Lipid Biosynthesis Perturbation Impairs Endoplasmic Reticulum-Associated Degradation
Samantha M. TurkChristopher J. IndovinaDanielle OvertonAvery M. RunnebohmCade J OrchardEllen M. DossKyle A. RichardsCourtney Broshar IrelanMahmoud M. DaraghmiConnor G BaileyJacob M MillerJulia M NiekampSamantha K. GosserMary E Tragesser-TiñaKieran P. ClaypoolSarah EngleBryce W. BuchananKelsey A. WoodruffJes OlesenPhilip J. SmaldinoEric M. Rubenstein
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ABSTRACT The relationship between lipid homeostasis and protein homeostasis (proteostasis) is complex and remains incompletely understood. We conducted a screen for genes required for efficient degradation of Deg1 -Sec62, a model aberrant translocon-associated substrate of the endoplasmic reticulum (ER) ubiquitin ligase Hrd1, in Saccharomyces cerevisiae . This screen revealed that INO4 is required for efficient Deg1 -Sec62 degradation. INO4 encodes one subunit of the Ino2/Ino4 heterodimeric transcription factor, which regulates expression of genes required for lipid biosynthesis. Deg1 -Sec62 degradation was also impaired by mutation of genes encoding several enzymes mediating phospholipid and sterol biosynthesis. The degradation defect in ino4 Δ yeast was rescued by supplementation with metabolites whose synthesis and uptake are mediated by Ino2/Ino4 targets. Stabilization of a panel of substrates of the Hrd1 and Doa10 ER ubiquitin ligases by INO4 deletion indicates ER protein quality control is generally sensitive to perturbed lipid homeostasis. Further, loss of INO4 sensitized yeast to proteotoxic stress, suggesting a broad requirement for lipid homeostasis in maintaining proteostasis. Abundance of the ER ubiquitin-conjugating enzyme Ubc7 was reduced in the absence of INO4 , consistent with a model whereby perturbed lipid biosynthesis alters the abundance of critical protein quality control mediators, with broad consequences for ER proteostasis. A better understanding of the dynamic relationship between lipid homeostasis and proteostasis may lead to improved understanding and treatment of several human diseases associated with altered lipid biosynthesis.Keywords:
Proteostasis
Endoplasmic-reticulum-associated protein degradation
Ubiquitin-Protein Ligases
Endoplasmic-reticulum-associated protein degradation
Protein Degradation
Ubiquitin-Protein Ligases
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Protein folding stress in the endoplasmic reticulum (ER) may lead to activation of the unfolded protein response (UPR), aimed to restore proteostasis in the ER. Previously, we demonstrated that UPR activation is an early event in Alzheimer disease (AD) brain. In our recent work we investigated whether activation of the UPR is employed to enhance the capacity of the ubiquitin proteasome system or autophagy in neuronal cells. We showed that the levels, composition and activity of the proteasome are not regulated by the UPR. In contrast, UPR activation enhances autophagy and LC3 levels are increased in neurons displaying UPR activation in AD brain. Our data suggest that autophagy is the major degradational pathway following UPR activation in neuronal cells and indicate a connection between UPR activation and autophagic pathology in AD brain.
