Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress
Ryosuke NakatoYu OhkuboAkari KonishiMari ShibataYuki KanekoTakao IwawakiTomohiro NakamuraStuart A. LiptonTakashi Uehara
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Abstract Protein S -nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S -nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S -nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S -nitrosylate the ER stress sensors IRE1α and PERK. While S -nitrosylation of IRE1α inhibited its ribonuclease activity, S -nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S -nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S -nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.Keywords:
S-Nitrosylation
Nitrosylation
Endoplasmic-reticulum-associated protein degradation
Endoplasmic Reticulum Associated Degradation (ERAD) clears misfolded or incorrectly processed proteins from the ER. One family of ER resident proteins that are involved in ERAD and exhibit disulfide redox, isomerization, and chaperone activity is the Protein Disulfide Isomerase (PDI) family. In humans there are twenty PDI homologs, whereas in Saccharomyces cerevisiase there are five. To address substrate specificity among the PDI family members and their mechanisms of action during ERAD, we investigated the contributions of distinct yeast PDIs on the ERAD of model substrates that either contain disulfide bonds or lack cysteines. Through the use of a yeast expression system for Apolipoprotein B (ApoB), which is disulfide‐rich, we discovered that Pdi1 interacts with ApoB and facilitates degradation through its chaperone activity. In contrast, Pdi1's redox activity was required for the ERAD of CPY*, an ERAD substrate containing five disulfide bonds. Distinct effects of mammalian PDI homologues on ApoB degradation were then observed in hepatic cells. These data indicate that PDIs contribute to ERAD through different mechanisms.
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Nitrosylation
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Abstract Background P2X7 receptor (P2X7R) is an ATP-gated nonselective cationic channel playing important roles in a variety of physiological functions, including inflammation, and apoptotic or necrotic cell death. An extracellular domain has ten cysteine residues forming five intrasubunit disulfide bonds, which are needed for the P2X7R trafficking to the cell surface and the recognition of surface epitopes of apoptotic cells and bacteria. However, the underlying mechanisms of redox/ S -nitrosylation of cysteine residues on P2X7R and its role in P2X7R-mediated post-status epilepticus (SE, a prolonged seizure activity) events remain to be answered. Methods Rats were given pilocarpine (380 mg/kg i.p.) to induce SE. Animals were intracerebroventricularly infused N ω -nitro- l -arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) 3 days before SE, or protein disulfide isomerase (PDI) siRNA 1 day after SE using an osmotic pump. Thereafter, we performed Western blot, co-immunoprecipitation, membrane fraction, measurement of S -nitrosylated (SNO)-thiol and total thiol, Fluoro-Jade B staining, immunohistochemistry, and TUNEL staining. Results SE increased S -nitrosylation ratio of P2X7R and the PDI-P2X7R bindings, which were abolished by L-NAME and PDI knockdown. In addition, both L-NAME and PDI siRNA attenuated SE-induced microglial activation and astroglial apoptosis. L-NAME and PDI siRNA also ameliorated the increased P2X7R surface expression induced by SE. Conclusions These findings suggest that PDI-mediated redox/ S -nitrosylation may facilitate the trafficking of P2X7R, which promotes microglial activation and astroglial apoptosis following SE. Therefore, our findings suggest that PDI-mediated regulations of dynamic redox status and S -nitrosylation of P2X7R may be a critical mechanism in the neuroinflammation and astroglial death following SE.
