanabenz (Tsaytler et al, 2011), Sephin1 (Das et al, 2015), Raphin1 (Krzyzosiak et al, 2018)), or block (PERK inhibitors (Atkins et al, 2013), ISRIB (Sidrauski et al, 2013)) eIF2α phosphorylation or its downstream signaling have been identified (Costa-Mattioli & Walter, 2020; Luh & Bertolotti, 2020). Guanabenz is an approved drug, initially developed as an α2-adrenergic agonist to treat hypertension (Holmes et al, 1983). It has recently shown efficacy in a phase 2 clinical trial in amyotrophic lateral sclerosis (Bella et al, 2021), 10 years after its activity as a proteostatic compound was revealed (Tsaytler et al, 2011). Like Guanabenz, Sephin1, a non-adrenergic guanabenz derivative (Tsaytler et al, 2011; Das et al, 2015; Krzyzosiak et al, 2018), as well as Raphin1, prolong eIF2α phosphorylation and protect cells and mice from protein misfolding stress and associated diseases (Luh & Bertolotti, 2020). Sephin1 has passed through a favorable phase 1 clinical trial (https://clinicaltrials.gov/ct2/show/NCT03610334). The development of PERK inhibitors has stopped due to on-target pancreatic toxicity (Atkins et al, 2013). ISRIB has shown benefit in mouse models of diverse diseases (Costa-Mattioli & Walter, 2020) and the development of its derivatives is evaluated for vanishing white matter disease (Wong et al, 2019). Because HF has progressed to phase 2 clinical trials for the treatment of HIV-Related Kaposi's Sarcoma and Duchenne muscular dystrophy treatment (www.clinicaltrials.gov), it is a potentially attractive compound to explore the benefit of ISR modulation both experimentally and clinically. Here, we dissected the mechanism of action of HF and revealed that although HF induces the two canonical ISR adaptations consisting of attenuation of bulk protein synthesis and gene expression reprogramming, this response is surprisingly atypical because translation attenuation occurs independently of GCN2 and eIF2α phosphorylation. We found that these changes following HF treatment are all rescued upon proline addition, demonstrating that they result from EPRS inhibition. The knowledge that the observed activities of HF are all on-target provides the molecular basis to select specific disease conditions for optimal responsiveness to the compound, as exemplified here with the increased sensitivity of proline-dependent cancer cells to HF treatment. Results Atypical ISR induction by HF To comprehensively characterize the activities of HF, we started by conducting a dose-response treatment of HeLa cells and monitored induction of key ISR markers. As anticipated (Sundrud et al, 2009; Keller et al, 2012; Misra et al, 2021), HF induced an ISR response manifested by increased phosphorylation of eIF2α and increased levels of ATF4, R15A (Fig 1A and B). Surprisingly, ATF4 and R15A were induced from 12.5 nM to 312.5 nM but no longer with higher concentrations of HF. Under these conditions, eIF2α phosphorylation continued to increase (Fig 1A and B) either because of the loss of R15A expression or because of persistent kinase activation or both. In contrast to HF, tunicamycin, which activates the PERK branch of the ISR, induced phosphorylation of eIF2α and increased ATF4 and R15A levels in a dose-dependent manner (Fig 1C). The blunted ISR at high concentrations of HF was also present in other studies (Sundrud et al, 2009; Keller et al, 2012; Misra et al, 2021), but the mechanism underlying this phenomenon remains unknown, a knowledge gap which motivated this investigation. We next examined whether the lack of induction of ATF4 and R15A seen at concentrations of HF above 312.5 nM was a complete loss or a kinetic delay. Thus, we conducted time course experiments at 62.5 nM, a concentration of HF leading to typical ISR induction, as well as 312.5 nM, a concentration where ISR induction was dampened (Fig 1A). Prolonged treatment with 312.5 nM HF did not rescue the high induction of ATF4 observed at 62.5 nM (Fig 1D). As previously reported (Misra et al, 2021), mTORC1 signaling was elevated following HF, as manifested by an increased phosphorylation of the ribosomal S6 kinase (Fig EV1). This is because mTORC1 is activated due to an increased abundance of amino acids (Misra et al, 2021), possibly as consequence of ISR induction. Figure 1. HF induces an atypical blunted ISR Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with indicated concentrations of halofuginone (HF) for 5 h. Quantification of ATF4 and P-eIF2α from experiments as in (A). Data are mean ± SD (n = 3 biological replicates). *P ≤ 0.0414, **P ≤ 0.0019, ****P < 0.0001, as determined by one-way ANOVA with Dunnett’s multiple comparison test. Similar to (A), but with HeLa cells treated with indicated concentrations of tunicamycin for 5 h. Similar to (A), but using lysates from HeLa cells treated with 62.5 nM and 312.5 nM HF for indicated times. Relative abundance of the indicated mRNAs detected by qPCR in lysates from HeLa cells treated with indicated concentrations of HF for 5 h. Data are mean ± SD (n = 3 biological replicates). *P ≤ 0.0388, **P ≤ 0.0046, ***P ≤ 0.0008, as determined by one-way ANOVA with Dunnett’s multiple comparison test. Data information: Representative results of at least three independent experiments are shown in each panel. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. mTORC1 activation upon HF treatment Representative immunoblots of indicated proteins in lysates from HeLa cells treated for 5 h with indicated concentrations of HF or 200 nM Rapamycin for 3 h. Download figure Download PowerPoint We next monitored changes in abundance of ISR mRNA targets (Wek, 2018). As previously observed with other ISR inducers (Dey et al, 2010; Schneider et al, 2020; Misra et al, 2021), HF caused a dose-dependent increase of Atf4 and R15a transcripts (Fig 1E). Expression of asparagine synthetase (ASNS) is controlled transcriptionally by ATF4 (Wek, 2018). HF increased accumulation of Asns transcripts at 62.5 and 312.5 nM (Fig 1E) but this did not occur at higher concentrations (Fig 1E). A similar pattern was observed for Pycr1, another ATF4 target (Nilsson et al, 2014) encoding a proline biosynthetic enzyme (Fig 1E). Thus, the dose-response of Asns and Pycr1 mRNA to HF precisely mirrored that of its transcriptional factor ATF4, peaking at 62.5 nM (Fig 1A and E). These detailed time course and dose-response analyses reveal that HF induces an atypical ISR, as this response is blunted downstream of eIF2α phosphorylation at high concentrations of the compound. ISR-dependent HF activities Because HF was recently reported to display some ISR-independent activities (Kim et al, 2020), we next examined if the activities observed here were ISR-related or off-target. eIF2α phosphorylation in response to various stresses is completely abolished in cells lacking all four eIF2α kinases (4KO; Taniuchi et al, 2016). However, signal-specific induction of eIF2α phosphorylation is restored upon reintroduction of the cognate kinase in the 4KO cells (Taniuchi et al, 2016). This provides a robust single eIF2α kinase system to examine ISR sensing. We monitored