Proteostasis
Endoplasmic-reticulum-associated protein degradation
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Proteostasis
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Endoplasmic-reticulum-associated protein degradation
ATF6
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The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys48-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-α and CD3-δ. The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys48-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-α and CD3-δ. When a newly synthesized protein molecule is translocated into the ER, 1The abbreviations used are: ERendoplasmic reticulumERADER-associated degradationE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeE3ubiquitin ligaseTCRT-cell receptorCFTRcystic fibrosis transmembrane conductance regulatorHMGR3-hydroxy-3-methylglutaryl-coenzyme A reductaseCHIPC terminus of the Hsc70-interacting protein*mutantAMFRautocrine motility factor receptorGFPgreen fluorescent proteinGSTglutathione S-transferase.1The abbreviations used are: ERendoplasmic reticulumERADER-associated degradationE1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeE3ubiquitin ligaseTCRT-cell receptorCFTRcystic fibrosis transmembrane conductance regulatorHMGR3-hydroxy-3-methylglutaryl-coenzyme A reductaseCHIPC terminus of the Hsc70-interacting protein*mutantAMFRautocrine motility factor receptorGFPgreen fluorescent proteinGSTglutathione S-transferase. there is a fair chance that it may never reach its final destination as a functional molecule, since a significant proportion of newly synthesized proteins is degraded via the endoplasmic reticulum-associated degradation (ERAD) pathway (1Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Google Scholar). In particular, proteins that misfold along the folding pathway or cannot be appropriately folded as a result of mutations are degraded via this route. The cystic fibrosis transmembrane conductance regulator (CFTR) and its common mutation ΔF508 in cystic fibrosis serve as an example in this context (2Ward C.L. Omura S. Kopito R.R. Cell. 1995; 83: 121-127Google Scholar). In addition, proteins that lack their oligomerization partner(s) are prone to degradation, e.g. individual subunits of the T-cell receptor like TCR-α and CD3-δ (3Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Google Scholar). Finally, ERAD also functions in the homeostatic regulation of metabolic pathways to degrade proteins whose activity needs to be attenuated at a certain metabolic state. Examples include 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) (4Gardner R.G. Shearer A.G. Hampton R.Y. Mol. Cell. Biol. 2001; 21: 4276-4291Google Scholar), which is further described below, and apolipoprotein B (5Fisher E.A. Zhou M. Mitchell D.M. Wu X. Omura S. Wang H. Goldberg A.L. Ginsberg H.N. J. Biol. Chem. 1997; 272: 20427-20434Google Scholar).Degradation of proteins from the ER requires dislocation of the substrate from the ER to the cytosol followed by proteolysis via the ubiquitin-proteasome pathway. The dislocation process is thought to require components of the translocon channel, including Sec61α (6Wiertz E.J. Tortorella D. Bogyo M. Yu J. Mothes W. Jones T.R. Rapoport T.A. Ploegh H.L. Nature. 1996; 384: 432-438Google Scholar, 7Zhou M. Schekman R. Mol. Cell. 1999; 4: 925-934Google Scholar, 8Pilon M. Schekman R. Romisch K. EMBO J. 1997; 16: 4540-4548Google Scholar), as well as a complex of proteins designated CDC48/p97-Ufd1-Npl4 (9Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Google Scholar, 10Rabinovich E. Kerem A. Frohlich K.U. Diamant N. Bar-Nun S. Mol. Cell. Biol. 2002; 22: 626-634Google Scholar, 11Ye Y. Meyer H.H. Rapoport T.A. Nature. 