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Up-regulation of protein disulfide isomerase (PDI) is an adaptive response to accumulation of misfolded proteins in the endoplasmic reticulum (ER) that helps protect neurons from apoptosis resulting from ER stress. After determining that exposure to a nitric oxide (NO) donor or activation of neuronal NO synthase (nNOS) expressed in HEK-293T cells led to PDI S -nitrosylation, Uehara et al. showed that S -nitrosylated PDI was present in the brains of people who had had Parkinson's disease or Alzheimer's disease. S -nitrosylation impaired the ability of PDI to act as a chaperone (assayed by inhibition of guanidinium-dependent rhodanese aggregation) and an isomerase (assayed by renaturation of an inactive form of RNase A with scrambled disulfide bonds). When coexpressed with synphilin-1 in a dopaminergic neuroblastoma cell line, PDI inhibited the development of synphilin-1-dependent Lewy-body-like inclusions, a protective effect that was attenuated by NO. Exposure of cultured cortical neurons to N -methyl-D-aspartate (NMDA) led to NOS-sensitive accumulation of S -nitrosylated PDI, accumulation of polyubiquitinated proteins, and apoptosis; PDI overexpression decreased the number of polyubiquitinated and apoptotic cells and attenuated NMDA-dependent activation of the unfolded protein response. In neuroblastoma cells, PDI overexpression inhibited cell death in response to ER stress, inhibition of the proteasome, or overexpression of a protein that induces the unfolded protein response, protective effects that were reversed by exposure to a NO donor. Thus, the authors conclude that, in neurodegenerative disorders, S -nitrosylation of PDI by NO attenuates its ability to protect neurons from the neurotoxic effects of ER stress. T. Uehara, T. Nakamura, D. Yao, Z.-Q. Shi, Z. Gu, Y. Ma, E. Masliah, Y. Nomura, S. A. Lipton, S -nitrosylated protein-disulphide isomerase links protein misfolding to neurodegeneration. Nature 441 , 513-517 (2006). [PubMed]
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The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway. The endoplasmic reticulum (ER) has a strict protein quality control system. Misfolded proteins generated in the ER are degraded by the ER-associated degradation (ERAD). Yeast Mnl1p consists of an N-terminal mannosidase homology domain and a less conserved C-terminal domain and facilitates the ERAD of glycoproteins. We found that Mnl1p is an ER luminal protein with a cleavable signal sequence and stably interacts with a protein-disulfide isomerase (PDI). Analyses of a series of Mnl1p mutants revealed that interactions between the C-terminal domain of Mnl1p and PDI, which include an intermolecular disulfide bond, are essential for subsequent introduction of a disulfide bond into the mannosidase homology domain of Mnl1p by PDI. This disulfide bond is essential for the ERAD activity of Mnl1p and in turn stabilizes the prolonged association of PDI with Mnl1p. Close interdependence between Mnl1p and PDI suggests that these two proteins form a functional unit in the ERAD pathway. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; MHD, mannosidase homology domain; EDEM, ER degradation enhancing α-mannosidase-like protein; DTT, dithiothreitol; CPY, caboxypeptidase Y; CPY*, a mutated version of caboxypeptidase Y; PMSF, phenylmethylsulfonyl fluoride; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; PDI, protein disulfide isomerase; WT, wild type. 