induction of eIF2α phosphorylation by HF in wild-type mouse embryonic fibroblasts (MEFs), 4KO cells, and in 4KO cells complemented by each of the four human eIF2α kinases (Taniuchi et al, 2016). HF induced eIF2α phosphorylation in WT cells but not in the 4KO cells and this was rescued upon complementation with GCN2 in the 4KO cells, but no other eIF2α kinases (Fig 2A). This demonstrates that eIF2α phosphorylation by HF is entirely dependent on GCN2 in mouse cells. To validate these findings, we knocked down GCN2 in HeLa cells (Fig 2B). siRNA targeting Gcn2 effectively eliminated GCN2 and as a consequence, cells were unable to increase eIF2α phosphorylation upon HF treatment (Fig 2B). In the 4KO cells treated up to 39 μM of HF, there was no detectable increase in eIF2α phosphorylation, in contrast to the 4KO cells complemented by GCN2 (Fig 2C). These results reveal that induction of eIF2α phosphorylation by HF is entirely dependent on GCN2. Figure 2. GCN2 mediates eIF2α phosphorylation by HF Representative immunoblots of indicated proteins in lysates from mouse embryonic fibroblasts (MEFs) of indicated genotype after treatment with 12.5 nM or 200 nM HF for 5 h. Similar to (A), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF for 5 h. Similar to (A), but with high HF concentrations. Data information: Representative results of at least three independent experiments are shown in each panel. Download figure Download PowerPoint We next used quantitative proteomics with tandem mass tag mass spectrometry as an unbiased approach to investigate the molecular basis for the unusual disconnect between high eIF2α phosphorylation and ATF4 induction at high HF concentrations. Discrete changes were observed in HeLa cells treated with 12.5 nM of HF for 5 h, with only 12 proteins found increased by the treatment (P ≤ 0.05, fold change ≥ 1.5), ATF4 showing the highest induction (Fig 3A and Dataset EV1). This validated the approach and demonstrated the selectivity of HF as an ISR inducer. Induction of ATF4 increased 3-fold after 5 h treatment with 12.5 nM of HF, and only 1.5-fold with 312.5 nM HF in these quantitative proteomic analyses performed in HeLa cells (Fig 3A). ATF4 also increased in HF-treated wild-type mouse embryonic fibroblasts (eIF2αS/S) but not in eIF2αA/A cells that are incapable of phosphorylating eIF2α (Scheuner et al, 2001; Fig 3B and C). As in HeLa cells, ATF4 induction was lower in eIF2αS/S cells treated with 200 nM HF than in cells treated with 12.5 nM (Fig 3B). This demonstrates that HF induces ATF4 through the canonical GCN2-to-eIF2α signaling pathway but this induction is dampened at high concentrations of HF. Figure 3. Global quantitative proteomic analyses of cell response to HF Tandem mass tag mass spectrometry analyses performed in triplicate from HeLa cells treated with 12.5 or 312.5 nM HF for 5 h. Similar to (A), but using eIF2αS/S MEF cells treated with 12.5 or 200 nM HF for 5 h. Similar to (B), but using eIF2αA/A cells. Data information: Results are presented as pairwise comparisons. Data points in magenta represent collagen proteins. Vertical dashed lines indicate Log2(Fold change) = 0.58 and that corresponds to 1.5-fold change. Download figure Download PowerPoint Whilst only a few proteins increased in abundance upon treatment of HeLa or MEF cells with 12.5 nM HF, 312.5 nM HF decreased the levels of 188 proteins (P ≤ 0.05, fold change ≥ 1.5) in HeLa, 204 in eIF2αS/S and 195 in eIF2αA/A cells (Fig 3 and Dataset EV1). Gene ontology analyses were performed (Dataset EV2). Amongst the different categories, enrichments for genes involved in collagen fibril organization, including various types of collagens were observed (Dataset EV1). Decreased collagen expression could provide the molecular basis for HF antifibrotic activities (Pines & Spector, 2015). This suggested that some of the medically relevant activities of HF may be a consequence of the decreased abundance of downregulated proteins. We then searched for the underlying mechanism. High concentrations of HF decrease R15B Amongst the proteins downregulated at high HF concentration was R15B (Fig 4A). This was unexpected because R15B is resistant to the translation attenuation resulting from eIF2α phosphorylation (Andreev et al, 2015; Schneider et al, 2020). Because the decreased abundance of R15B (Fig 4A) could explain the unusually high increase in eIF2α phosphorylation observed at high HF concentrations (Fig 1A), we investigated it further. First, we confirmed that R15B was detectable up to 312.5 nM HF (Fig 4B). However, in cells treated for 5 h at higher concentrations than 312.5 nM HF, R15B was essentially depleted (Fig 4B). Surprisingly, this decrease was GCN2-independent and even exacerbated in GCN2-depleted cells (Fig 4C). Thus, we next examined whether the depletion of R15B observed at high concentrations of HF resulted from an increased degradation. As previously reported, R15B (Jousse et al, 2003) and ATF4 (Rutkowski et al, 2006) are unstable proteins. Both R15B and ATF4 are targeted for degradation by the cullin-RING E3 ligase β-TRCP (Lassot et al, 2001; Coyaud et al, 2015). The activity of such ligases is controlled by NEDD8-activating enzyme, which can be inhibited by the small-molecule inhibitor MLN4924 (Soucy et al, 2009). Both R15B and ATF4 proteins were stabilized in cells treated with MLN4924 (Fig EV2A–C), confirming that they are targeted for degradation by a cullin-RING ligase. The decreased abundance of ATF4 and R15B in cells treated with high concentrations of HF was not affected by treatment with MLN4924 (Fig 4D) indicating that it does not result from increased degradation. To confirm these observations, we treated cells with the proteasome inhibitor MG-132. Proteasome inhibition resulted in a marked accumulation of ATF4 and a slight increase in R15B in absence of HF (Fig 4E). However, MG-132 did not prevent the loss of ATF4 and R15B observed after treatment with high HF concentrations, above 312.5 nM (Fig 4E). Thus, the loss of ATF4 and R15B at high HF concentrations does not result from increased degradation. Figure 4. HF decreases R15B abundance independently of GCN2 and protein degradation Fold change of ATF4 and R15B after 5 h treatment with 12.5 or 312.5 nM HF relative to untreated cells. Data obtained from the quantitative proteomic analyses shown in Fig 3A. Representative immunoblots of indicated proteins in lysates from HeLa cells after 5 h treatment with indicated concentrations of HF. Similar to (B), but using HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without the Nedd8-activating enzyme inhibitor MLN4924 (1 μM). Representative immunoblots of lysates from HeLa cells treated with indicated concentrations of HF for 5 h with or without proteasome inhibitor MG-132 (10 μM). Data information: Representative results of at least three independent experiments are shown in each panel. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. The Nedd8-activating enzyme inhibitor MLN4924 stabilizes ATF4 and R15B Representative immunoblots of the indicated proteins in lysates from HeLa cells treated with cycloheximide (20 μg/ml) with or without the inhibitor of the NEDD8-activating enzyme MLN4924 (1 μM) for indicated times. Quantification of R15B and ATF4 in cells treated with MLN4924 (1 μM) for 120 min. Data are mean ± SEM (n = 3 biological replicates). *P = 0.0369, as determined by unpaired t-test. Quantification of experiments such as the ones shown in (A). t1/2 values were calculated with results from 3 independent experiments. Data are mean ± SEM (n = 3 biological replicates). Download figure Download PowerPoint Translational changes upon HF treatment To gain further mechanistic insights into HF response, we next focused on translation. We first took advantage of a published ribosome profiling dataset (Misra et al, 2021) and performed metagene analyses to characterize the translational response to HF at a global level. The distribution of ribosomes on mRNAs at a genome-wide level in control cells appeared as expected (Vorontsov et al, 2021), with a gradient of footprint density toward the 3’ end attesting high translation activity (Fig 5A). A low ribosome density after stop codons revealed efficient translation termination in all conditions (Fig 5A). HF treatment caused a striking genome-wide redistribution of ribosomes, with an increased density at the beginning of the ORFs and a decreased density toward the 3’ end (Fig 5A). This difference was confirmed by a polarity score analysis (Fig 5B) and may be explained by the fact that HF causes ribosome pausing at proline codons (Misra et al, 2021), as this would result in fewer ribosomes reaching the end of the transcript. Following treatment with HF, translation of some mRNAs decreased, others increased, whilst the majority remained unchanged (Misra et al, 2021). We next performed metagene analyses of ribosome footprints in the differentially regulated groups of transcripts. The increased 5’ end occupancy on the ORFs observed at the global level after HF was not seen on transcripts that were preferentially translated upon HF (Fig 5C). The translationally repressed mRNAs displayed an increased ribosome density in their 5’ UTR (Fig 5D). This has been seen at a genome-wide levels in yeast exposed to amino acid starvation (Ingolia et al, 2009; Schuller et al, 2017) and is consistent with the notion that uORF translation in 5’ UTR often represses translation of the main ORFs (Hinnebusch et al, 2016). We then focused on Atf4 translation. High ribosome occupancy was observed on the uORFs but not on the main ORF of Atf4 in untreated cells (Fig 5E). HF (100 nM) increased occupancy of ribosomes on the main ORF (Fig 5E), in agreement with the increased expression of the protein observed here (Fig 1A). Figure 5. Global analyses of translational changes upon HF treatment Metagene analysis plot showing average ribosome occupancy from all genes aligned at start codons in MEF cells untreated (UT, 3 replicates in blue) or treated with 100 nM HF (3 replicates in red) for 6 h. The vertical dashed lines, from left to right, separate the length-normalized transcripts into the (i) 5′UTR, (ii) CDS and (iii) 3′UTR. All genes were included (8,712–7,889 transcripts depending on the sample). Polarity scores distribution for all genes. Negative values correspond to footprint enrichment at the 5′ end of a CDS. Metagene analysis plot for genes identified with increased translation efficiency following HF treatment (180–167 transcripts). Similar to (C), but for genes with repressed translational efficiency following HF treatment. (335–293 transcripts). Plot representing ribosome density across the Atf4 mRNA in untreated cells (UT1-3, in blue) and treated with HF (HF1-3, in red). Black lines represent introns, intermediate-sized blocks denote UTRs, and thick blocks represent CDS. uORF1 and uORF2, split across an intron, are represented. Note that the 6 tracks representing ribosome density on Atf4 mRNA have been scaled such that the maximum value in each track has a fixed height. Data information: 5A–E were analyzed from (Misra et al, 2021). Download figure Download PowerPoint From these diverse analyses, the genome-wide shift of ribosome density toward the 5’ end of coding regions observed at a global level is the most intriguing because it is reminiscent to the redistribution caused in yeast upon depletion of a translation elongation factor (Schuller et al, 2017). This suggests that HF causes an elongation defect. This was unexpected because the GCN2-dependent eIF2α phosphorylation observed upon HF treatment (Figs 1 and 2) is expected to cause attenuation of translation initiation. HF decreases translation in a GCN2- and eIF2α-independent manner We next measured translation in cells treated with various concentrations of HF. Treatment of HeLa cells with 12.5, 62.5, and 312.5 nM HF decreased translation by ~20, 40, and 85%, respectively (Fig 6A). For comparison, the ISR-dependent translation attenuation induced by tunicamycin was ~30%, in contrast to the general translation inhibitor cycloheximide that blocked translation completely (Fig 6B). This suggested that the 40 and 85% decrease in translation at high HF concentrations may be ISR-independent. Thus, we next analyzed the consequence of HF treatment in the 4KO cells completely defective in ISR sensing. Surprisingly, a dose-dependent translation attenuation was detected in the 4KO cells at 12.5 and 200 nM HF (Fig 6C). Quantification of four replicates revealed that translation attenuation in the 4KO or 4KO+GCN2 cells was not statistically significant (Figs 6C and EV3). This was unexpected. Thus, we performed similar experiments in eIF2αA/A cells. The dose-dependent decrease in translation upon HF treatment was not significantly different in eIF2αS/S and eIF2αA/A cells (Figs 6D and EV3). In contrast, translation attenuation induced by tunicamycin was completely abolished in the eIF2αA/A cells (Fig 6E), as expected (Scheuner et al, 2001). This confirms our ability to detect ISR-dependent translational changes and reveals that the translation attenuation upon HF treatment is atypically ISR-independent in MEFs, requiring neither GCN2 nor eIF2α phosphorylation. This conclusion was corroborated in human cells, with translation attenuation upon HF treatment being resistant to GCN2 knockdown (Fig 6F). Figure 6. Translation attenuation in response to tRNA synthetase inhibitors is independent of the ISR Newly synthesized proteins pulse-labeled with 35S-methionine for 10 min in HeLa cells pre-treated with indicated compounds for 2.5 h, except (E) (Bottom) Coomassie-stained gel. Newly synthesized proteins in HeLa cells treated with 2.5 μg/ml Tm or 50 μg/ml cycloheximide (CHX). Newly synthesized proteins in 4KO or 4KO+GCN2 MEF cells treated with 12.5 or 200 nM HF. Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with 12.5 or 200 nM HF. Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h. Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF. Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM). Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM). Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h