2001; 414: 652-656Google Scholar). Ubiquitination also plays an essential role in dislocation as illustrated by the inhibition of protein dislocation when the ubiquitination machinery is disrupted (9Jarosch E. Taxis C. Volkwein C. Bordallo J. Finley D. Wolf D.H. Sommer T. Nat. Cell Biol. 2002; 4: 134-139Google Scholar, 13Shamu C.E. Flierman D. Ploegh H.L. Rapoport T.A. Chau V. Mol. Biol. Cell. 2001; 12: 2546-2555Google Scholar, 14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar, 15de Virgilio M. Kitzmuller C. Schwaiger E. Klein M. Kreibich G. Ivessa N.E. Mol. Biol. Cell. 1999; 10: 4059-4073Google Scholar, 16Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 246-255Google Scholar).The coupling of ubiquitin chains to proteins involves three enzymes. A ubiquitin-activating enzyme (E1) activates ubiquitin in an ATP-dependent manner. Subsequently one of a second set of enzymes, designated E2, conjugates the activated ubiquitin through a thiol ester bond to its essential cysteine residue. Finally, with the aid of a third set of enzymes, E3 ubiquitin ligases, the ubiquitin molecules are successively transferred from the E2 onto one or more lysine residues or the N terminus of the protein destined for degradation (1Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Google Scholar, 17Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Google Scholar). It is thought that E3 ubiquitin ligases or the combinations of E2/E3 enzymes provide specificity to the ubiquitination of protein targets. Thus, the identification of E3 ubiquitin ligases that are involved in the elimination of proteins associated with the ER should greatly advance our understanding of the regulation of this process.At present, three classes of E3 ligases are recognized. The first group has a HECT domain, named after the E6-AP C terminus, and the second group, to which the CHIP E3 ligase belongs, contains a so-called U-box (1Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Google Scholar, 16Tsai B. Ye Y. Rapoport T.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 246-255Google Scholar). The third group that seems to expand the fastest is that of the RING finger-containing E3 ligases. The RING motif consists of a series of eight conserved cysteines and histidines, which bind two zinc atoms and form a structure of “cross-braced” rings. The middle two residues in the motif comprise either one or two histidines, resulting in three subclasses of RING finger motifs: classical or RING-HC, RING-CH, and RING-H2. To date, all characterized examples of these variants have been shown to possess E3 ligase activity in vitro (18Joazeiro C.A. Weissman A.M. Cell. 2000; 102: 549-552Google Scholar, 19Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Google Scholar, 20Freemont P.S. Curr. Biol. 2000; 10: R84-R87Google Scholar, 21Coscoy L. Ganem D. Trends Cell Biol. 2003; 13: 7-12Google Scholar). For some RING finger E3 ligases, it was found that the RING finger structure binds the E2. However, the exact mechanism by which the RING finger-containing E3 ligase catalyzes the transfer of ubiquitin to the target proteins is yet unknown.One of the best characterized RING-H2 finger-containing E3 ligases involved in ERAD in yeast is Hrd1p (22Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Google Scholar), also designated Der3p (23Bordallo J. Plemper R.K. Finger A. Wolf D.H. Mol. Biol. Cell. 1998; 9: 209-222Google Scholar). This protein was identified by Hampton and co-workers (22Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Google Scholar) in search for factors that take part in the degradation of Saccharomyces cerevisiae Hmg2p, one of the yeast isozymes of HMGR. HMGR is the rate-limiting enzyme in the mevalonate pathway in which sterols and a myriad of essential isoprenoids are synthesized. In mammalian as well as in yeast cells, the intracellular levels of HMGR are tightly regulated by the cellular demands for mevalonate-derived sterol and non-sterol metabolites (24Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Google Scholar, 25Roitelman J. Simoni R.D. J. Biol. Chem. 1992; 267: 25264-25273Google Scholar, 26Gardner R.G. Shan H. Matsuda S.P. Hampton R.Y. J. Biol. Chem. 2001; 276: 8681-8694Google Scholar). This feedback control involves alteration of enzyme stability (24Goldstein J.L. Brown M.S. Nature. 