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; MHD, mannosidase homology domain; EDEM, ER degradation enhancing α-mannosidase-like protein; DTT, dithiothreitol; CPY, caboxypeptidase Y; CPY*, a mutated version of caboxypeptidase Y; PMSF, phenylmethylsulfonyl fluoride; AMS, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid; PDI, protein disulfide isomerase; WT, wild type. is the first organelle in the secretory pathway of eukaryotic cells and provides an optimum environment for maturation of newly synthesized secretory and membrane proteins. Protein folding/assembly in the ER is aided by molecular chaperones and folding enzymes. Molecular chaperones in the ER assist folding of newly synthesized proteins and prevent them from premature misfolding and/or aggregate formation (1Buck T.M. Wright C.M. Brodsk J.L. Semin. Cell Dev. Biol. 2007; 18: 751-761Crossref PubMed Scopus (65) Google Scholar, 2Anelli T. Sitia R. EMBO J. 2008; 27: 315-327Crossref PubMed Scopus (454) Google Scholar). Protein folding in the ER is often associated with formation of disulfide bonds, which contribute to stabilization of native, functional states of proteins. Disulfide bond formation could be a rate-limiting step of protein folding both in vitro and in vivo (3Creighton T.E. Zapun A. Darby N.J. Trends. Biotech. 1995; 13: 18-23Abstract Full Text PDF PubMed Scopus (89) Google Scholar, 4Molinari M. Helenius A. Nature. 1999; 402: 90-93Crossref PubMed Scopus (271) Google Scholar), and the ER has a set of folding enzymes including protein-disulfide isomerase (PDI) and its homologs that catalyze disulfide bond formation (5Sevier C.S. Kaiser C.A. Antioxid. Redox. Signal. 2006; 8: 797-811Crossref PubMed Scopus (91) Google Scholar, 6Appenzeller-Herzog C. Ellgaard L. Biochim. Biophys. Acta. 2008; 1783: 535-548Crossref PubMed Scopus (302) Google Scholar). In parallel, protein folding/assembly in the ER relies on the inherent failsafe mechanism, i.e. the ER quality control system, to ensure that only correctly folded and/or assembled proteins can exit the ER. Misfolded or aberrant proteins are retained in the ER for refolding by ER-resident chaperones, whereas terminally misfolded proteins are degraded by the mechanism known as ER-associated degradation (ERAD). The ERAD consists of recognition and processing of aberrant substrate proteins, retrotranslocation across the ER membrane, and subsequent proteasome-dependent degradation in the cytosol. More than 20 different components have been identified to be involved in this process in yeast and mammals (7Nakatsukasa K. Brodsky J.L. Traffic. 2008; 9: 861-870Crossref PubMed Scopus (232) Google Scholar). The majority of proteins synthesized in the ER are glycoproteins, in which N-linked glycans are not only important for folding but also crucial for their ERAD if they fail in folding. Specifically, trimming of one or more mannose residues of Man9GlcNAc2 oligosaccharide and recognition of the modified mannose moiety represent a key step for selection of terminally misfolded proteins for disposal (8Jakob C.A. Burda P. Roth J. Aebi M. J. Cell Biol. 1998; 142: 1223-1233Crossref PubMed Scopus (299) Google Scholar). A mannosidase I-like protein, Mnl1p/Htm1p (yeast), and EDEM (mammals, ER degradation enhancing α-mannosidase-like protein) were identified as candidates for lectins that recognize ERAD substrates with modified mannose moieties (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar, 11Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar). Both Mnl1p and EDEM contain an N-terminal mannosidase homology domain (MHD), which lacks cysteine residues conserved among α1,2-mannosidase family members and is proposed to function in recognition of mannose-trimmed carbohydrate chains (supplemental Fig. S1). However, whether Mnl1p or EDEM indeed functions as an ERAD-substrate-binding lectin or has a mannosidase activity is still in debate (11Hosokawa N. Wada I. Hasegawa K. Yorihuzi T. Tremblay L.O. Herscovics A. Nagata K. EMBO Rep. 2001; 2: 415-422Crossref PubMed Scopus (380) Google Scholar, 12Hirao K. Natsuka Y. Tamura T. Wada I. Morito D. Natsuka S. Romero P. Sleno B. Tremblay L.O. Herscovics A. Nagata K. Hosokawa N. J. Biol. Chem. 2006; 281: 9650-9658Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 13Olivari S. Cali T. Salo K.E. Paganetti P. Ruddock L.W. Molinari M. Biochem. Biophys. Res. Commun. 