The regulation of unfolded protein response (UPR) sensors has been studied for over 20 years, but IRE1β has remained a mystery. IRE1β is highly expressed in goblet cells: specialized epithelial cells that secrete mucus. Two articles in this issue show that IRE1β is held in an inactive state by the goblet cell-specific chaperone AGR2; upon dissociation of AGR2, IRE1β dimerizes and becomes active. This mechanism is reminiscent of the dynamic control by BiP of three other UPR sensors: IRE1α, PERK and ATF6.

Alzheimer's disease, Parkinson's, Huntington's, amyotrophic lateral sclerosis (ALS) and prion disorders are devastating neurodegenerative diseases of increasing prevalence in aging populations. Although clinically different, they share similar molecular features: the accumulation of one or two proteins in abnormal conformations inside or outside neurons. Enhancing protein quality control systems could be a useful strategy to neutralize the abnormal proteins causing neurodegenerative diseases. This review emphasizes the subcellular location of protein deposits in neurodegenerative diseases and the need to tailor strategies aimed at boosting protein quality control systems to the affected subcellular compartment. Inhibition of a protein phosphatase terminating the unfolded protein response will be discussed as a strategy to protect from diseases associated with misfolded proteins in the endoplasmic reticulum.

This protocol describes how to implement a set of rules for robust and quantitative analysis of motor performance in mice. qMotor can be used to assess early disease onset, before paralysis, and disease progression in diverse mouse models, with a smaller number of animals than in previous protocols. qMotor can be used for rapid and robust evaluation of potential therapeutic treatments in diverse mouse disease models. Phenotypic analysis of mouse models of human diseases is essential to understanding the underlying disease mechanisms and to developing therapeutics. Many models of neurodegenerative diseases are associated with motor dysfunction, a powerful readout for the disease. We describe here a set of measures to quantitatively monitor early disease onset and progression. We named this set of rules qMotor because it enables sensitive, robust and quantitative measurement of motor performance in 3 d. qMotor can be used to assess early disease onset, before paralysis, as well as disease progression in diverse mouse models, and can be exploited to define robust and humane experimental end points, thereby reducing animal suffering. As an example, we apply qMotor to SOD1G93A transgenic mice. Early studies with the original transgenic SOD1G93A mice in the hybrid background (B6SJL-Tg(SOD1-G93A) have been criticized because of high noise in this mixed background and because of inadequate study designs. We applied qMotor in SOD1G93A transgenic mice in an inbred C57BL/6J background, hereafter called iSOD1G93A mice, and show a remarkably robust and consistent phenotype in this line that we use to evaluate a therapeutic approach. qMotor is a protocol generically applicable to different mouse models.