1990; 343: 425-430Google Scholar, 27Gardner R.G. Hampton R.Y. J. Biol. Chem. 1999; 274: 31671-31678Google Scholar). Thus, when the demands are high, HMGR protein is stable. When the requirements for these metabolites have been satisfied, the enzyme is rapidly degraded. Studies in yeast, as well as more recent experiments in mammalian cells, have unequivocally shown that the degradation of HMGR involves its regulated ubiquitination and eventual elimination by the 26 S proteasome (22Hampton R.Y. Gardner R.G. Rine J. Mol. Biol. Cell. 1996; 7: 2029-2044Google Scholar, 28Ravid T. Doolman R. Avner R. Harats D. Roitelman J. J. Biol. Chem. 2000; 275: 35840-35847Google Scholar). Hrd1p/Der3p, as an E3 ubiquitin ligase, was shown to be involved in this metabolically regulated degradation of yeast HMGR (29Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Google Scholar). Moreover, it has been demonstrated that Hrd1p, which was independently isolated by Wolf et al. (23Bordallo J. Plemper R.K. Finger A. Wolf D.H. Mol. Biol. Cell. 1998; 9: 209-222Google Scholar) as Der3p, is also involved in ERAD of other ER proteins, including CPY* and Sec61-2p.Hrd1p/Der3p is a multispanning membrane protein with its C-terminal RING-H2 finger domain located in the cytoplasm. In yeast, Hrd1p is found in a stoichiometric complex with Hrd3p, a lumen-oriented ER membrane protein that stabilizes Hrd1p and modulates its ligase activity (30Gardner R.G. Swarbrick G.M. Bays N.W. Cronin S.R. Wilhovsky S. Seelig L. Kim C. Hampton R.Y. J. Cell Biol. 2000; 151: 69-82Google Scholar). The enzyme predominantly uses Ubc7p as an E2 but also cooperates with Ubc6p and Ubc1p in ERAD (29Bays N.W. Gardner R.G. Seelig L.P. Joazeiro C.A. Hampton R.Y. Nat. Cell Biol. 2001; 3: 24-29Google Scholar, 31Friedlander R. Jarosch E. Urban J. Volkwein C. Sommer T. Nat. Cell Biol. 2000; 2: 379-384Google Scholar).Another yeast E3 ligase involved in ERAD is Doa-10 (32Swanson R. Locher M. Hochstrasser M. Genes Dev. 2001; 15: 2660-2674Google Scholar), which contains a RING finger of the RING-CH type at its N terminus, and is ER-localized. It degrades the transcription factor MATα2 and a number of other ERAD substrates that are not served by the Hrd1p/Der3p complex. A third yeast E3 ligase implicated in degradation of ER quality control substrates is the HECT domain-containing Rsp5p (33Haynes C.M. Caldwell S. Cooper A.A. J. Cell Biol. 2002; 158: 91-101Google Scholar), which also seems to assist in degradation of proteins in the ER, especially in times of ER stress.In the mammalian cell, the number of E3 ligases involved in ERAD is also rapidly expanding. gp78, previously known as the autocrine motility factor receptor (AMFR), was identified as an ER-localized E3 ubiquitin ligase that can mediate the degradation of the ERAD substrates CD3-δ and apolipoprotein B100 (34Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Google Scholar, 35Liang J.S. Kim T. Fang S. Yamaguchi J. Weissman A.M. Fisher E.A. Ginsberg H.N. J. Biol. Chem. 2003; 278: 23984-23988Google Scholar). CHIP is a cytosolic U-box-containing E3 ligase that can target CFTR for degradation from the ER in an Hsp/Hsc70-dependent way (36Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Google Scholar). CHIP also catalyzes degradation of glucocorticoid hormone receptor (37Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Hohfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Google Scholar) via a process that requires Hsp90. Finally, F-box2 protein, the substrate-recognizing subunit of an SCF (Skp, Cullin, F-box) E3 ligase complex localized in the cytosol, binds to N-glycans of proteins in the ER and assists in their degradation (38Yoshida Y. Chiba T. Tokunaga F. Kawasaki H. Iwai K. Suzuki T. Ito Y. Matsuoka K. Yoshida M. Tanaka K. Tai T. Nature. 2002; 418: 438-442Google Scholar).In this study, we characterized the structure and function of the recently identified human homologue of yeast Hrd1p/Der3p E3 ligase, designated HRD1. Expression and subcellular localization of human HRD1 were addressed, and the overall membrane topology of human HRD1 was determined using deglycosylating enzymes and proteinase K digestions. In vitro ubiquitination assays were performed to establish whether ubiquitin linkage by HRD1 is Lys48-specific. The anticipated function of human HRD1 in degradation of HMGR was investigated as well as its role in degradation of other ERAD substrates.EXPERIMENTAL PROCEDURESMaterials—Unless noted otherwise, all reagents were obtained from Sigma. Geneticin (G418 sulfate) was procured from Invitrogen. 