2006; 349: 1278-1284Crossref PubMed Scopus (142) Google Scholar, 14Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 15Clerc S. Hirsch C. Oggier D.M. Deprex P. Jakob C. Sommer T. Aebi M. J. Cell Biol. 2009; 184: 159-172Crossref PubMed Scopus (199) Google Scholar), and Yos9p was suggested to take the role of ERAD-substrate binding lectin (14Quan E.M. Kamiya Y. Kamiya D. Denic V. Weibezahn J. Kato K. Weissman J.S. Mol. Cell. 2008; 32: 870-877Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 16Bhamidipati A. Denic V. Quan E.M. Weissman J.S. Mol. Cell. 2005; 16: 741-751Abstract Full Text Full Text PDF Scopus (189) Google Scholar, 17Kim W. Spear E.D. Ng D.T. Mol. Cell. 2005; 16: 753-764Abstract Full Text Full Text PDF Scopus (149) Google Scholar, 18Szathmary R. Bielmann R. Nita-Lazar M. Burda P. Jakob C.A. Mol. Cell. 2005; 16: 765-775Abstract Full Text Full Text PDF Scopus (164) Google Scholar). Mnl1p, but not EDEM, has a large C-terminal extension, which does not show any homology to known functional domains and is conserved only among fungal Mnl1p homologs (supplemental Fig. S1). After recognition of the modified mannose signal for degradation, aberrant proteins are maintained or converted to be retrotranslocation competent by ER chaperones including BiP (19Nishikawa S. Fewell S.W. Kato Y. Brodsky J.L. Endo T. J. Cell Biol. 2001; 153: 1061-1070Crossref PubMed Scopus (255) Google Scholar). PDI was also indicated to be involved in these steps in the ERAD by, for example, its possible chaperone-like functions (20Gilbert H.F. J. Biol. Chem. 1997; 272: 29399-29402Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 21Klappa P. Hawkins H.C. Freedman R.B. Eur. J. Biochem. 1997; 238: 38-42Google Scholar, 22Gillece P. Luz J.M. Lennarz W. de la Cruz F.J. Römisch K. J. Cell Biol. 1999; 147: 1443-1456Crossref PubMed Scopus (151) Google Scholar, 23Nørgaard R. Westphal V. Tachibana C. Alsøe L. Horst B. Winther J.R. J. Cell Biol. 2001; 152: 553-562Crossref PubMed Scopus (105) Google Scholar). The yeast PDI, Pdi1p, contains four thioredoxin-like domains, two of which have a CGHC motif as active sites, followed by a C-terminal extension containing the ER retention signal. During its catalytic cycle, PDI transiently forms a mixed disulfide intermediate with its substrate through an intermolecular disulfide bond between the cysteine residues of the active site of PDI and the substrate molecule. Here we report identification of PDI as an Mnl1p-interacting protein. Stable interactions between the C-terminal domain of Mnl1p and PDI involve intermolecular disulfide bonds. Stably interacting PDI is required for formation of the functionally essential intramolecular disulfide bond in the MHD of Mnl1p, which in turn stabilizes and prolongs the Mnl1p-PDI interactions. Possible roles for those stable interactions between Mnl1p and PDI in the ERAD will be discussed. Strains, Plasmids, and Culturing Conditions—Yeast strains used in this study are W303-1A (MATa ade2 ura3 leu2 trp1 his3 ade2 can1), SEY6210 (MATa ura3 leu2 trp1 his3 lys2 suc2) (24Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (722) Google Scholar), SNY1079 (MATa mnl1::HIS3 ura3 leu2 trp1 his3 lys2 suc2) (24Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (722) Google Scholar), SNY1080 (MATa prc1-1 mnl1::HIS3 ura3 leu2 trp1 his3 lys2 suc2) (25Nishikawa S. Endo T. J. Biol. Chem. 1997; 272: 12889-12892Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and PBY3–9B (MATa sec11-7 ura3 leu2 his4) (26Böhni P.C. Deshaies R.J. Schekman R.W. J. Cell Biol. 1988; 106: 1035-1042Crossref PubMed Scopus (104) Google Scholar). KRY94 (MATa pdi1::HIS3 ade2 ura3 leu2 trp1 his3 ade2 can1 pPDI-URA3) (27LaMantia M.L. Lennarz W.J. Cell. 1993; 74: 899-908Abstract Full Text PDF PubMed Scopus (181) Google Scholar) is a gift from K. Römisch (Universität des Saarlandes). Yeast cells were grown in YPD (1% yeast extract, 2% polypeptone, and 2% glucose) or SCD (0.67% yeast nitrogen base without amino acids, 2% glucose, 0.5% casamino acids) with appropriate supplements. The mnl1::CgTRP1 allele was constructed as follows. A DNA fragment containing the Candida glabrata TRP1 gene (CgTRP1) was amplified by PCR using pCgTRP1 (28Kitada K. Yamaguchi E. Arisawa M. Gene (Amst.). 