<title>Abstract</title> Polyglutamine expansion in Huntingtin (HTT) causes its aggregation and progressive loss of striatal neurons in Huntington’s disease (HD). HD is a mostly adult-onset neurodegenerative disorder with no disease-modifying therapies. Here we found that human striatal aging is associated with a global upregulation of genes involved in translation, including the translation and proteostasis regulator PPP1R15B (R15B). We used the R15B inhibitor Raphin1 to investigate if the age-associated changes could modify HD pathology. R15B inhibition rescued early learning and late motor deficits in HDYAC128 mice. In striatal medium spiny neurons directly reprogrammed from fibroblasts of symptomatic HD patients (HD-MSNs), Raphin1 reduced the formation of mutant HTT aggregates and neuronal death. Genetic knockdown of R15B also protected HD-MSNs from neurodegeneration whereas its overexpression exacerbated disease phenotypes. Moreover, both human striatum and reprogrammed MSNs exhibited age-dependent decline of miR-196a, a microRNA that directly targets non-conserved sites in human R15B 3’UTR and overexpressing miR-196a lowered mutant HTT aggregation. This work identifies age-dependent alterations in miR-196a and its target R15B and demonstrates the therapeutic potential of reversing these changes in diverse models and readouts of HD. We propose miR-196a and R15B as disease-modifying targets in HD.

IRE1 proteins mediate cellular responses to accumulation of malfolded proteins in the endoplasmic reticulum in the yeast and mammalian unfolded protein responses. A sensitive in vivo u.v. crosslinking assay showed that IRE1 proteins are intimately associated with RNA in mammalian cells. The IRE1-associated RNA fragments recovered by this assay were different in stressed and unstressed cells. The amount of RNA associated with IRE1 that could be revealed by end-labeling with T4 kinase was greater in IRE1-containing complexes isolated from stressed cells. Furthermore, the RNA fragments recovered from complexes found in stressed cells were shorter than those from unstressed cells, revealing a dynamic change in the IRE1-RNA complex during the UPR. Formation of the complex between IRE1 and RNA was dependent on both the kinase and endonuclease domains of IRE1, and involved pre-existing RNA species. When viewed in the context of the known importance of Ire1p-HAC1 mRNA interactions to the yeast unfolded protein response, these findings suggest that full-length mammalian IRE1s also engage RNA molecules as downstream effectors.

Phosphorylation of the translation initiation factor eIF2α is an adaptive signaling event that is essential for cell and organismal survival from yeast to humans. It is central to the integrated stress response (ISR) that maintains cellular homeostasis in the face of threats ranging from viral infection, amino acid, oxygen, and heme deprivation to the accumulation of misfolded proteins in the endoplasmic reticulum. Phosphorylation of eIF2α has broad physiological, pathological, and therapeutic relevance. However, despite more than two decades of research and growing pharmacological interest, phosphorylation of eIF2α remains difficult to detect and quantify, because of its transient nature and because substoichiometric amounts of this modification are sufficient to profoundly reshape cellular physiology. This review aims to provide a roadmap for facilitating a robust evaluation of eIF2α phosphorylation and its downstream consequences in cells and organisms.

Abstract Huntington's disease is a neurodegenerative disease caused by a polyglutamine (polyQ) expansion in Huntingtin, which provokes aggregation of a proteolytic amino‐terminal fragment of the affected protein encompassing the polyQ expansion. Accumulation of mutant Huntingtin somehow triggers cellular dysfunction and leads to a progressive degeneration of striatal neurons. Despite considerable efforts, the function of Huntingtin as well as the precise molecular mechanisms by which the expanded polyQ elicits cellular dysfunction remain unclear. In addition, no treatment is available to prevent, cure, or even slow down the progression of this devastating disorder. Antibodies are valuable tools to understand protein function and disease mechanisms. Here, we have identified the epitope recognized by the mAb 2B4, a broadly used antibody generated against the amino‐terminal region of Huntingtin, which detects both aggregated and soluble Huntingtin. The 2B4 antibody specifically recognizes amino acids 50–64 of human Huntingtin but not the murine homologous region. Furthermore, the 2B4 epitope resides within the proline‐rich region of Huntingtin, which is critical for polyQ aggregation and toxicity. These properties suggest that the 2B4 antibody might be useful in antibody‐based therapeutic strategies.

Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Here, we generated a series of R15 truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. G-actin, which has been proposed to confer selectivity to R15 phosphatases, does not bind these regions, indicating that it is not required for substrate binding. Fragments containing the substrate-binding regions but lacking the PP1-binding motif trapped the phospho-substrate and caused accumulation of phosphorylated eIF2α in unstressed cells. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1-binding region, exhibit wild-type activity. This work identifies the substrate-binding region in R15s, that functions as a phospho-substrate trapping mutant, thereby defining a key region of R15s for follow up studies.

Nature 485, 507–511 (2012); doi:10.1038/nature11058 It has been brought to our attention that there is an error in Supplementary Fig. 1b, owing to incorrect assembly of the image. The correct panel and figure legend (and the raw data used to generate Supplementary Fig. 1b) are shown in the Supplementary Information to this Corrigendum.

Abstract Phosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A 325-554 and R15B 340-639 . Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A 325-674 and R15B 340-713 , encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.

Proteasome degradation is essential, but the intrinsic features of a protein that signals its destruction remain incompletely understood. In this issue of Molecular Cell, Geffen et al., 2016Geffen Y. Appleboim A. Gardner R.G. Friedman N. Sadeh R. Ravid T. Mol. Cell. 2016; 63 (this issue): 1055-1065Google Scholar report an unbiased and proteome-wide method that provided insights into the protein destruction signals and pathways. Proteasome degradation is essential, but the intrinsic features of a protein that signals its destruction remain incompletely understood. In this issue of Molecular Cell, Geffen et al., 2016Geffen Y. Appleboim A. Gardner R.G. Friedman N. Sadeh R. Ravid T. Mol. Cell. 2016; 63 (this issue): 1055-1065Google Scholar report an unbiased and proteome-wide method that provided insights into the protein destruction signals and pathways. Proteins are essential effectors of cellular functions, and a balance between protein synthesis and degradation is vital. When proteins are no longer needed, defective, or no longer functional, they have to be degraded (Hershko and Ciechanover, 1998Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6887) Google Scholar). The 26S proteasome is a multisubunit proteolytic complex that degrades proteins into small peptides, which will be further processed in amino acids by intracellular peptidases (Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1279) Google Scholar) (Figure 1). The proteasome thereby regulates the half-life of many cellular proteins and most cellular functions (Amm et al., 2014Amm I. Sommer T. Wolf D.H. Biochim. Biophys Acta. 2014; 1843: 182-196Crossref PubMed Scopus (300) Google Scholar). Protein degradation needs to be tightly regulated because untimely or uncontrolled proteolysis would be deleterious. Indeed, perturbation of proteasomal degradation is associated with diverse diseases such as cancer and neurodegeneration (Schneider and Bertolotti, 2015Schneider K. Bertolotti A. J. Cell Sci. 2015; 128: 3861-3869Crossref Scopus (44) Google Scholar, Tanaka and Matsuda, 2014Tanaka K. Matsuda N. Biophys. Acta. 2014; 1843: 197-204Crossref Scopus (123) Google Scholar). A key element of control in protein degradation consists of modification of the target protein with the covalent attachment of a small protein, ubiquitin, usually in the form of polyubiquitin chains (Hershko and Ciechanover, 1998Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6887) Google Scholar) (Figure 1). Ubiquitination requires the concerted action of three different enzymes: E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase (Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1279) Google Scholar). An important commitment step in proteasomal degradation consists of unfolding the proteins to be degraded to enable their entry into the proteolytic chamber of the proteasome (Finley, 2009Finley D. Annu. Rev. Biochem. 2009; 78: 477-513Crossref PubMed Scopus (1279) Google Scholar). Because folded proteins cannot enter the narrow catalytic core of the proteasome, efficient protein degradation by the proteasome requires, in addition to ubiquitin, a disordered region in the target protein, which serves as an initiation site for proteolysis (Schrader et al., 2009Schrader E.K. Harstad K.G. Matouschek A. Nat. Chem. Biol. 2009; 5: 815-822Crossref PubMed Scopus (224) Google Scholar). The E3 ligases recognize target proteins, providing the specificity in the ubiquitin-proteasome system (UPS) (Komander and Rape, 2012Komander D. Rape M. Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2236) Google Scholar) (Figure 1). Since the discovery of ubiquitin (Hershko and Ciechanover, 1998Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6887) Google Scholar), hundreds of E3 ligases and some of their respective targets have been discovered (Komander and Rape, 2012Komander D. Rape M. Annu. Rev. Biochem. 