25-Hydroxycholesterol was purchased from Steraloids. Immobilized recombinant Protein A was obtained from RepliGen, and Protein A- and G-Sepharose were from Amersham Biosciences. Proteinase K was purchased from Invitrogen. MicroBCA protein reagent and SuperSignal® chemiluminescence substrate were from Pierce, and the ECL+ chemiluminescence kit was from Amersham Biosciences. MG-132 proteasome inhibitor was purchased from Calbiochem or Peptide Institute (Osaka, Japan). Compactin was a kind gift from Robert Simoni, Stanford University, and mevalonolactone was bought from Fluka (Buchs, Switzerland). Lipoprotein-deficient fetal calf serum (d ≥ 1.25) was prepared by ultracentrifugation as described previously (39Goldstein J.L. Basu S.K. Brown M.S. Methods Enzymol. 1983; 98: 241-260Google Scholar).Plasmids—Several entries comprising the human homologue of S. cerevisiae Hrd1p, HRD1, are present in the National Center for Biotechnology Information (NCBI) gene data base (i.e. accession numbers AAL26903, AAH30530, NM_032431, XP_045498, NP_757385, and NP_115807), resulting from independent cloning and sequencing of the gene by different researchers. Some major and minor variations in the gene can be identified: the KIAA1810 clone (protein accession number BAB47439), for which the cDNA was produced from human fetal brain tissue, lacks two exons compared with the rest of the entered sequences. Entries NP_115807, XP_045498, and AAL26903 on one hand and NP_757385 and AAH30530 on the other represent two other splice variants, designated Isoform a (“short”) and Isoform b (“long”), respectively. This splice variation results in one additional codon in the long isoform, encoding an alanine residue at amino acid position 413. The work described in this report was performed with the short isoform (identical to entries NP_115807, XP_045498, and AAL26903), which was cloned as described below.The KIAA1810 cDNA, cloned in pBluescript vector, was obtained from the HUGE sequencing project (40Kikuno R. Nagase T. Waki M. Ohara O. Nucleic Acids Res. 2002; 30: 166-168Google Scholar). To produce an expression construct, the HRD1-encoding open reading frame was cut out using KpnI and BspLU11I restriction enzymes. The BspLU11I-cut side was made blunt, and the fragment was cloned into KpnI/EcoRV sites of a pcDNA3.1/hygro(+) vector. The KIAA1810 open reading frame lacks two exons (encoding 51 amino acids) relative to other human HRD1 entries in the NCBI data base (accession numbers AAL26903, AAH30530, XP_045498, NP_757385, and NP_115807). The missing region was isolated from HeLa cell cDNA by PCR and cloned into the KIAA1810 open reading frame. A mutation of the first or second cysteine of the RING finger into an alanine was accomplished with the QuikChange site-directed mutagenesis kit (Stratagene), resulting in a product designated HRD1-C1A or HRD1-C2A, respectively. The human HRD1 open reading frame and its RING finger mutant were also cloned into the EcoRI/KpnI sites of the pcDNA3.1 Myc/His A(-) vector and into a FLAG tag-containing pLNCX vector (Clontech). The resulting constructs contained a C-terminal Myc and His tag or a FLAG tag, respectively. pTCR-α-Neo was kindly provided by Dr. Ron Kopito (Stanford University), and pLZRS-based retroviruses that express TCR-α were a kind gift from Dr. Mirjam Heemskerk (Leiden University Medical Center, Leiden, The Netherlands). Expression plasmids pCIneo-gp78 and pCIneo-gp78R2m were a kind gift from Dr. Allan Weissman (NCI, National Institutes of Health, Bethesda, MD). The human CD3-δ gene was isolated from pCD1-CD3-δ (a gift from Dr. Peter van den Elsen, Leiden University Medical Center) by PCR and cloned into pcDNA3.1/hygro(+) vector using XhoI and XbaI sites.Antibodies—Polyclonal rabbit antiserum against human HRD1 was produced using a purified fragment of the C-terminal 228 amino acids of the protein fused N-terminally to maltose-binding protein. Anti-Myc antibodies were from Roche Applied Science (immunoblot and immunoprecipitation) or Invitrogen (immunoprecipitation). Anti-FLAG monoclonal antibodies, clone M2, were purchased from Sigma. Anti-GFP antibodies were from Invitrogen or were a kind gift from Dr. Jaques Neefjes (Netherlands Cancer Institute, Amsterdam). Anti-US11 antiserum was produced in rabbits as described previously (14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar). Monoclonal antibodies against human transferrin receptor (clone H68.4) were purchased from Zymed Laboratories Inc. HMGR was immunoprecipitated with specific antiserum, which was described earlier (41Roitelman J. Olender E.H. Bar-Nun S. Dunn Jr., W.A. Simoni R.D. J. Cell Biol. 1992; 117: 959-973Google Scholar), directed against peptides derived from the HMGR membrane domain. Antibodies against TCR-α (H28-710 monoclonal antibody) were kindly provided by Dr. Ron Kopito (Stanford University), and polyclonal rabbit antibodies against CD3-δ and TCR-α (42Lew A.M. Maloy W.L. Koning F. Valas R. Coligan J.E. J. Immunol. 