1995; 165: 203-206Crossref PubMed Scopus (128) Google Scholar) as a template with primers 5′-GAAGACGATGCGTACTCATTCACTTCTAAAGAACTTAAGGGTTGTAAAACGACGGCCAGT-3′ and 5′-GTGGGGGAAACTCCGGAGGACTAAAGTTCCACCTTTCAGGCACAGGAAACAGCTATGACC-3′. The amplified DNA fragment, flanked by 40 base pairs each of the upstream and downstream sequences of the MNL1 gene, was introduced into KRY94, and Trp+ transformants were selected. Disruption of the MNL1 gene was confirmed by PCR, and the resulting strain was named W303-1A mnl1ΔpdiΔ/pPDI-URA3. Plasmids expressing C-terminally FLAG-tagged Mnl1p (Mnl1p-FLAG) was generated as follows. The MNL1 gene was amplified by PCR using yeast genomic DNA as a template with primers 5′-GCGCTCGAGTGACCGATCCACCCTTTAAG-3′ and 5′-GCGGAGCTCCTTTCCTCAATAGTGGTGTA-3′. The amplified 3.3-kilobase pair DNA fragment was digested with SacI and XhoI and inserted into the SacI-XhoI sites of pRS316 (29Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) to give pSNA27. A BglII site was inserted between the 796th codon and the stop codon of the MNL1 gene by oligonucleotide-directed mutagenesis to give pKHY1. A DNA fragment for the 3×FLAG tag sequence was amplified by PCR with primers 5′-GGCGAATTGGGATCCGGGCCCGAC-3′ and 5′-CGCGGATCCGTCGACGGGGGGCCTCTT-3′ using pTYE247 (30Yoshihisa T. Yunoki-Esaki K. Ohshima C. Tanaka N. Endo T. Mol. Biol. Cell. 2003; 14: 3266-3279Crossref PubMed Scopus (133) Google Scholar) as a template. The amplified DNA fragment was digested with BglII and inserted into the BglII site of pKHY1 to give pKHY3. The 3.4-kb SacI-XhoI fragment of pKHY3 was introduced into the SacI-XhoI sites of pYO326 (31Qadota H. Ishii I. Fujiyama A. Ohya Y. Anraku Y. Yeast. 1992; 8: 735-741Crossref PubMed Scopus (53) Google Scholar) to give pMAY5. A series of the Cys → Ser Mnl1p mutants, the Ala substitution mutants for the conserved residues in the C-terminal domain of Mnl1p, and the ΔC Mnl1p mutant were constructed by oligonucleotide-directed mutagenesis using pMAY5 as a template. pPDI-TRP1 and pPDI-S1S2 plasmids are provided from W. J. Lennarz (Stony Brook University). pPDI-S5S6 and a series of S3S4 mutants of PDI were constructed by oligonucleotide-directed mutagenesis using pPDI-TRP1 as a template. pPDI-TRP1, pPDI-S1S2, or pPDI-S5S6 was introduced into W303-1A mnl1ΔpdiΔ/pPDI-URA3, and then pPDI-URA was removed by growing the transformants on medium containing 1 mg/ml 5-fluoroorotic acid. Preparation of Microsomes and Crude Membrane Fractions—The microsomes were prepared as described by McCracken and Brodsky (32McCracken A.A. Brodsky J.L. J. Cell Biol. 1996; 132: 291-298Crossref PubMed Scopus (343) Google Scholar). Crude membrane fractions were prepared as described below. Yeast cells (1.5 × 107 cells) collected from exponentially growing cell cultures were suspended in 1 ml of 0.1 m Tris-SO4, pH 9.4, and 10 mm DTT and immediately collected by centrifugation at 9,500 × g for 30 s at 4 °C. The cells were converted to spheroplasts by incubating in 1 ml of 20 mm Tris-HCl, pH 7.4, 1.2 m sorbitol, and 0.02 mg/ml Zymolyase 20T (Seikagaku Corporation) for 15 min at 30 °C. The spheroplasts were suspended in 100 μl of 100 mm sorbitol, 50 mm potassium acetate, 2 mm EDTA, 1 mm PMSF, 10 mm Hepes-KOH, pH 7.4, and 10 mm DTT and disrupted by vortexing for 1 min with glass beads (∼100 mg) for two cycles with an 1.5-min interval on ice. In Fig. 1D, DTT was omitted from this solution. Cell homogenates were diluted 5-fold with the same solution as used in homogenization and centrifuged at 550 × g for 5 min at 4 °C to remove cell debris. The supernatant was centrifuged at 15,000 × g for 15 min at 4 °C, and the resulting pellet was used as the crude membrane fraction. Immunoprecipitation—Microsomes or crude membranes were suspended in 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl, 1% Nonidet P-40, and protease inhibitor mixture for use with mammalian cell and tissue extracts (PiC; Sigma-Aldrich), incubated on ice for 1 h and centrifuged at 15,000 × g for 5 min at 4 °C to remove insoluble materials. The