2012; 81: 203-229Crossref PubMed Scopus (2236) Google Scholar). A number of degradation signals have also been previously identified (Ravid and Hochstrasser, 2008Ravid T. Hochstrasser M. Nat. Rev. Mol. Cell Biol. 2008; 9: 679-690Crossref PubMed Scopus (610) Google Scholar). Varshavsky and colleagues discovered 20 years ago that an important determinant of protein stability is encoded by its amino-terminal residue, leading them define the N-end rule (Bachmair et al., 1986Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1372) Google Scholar). Varshavsky also coined the term “degron” to define transferable sequences that destabilize proteins (Varshavsky, 1991Varshavsky A. Cell. 1991; 64: 13-15Abstract Full Text PDF PubMed Scopus (122) Google Scholar). However, our understanding of the distinguishing features of a protein that needs be degraded remains incomplete. In this issue of Molecular Cell, Geffen et al., 2016Geffen Y. Appleboim A. Gardner R.G. Friedman N. Sadeh R. Ravid T. Mol. Cell. 2016; 63 (this issue): 1055-1065Google Scholar used an unbiased and high-throughput approach to identify the degradation signals—or degrons in the entire yeast proteome. Using an elegant, simple, and robust reporter-based assay that exploits the power of yeast genetics, combined with deep sequencing, these authors were able to identify and evaluate hundreds of different degrons (Figure 2A). The authors used the enzyme Ura3 as a reporter because it can be used for both positive and negative selection and fused fragments of yeast cDNAs at its C terminus. The initial high-complexity library comprised 25,000 transcriptome-derived fragments, with all possible reading frames represented (Figure 2A), facilitating in this way the discovery of native protein-derived degrons. The reporter stability was used to perform a negative selection: Ura3p catalyzes the conversion of 5FOA into a cytoxic product. In the presence of 5FOA, yeast with lower levels of reporter were able to grow. Conversely, stable reporter proteins impaired cell growth. Therefore, this method uses growth rate to quantitatively measure degron potency (Figure 2A). The Geffen screen of the high-complexity library led to the identification 446 putative native degrons (Figure 2B). This suggests that there must be complex layers of regulation, which must at least in part involve E3 ligases. There are ∼80 genes encoding E3 ubiquitin ligases in yeast and ∼600 in human, and most of their substrates remain unknown. Thus, the Ravid group used their assay to look for E3-specific degrons (Figure 2B), using the E3 ligase Doa10 as an example. They expressed their reporter in wild-type and doa10Δ cells to isolate sequences stabilized in doa10Δ cells, which they further characterized to identify authentic Doa10 substrates. A degron in the enzyme enolase was identified in this way. Importantly, the enolase degron displays known features of other Doa10 degrons, thereby validating the robustness of the method. In a recent study aimed at identifying Doa10 substrates, the Michaelis group also identified Doa10 specific degrons (Maurer et al., 2016Maurer M.J. Spear E.D. Yu A.T. Lee E.J. Shahzad S. Michaelis S. G3 (Bethesda). 2016; 6: 1853-1866Crossref Scopus (36) Google Scholar). These methods can be used to systematically search for specific E3 substrates and degrons. This is exciting because, in the future, many E3 substrates could be identified in this way. Aiming at further characterizing the specificity of degrons, Geffen et al., 2016Geffen Y. Appleboim A. Gardner R.G. Friedman N. Sadeh R. Ravid T. Mol. Cell. 2016; 63 (this issue): 1055-1065Google Scholar analyzed the influence of subcellular compartment in degron potency. The authors added a nuclear localization signal (NLS) to their reporter construct to compare cytosolic and nuclear degradation. Interestingly, some degrons are more potent when expressed in the nucleus rather than in the cytosol. This observation supports the idea of compartment-specific degrons and further demonstrates that the method is a valuable tool to analyze proteolysis in diverse cellular contexts. The possibility of identifying the rules governing compartment-specific degradation is also exciting. At this point, it is not clear what determines degron potency. The identified degrons have variable length, secondary structure, and amino acid composition. It is reasonable to anticipate that there are other levels of complexity and regulation, which goes beyond the simplistic “sequence composition = degradation potency” vision. For example, post-translation modifications are known to affect protein stability. In a similar way as the Ravid group has examined E3-specific degron preferences by testing their library in a specific E3 knockout strain, it will be interesting to look at the contribution of post-translational modifications to different degrons potencies by expressing the reporter library in strains where specific kinases, phosphatases, etc. are knocked out. Because protein degradation is fundamental for cell fitness, an imbalance in protein degradation is associated with diverse human diseases (Schneider and Bertolotti, 2015Schneider K. Bertolotti A. J. Cell Sci. 2015; 128: 3861-3869Crossref Scopus (44) Google Scholar, Tanaka and Matsuda, 2014Tanaka K. Matsuda N. Biophys. Acta. 2014; 1843: 197-204Crossref Scopus (123) Google Scholar). A comprehensive understanding of the protein destruction code is important to begin to learn how perturbations of protein degradation contribute to diseases. The study by Geffen and coworkers is a step forward in this direction. The authors are funded by Medical Research Council (UK) MC_U105185860. We thank M. Hochstrasser and members of the Bertolotti laboratory for comments on the manuscript. We regret that space limitations restricted the number of citations. Mapping the Landscape of a Eukaryotic DegronomeGeffen et al.Molecular CellSeptember 8, 2016In BriefUsing a reporter-based competition assay, Geffen, Appleboim et al. measured the degradation potency of thousands of polypeptides. The assay was further used for identifying new quality control substrates of the ubiquitin system and for comparing cytosolic and nuclear degradation potency. Full-Text PDF Open Archive