1987; 138: 807-814Google Scholar) were a generous gift from Dr. Frits Koning (Leiden University Medical Center). Polyclonal antibodies against gp78 (34Fang S. Ferrone M. Yang C. Jensen J.P. Tiwari S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14422-14427Google Scholar) were kindly provided by Dr. Allan Weissman (NCI, National Institutes of Health). Ubiquitin was detected with a monoclonal antibody from Santa Cruz Biotechnology (clone P4D1). Horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies were from Jackson ImmunoResearch Laboratories.Cells—NIH-3T3 cells were grown in Medium A (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 10 mm Na-HEPES, pH 7.4, 2 mm glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin). To obtain stably transfected cells, an HEK 293 packaging cell line was transfected with pLNC-HRD1 or pLNC-HRD1-C2A. Cells were selected with 1 mg/ml Geneticin (Invitrogen), and recombinant retrovirus was collected from the supernatant. NIH-3T3 cells were transduced with the recombinant retroviruses, and expressing clones were isolated by limiting dilution. These cells were maintained in Medium A supplemented with 250 μg/ml Geneticin. HeLa cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mm glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin.Metabolic Labeling and Immunoprecipitation—Cells were transfected using jetPEI (Qbiogene Molecular Biology) or LipofectAMINE Plus™ (Invitrogen) according to the manufacturer's instructions. Where indicated, HMGR expression was up-regulated 14-16 h prior to radioactive labeling by refeeding the cells with Medium B (Dulbecco's modified Eagle's medium supplemented with 10% lipoprotein-deficient serum, 2 mm glutamine, 2 μm compactin, and 100 μm mevalonate). At 24 or 48 h after transfection cells were starved and metabolically labeled with 35S-amino acids as described previously (14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar) and chased for the times indicated. Lysates were made in Nonidet P-40 lysis mixture (50 mm Tris/HCl, pH 7.4, 5 mm MgCl2, and 0.5% (v/v) Nonidet P-40), and proteins were immunoprecipitated using Protein A- and G-Sepharose beads as described previously (14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar). Dried polyacrylamide gels were analyzed using phosphorimaging technology.PAGE and Immunoblotting—Transfected cells were harvested 24 or 48 h after transfection, lysed in a small volume of Nonidet P-40 lysis mixture, incubated for 30 min on ice, and centrifuged for 10 min at 14,000 × g. One volume of 2× sample buffer (40 mm Tris/HCl, pH 8.0, 4 mm EDTA, 8% (w/v) SDS, 40% (w/v) glycerol, 0.1% bromphenol blue) was added to the supernatant, and the samples were incubated at 95 °C for 5 min. Proteins were separated on polyacrylamide gels and blotted onto Optitran BAS-83 reinforced nitrocellulose membranes (Schleicher & Schuell) or polyvinylidene difluoride membrane (PerkinElmer Life Sciences). Immunodetected proteins were visualized using chemiluminescence. Quantifications were done using Quantity-One software (Bio-Rad).In Vitro Ubiquitination Assay—The glutathione S-transferase (GST)-HRD1-RING fusion protein was obtained from bacterial expression of a plasmid in which the nucleotide sequence coding for residues 272-343 of human HRD1 was inserted downstream of the BamHI site of the vector pGEX-4T1. The expressed protein was purified by affinity chromatography on glutathione-coupled gel beads. The GST-Hrd1p-RING fusion protein was similarly obtained by replacing the human HRD1 sequence with that coding for residues 331-413 of the S. cerevisiae Hrd1p. The ubiquitin-conjugating enzymes (E2) yeast Ubc7p, yeast Ubc4p, yeast Ubc2p, human UBC7, human UbcH5b, and human UBC2 were obtained as described previously (43Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Google Scholar, 44Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Google Scholar, 45Cook W.J. Martin P.D. Edwards B.F. Yamazaki R.K. Chau V. Biochemistry (Mosc.). 1997; 36: 1621-1627Google Scholar). Purified E1 was obtained by expression of a S. cerevisiae N-terminally poly-His-tagged UBA1 coding sequence in a Δuba1 strain (kindly provided by J. Dohlman) and subsequent purification by sequential nickel affinity and ubiquitin affinity chromatography. The concentrations of E1 and E2 were determined by measuring the amount of ubiquitin that forms a thiol ester bond with the enzymes. The concentration of GST fusion proteins was obtained from absorbance measured at 280 nm in 8 m urea using a molar extinction coefficient based on their tryptophan, tyrosine, and phenylalanine content.In vitro ubiquitination assays were carried out at 30 °C in a reaction mixture containing 25 mm HEPES, pH 7.5, 1 mm ATP, 10 mm MgCl2, 10 nm E1 enzyme, 1 μm GST-RING fusion protein, a 0.1 μm concentration of an E2 enzyme, and 50 μm ubiquitin. Reactions were terminated by the addition of SDS gel sample buffers, and protein components were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining.Immunofluorescence Assay—Immunofluorescence of transfected protein was performed as described in Kikkert et al. (14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar).In Vitro Translation and Proteinase K Digestion—Human HRD1 was transcribed in vitro with T7 RNA polymerase using an in vitro transcription system (Invitrogen), and the resulting RNA was translated using a Promega in vitro translation kit in the presence of 35S-labeled methionine (Amersham Biosciences) and canine pancreatic microsomal membranes. Translation reactions were performed at 30 °C for 90 min.For proteinase K digestions, microsomal membranes were centrifuged at 14,000 × g at 4 °C for 15 min and washed with 100 μl of KMH buffer (110 mm KAc, 2 mm MgAc, and 20 HEPES-KOH, pH 7.2). Proteinase K digestions were performed in 50 μl of KMH buffer or Nonidet P-40 lysis mixture for 30 min on ice at concentrations indicated. After digestion, 1 μl of 500 mm phenylmethylsulfonyl fluoride was added to the Nonidet P-40 samples, and 200 μl of KMH buffer containing 4 mm phenylmethylsulfonyl fluoride was added to the KMH buffer samples. The microsomes were centrifuged at 14,000 × g at 4 °C for 15 min and resuspended in 60 μl of Nonidet P-40 lysis mixture containing 1 μl of 500 mm phenylmethylsulfonyl fluoride. After lysis for 20 min, samples were cleared by centrifugation at 14,000 × g at 4 °C for 15 min. The supernatant was split and used for either direct loads or immunoprecipitation. Immunoprecipitations, SDS-PAGE, and phosphorimaging were performed as described previously (14Kikkert M. Hassink G. Barel M. Hirsch C. van der Wal F.J. Wiertz E. Biochem. J. 2001; 358: 369-377Google Scholar).RESULTSExpression, N-Linked Glycosylation, and Topology of Human HRD1—To gain information on the expression of human HRD1, a polyclonal antiserum was developed against human HRD1 by immunizing a rabbit with the C-terminal 228 amino acids of the HRD1 protein fused at the N terminus to maltose-binding protein. This antiserum specifically recognized a single protein of 81 kDa in HeLa cells (Fig. 1A, lane 1). We found that a 4-h treatment with tunicamycin resulted in a 3-4-fold increase in the amount of endogenous HRD1 protein in HeLa cells (Fig. 1A, lane 2). This is in agreement with the observation that elevated HRD1 mRNA levels occur under ER stress conditions (46Kaneko M. Ishiguro M. Niinuma Y. Uesugi M. Nomura Y. FEBS Lett. 2002; 532: 147-152Google Scholar). As judged from pulse-chase experiments, both endogenous (Fig. 1B) and transiently transfected human HRD1 protein (Fig. 1C) display a half-life of ∼15 h in HeLa cells (Fig. 1, B and C), which renders HRD1 a protein of relatively high stability. Fig. 1D shows subcellular localization of transfected human HRD1 in HeLa cells. Most HRD1 is localized in a typical lacy pattern, characteristic for the ER. This is confirmed by the co-localization with calnexin, an ER-resident chaperone (Fig. 1D). Besides the ER pattern, clustered protein was detected in some of the HeLa cells transiently transfected with human HRD1 (Fig. 1D, arrows). These clusters also co-localized with calnexin, suggesting that they may be ER membrane-derived (Fig. 1D). Staining with anti-vimentin antibodies did not show any vimentin “cages” characteristic of aggresomes (data not shown). We conclude that human HRD1 is a stable