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressive motor neuron disease, with 50 % of patients dying within 1.5 years of symptoms onset. The clinical manifestations are heterogeneous in ALS, as the region of onset of muscle weakness varies between individuals. Regardless of the site of onset, the symptoms of ALS begin in one discrete body region in 98 % of the cases. Subsequently, symptoms inevitably progress to regions contiguous to the site of onset where they appear with decreasing severity. These unique clinical features suggest that neurodegeneration in ALS is an orderly and propagating process. At the molecular level, it is now well recognized that protein misfolding plays a central role in both familial and sporadic ALS. Recently, it was found that mutant SOD1, the major component of the protein deposits in familial forms of ALS, propagates misfolding from cell to cell and replicates its misfolding conformation indefinitely, just like prions do. This phenomenon could provide the molecular basis of the focality and spreading of muscle weakness in ALS, as well as the cell autonomous and non-cell autonomous processes in ALS.KeywordsAmyotrophic Lateral SclerosisMotor Neuron DiseaseMutant SOD1Sporadic Amyotrophic Lateral SclerosisFamilial Amyotrophic Lateral SclerosisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

All intracellular proteins are subjected to exquisite quality control to assure maintenance of a healthy and functional proteome. Chaperones and proteolytic systems are integral components of the proteostasis networks, which monitor and assist proteins through the multiple steps they undergo through their cellular life (folding, trafficking, assembly, disassembly, refolding and degradation). Loss of protein homeostasis has been identified as one of the hallmarks of aging organisms and underlies the basis of severe age-related disorders such as neurodegenerative diseases. The recent better understanding of the proteostasis networks and their integration across cells, tissues and organs, has revealed them as novel possible targets for anti-aging interventions. In this session, we will review recent findings on age-related changes in the molecular components that participate in cellular homeostasis (chaperones (Frydman), the ubiquitin/proteasome system (Gonos) and autophagy (Cuervo)), the consequences of these changes (Santambrogio) and provide examples of current attempts to modulate the proteostasis networks in aging and age-related disorders (Bertolotti).

A broad range of age-related human diseases, including common and devastating neurodegenerative diseases, are caused by the deposition of misfolded proteins that occurs when cells become unable to withstand the pressure of misfolded proteins. Often, proteins, which are normally soluble, eventually misfold and aggregate late in life. The fact that protein aggregates build up later in life suggests that the cellular defense systems against misfolded proteins gradually fail with age. In the past 20–30 years, many components of protein quality control systems have been identified. The challenge that remains is to use this knowledge to identify strategies to correct the broad range of diseases that arise when protein quality control is overwhelmed. In this talk I will discuss our efforts to identify unbiased approaches that can be used to rescue cells from protein quality control catastrophes, such as targeting mechanisms by which cells maintain proteasome homeostasis.

Aggregation of misfolded proteins is a characteristic of several neurodegenerative diseases. The huntingtin amino-terminal fragment with extended polyglutamine repeat forms aggregates closely associated with chaperones both in the cytoplasm and the nucleus. Because each cellular compartment contains distinct chaperones and because the molecular mechanisms controlling polyglutamine aggregation are largely unknown, we decided to investigate the influence of different cellular environments on the aggregation of this pathological protein. Here, we show that aggregation of a protein containing a polyglutamine stretch of pathological length is abolished when its expression is targeted to the endoplasmic reticulum. Once retrogradely transported outside the endoplasmic reticulum, the aggregation-prone polyglutamine-containing protein recovers its ability to aggregate. When expressed in the mitochondria, a protein containing 73 glutamines is entirely soluble, whereas the nucleocytosolic equivalent has an extremely high tendency to aggregate. Our data imply that polyglutamine aggregation is a property restricted to the nucleocytosolic compartment and suggest the existence of compartment-specific cofactors promoting or preventing aggregation of pathological proteins.

Le controle de la transcription constitue un mode de regulation de l'expression des genes. L'initiation de la transcription necessite de nombreux facteurs dont tfiid qui reconnait les promoteurs des genes codant pour des proteines. Dans les cellules humaines, il existe plusieurs complexes tfiid de fonction distincte. Tous contiennent tbp et les htaf#i#is communs mais different par la presence de htaf#i#is specifiques. Au court de ce travail de these, htaf#i#i68, un nouveau htaf#i#i specifique a ete identifie et l'adnc correspondant ainsi qu'une partie du gene ont ete clones. Htaf#i#i68 est homologue a deux proteines nucleaires humaines tls/fus et ews qui sont les produits de genes sujets a des translocations dans les sarcomes humaines. Htaf#i#i68, ews et tls/fus sont trois htaf#i#is specifiques qui sont presents dans des complexes tfiid fonctionnellement distincts. Htaf#i#i68 et ews sont egalement associes a l'arn polymerase ii et htaf#i#i68 stimule la processivite de l'enzyme. Nous avons egalement cherche a caracteriser les mecanismes moleculaires par lesquels la proteine oncogenique derivee de ews dans les sarcomes d'ewing exerce son pouvoir transformant. Par ailleurs, l'analyse de htaf#i#i68 est un oncogene potentiel. Ainsi, au court de ce travail de these, nous avons caracterise de nouveaux facteurs de transcription impliques dans l'oncogenese. Ces facteurs htaf#i#i68, ews et tls/fus definissent une nouvelle famille de proteines que nous avons nommee la famille tet.