protein localized in the ER.Fig. 2A shows a schematic representation of the predicted membrane topology of human HRD1 based on its hydrophobicity plot (according to Kyte and Doolittle (47Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Google Scholar)) and predictions through several algorithms from the Expasy internet site (SOSUI (TUAT, Tokyo University of Agriculture and Technology), TMHMM (CBS, Copenhagen, Denmark), and others). The resulting model contains six putative transmembrane domains and a RING finger-containing C-terminal domain positioned in the cytosol. Two potential N-linked glycosylation sites (NX(T/S)) were found in the sequence that are indicated in the model as well. Since one N-linked gly
Endoplasmic-reticulum-associated protein degradation
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ABSTRACT The PERK arm of the unfolded protein response (UPR) regulates cellular proteostasis and survival in response to endoplasmic reticulum (ER) stress. However, the impact of PERK signaling on extracellular proteostasis is poorly understood. We define how PERK signaling influences extracellular proteostasis during ER stress using a conformational reporter of the secreted amyloidogenic protein transthyretin (TTR). We show that inhibiting PERK signaling impairs ER stress-dependent secretion of destabilized TTR by increasing its ER retention in chaperone-bound complexes. Interestingly, PERK inhibition promotes the ER stress-dependent secretion of TTR in non-native conformations that accumulate extracellularly as soluble oligomers. Pharmacologic or genetic TTR stabilization partially restores secretion of native TTR tetramers. However, PERK inhibition still increases the ER stress-dependent secretion of TTR in non-native conformations under these conditions, indicating that the conformation of stable secreted proteins can also be affected by inhibiting PERK. Our results define a role for PERK in regulating extracellular proteostasis during ER stress and indicate that genetic or aging-related alterations in PERK signaling can exacerbate ER stress-related imbalances in extracellular proteostasis implicated in diverse diseases.
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Endoplasmic-reticulum-associated protein degradation
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Protein Degradation
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Abstract The PERK arm of the unfolded protein response (UPR) regulates cellular proteostasis and survival in response to endoplasmic reticulum (ER) stress. However, the impact of PERK signaling on extracellular proteostasis is poorly understood. We define how PERK signaling influences extracellular proteostasis during ER stress using a conformational reporter of the secreted amyloidogenic protein transthyretin (TTR). We show that inhibiting PERK signaling impairs secretion of destabilized TTR during thapsigargin (Tg)-induced ER stress by increasing its ER retention in chaperone-bound complexes. Interestingly, PERK inhibition increases the ER stress-dependent secretion of TTR in non-native conformations that accumulate extracellularly as soluble oligomers. Pharmacologic or genetic TTR stabilization partially restores secretion of native TTR tetramers. However, PERK inhibition still increases the ER stress-dependent secretion of TTR in non-native conformations under these conditions, indicating that the conformation of stable secreted proteins can also be affected by inhibiting PERK. Our results define a role for PERK in regulating extracellular proteostasis during ER stress and indicate that genetic or aging-related alterations in PERK signaling can exacerbate ER stress-related imbalances in extracellular proteostasis implicated in diverse diseases.
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Tunicamycin
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RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
Endoplasmic-reticulum-associated protein degradation
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The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER) is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR), distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI) is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.
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