supernatant was diluted 10-fold with 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl, and PiC and incubated with anti-FLAG M2-agarose (Sigma-Aldrich) or anti-PDI antibody-bound protein G-Sepharose at 4 °C for more than 3 h. The immunoprecipitated materials were washed twice with 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl, and 0.1% Nonidet P-40 and eluted with sample buffer for SDS-PAGE without 2-mercaptoethanol. In Fig. 2E, 3 and 5B, spheroplasts were incubated in medium containing 1.2 m sorbitol for 30 min at 30 °C, and the proteins were precipitated by incubation with 10% trichloroacetic acid on ice for 10 min followed by centrifugation at 15,000 × g for 5 min at 4 °C. The precipitated materials were washed twice with cold acetone, and the proteins were suspended in 2% SDS, 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl, 35 mm iodoacetamide, and PiC and incubated at 94 °C for 5 min. Insoluble materials were removed by centrifugation at 15,000 × g for 5 min at 4 °C. The supernatant was diluted 10-fold with 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl, and PiC and incubated with anti-FLAG M2-agarose at room temperature for 1 h. The immunoprecipitates were washed twice with 20 mm Hepes-KOH, pH 7.4, 50 mm NaCl and eluted with sample buffer for SDS-PAGE without 2-mercaptoethenol. For SDS-PAGE under reducing conditions, 2-mercaptoethanol was added to the sample at 3% prior to the analyses.FIGURE 3Cys → Ser mutants of Mnl1p. A, schematic representation of Mnl1p with eight Cys residues. The signal sequence and the MHD are shown in black and gray boxes, respectively. B, spheroplasts prepared from cells expressing WT Mnl1p-FLAG and a series of Cys → Ser Mnl1p-FLAG mutants and from those with a vector alone (vec) were analyzed as in Fig. 2E. The open and filled circles indicate the PDI-Mnl1p-FLAG complexes formed by the intermolecular disulfide bonds involving C6 or C5, respectively. Reduced and oxidized forms of the Mnl1p-FLAG monomer are indicated as red and ox, respectively. C, proteins in the cell lysate prepared in the presence of iodoacetamide from WT (W303-1A mnl1ΔpdiΔ/pPDI1, pMAY5), pdi1-S1S2 (S1S2; W303-1A mnl1ΔpdiΔ/pPDI1-S1S2, pMAY5) or pdi1-S5S6 (S5S6; W303-1A mnl1ΔpdiΔ/pPDI1-S5S6, pMAY5) cells expressing Mnl1p-FLAG from a multicopy plasmid were analyzed by nonreducing SDS-PAGE and immunoblotting (IB) with anti-Mnl1p antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Interactions between the C-terminal domain of Mnl1p and PDI without intermolecular disulfide bonds. A, sequence alignment of Mnl1p (residues 600–667) with its fungal orthologs. The accession numbers are: Vanderwaltozyma polyspora, XP_001643561; C. glabrata, XP_446361; Ashbya gossyppi, NP_983706; Kluyveromyces lactis, XP_451695; Pichia guilliermondii, XP_001484730; and Pichia stipitis, XP_001383807. Identical and similar residues are denoted with double (**) and single asterisks (*), respectively. Amino acid residues denoted with filled circles were replaced with Ala. B, spheroplasts prepared from cells expressing wild type Mnl1p-FLAG and a series of Ala substitution mutants from a multicopy plasmid and from those with a vector alone (vec) were analyzed as in Fig. 2E. The open and filled circles indicate the PDI-Mnl1p-FLAG complexes formed by the intermolecular disulfide bonds. Reduced and oxidized forms of the Mnl1p-FLAG monomer are indicated as red and ox, respectively. Mutants that do not form a disulfide-linked Mnl1p-PDI complex are highlighted by black boxes. C, membrane fractions were prepared from cells expressing wild type Mnl1p-FLAG and a series of Ala substitution mutants from a multicopy plasmid and from those with a vector alone in the presence of 10 mm DTT and were subjected to analyses as in Fig. 4. IP, immunoprecipitation; IB, immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Protease Protection Assay—The microsomes were incubated with or without 0.2 mg/ml proteinase K in 20 mm Hepes-KOH, pH 7.4, 100 mm KCl, 300 mm mannitol in the presence or absence of 1% Triton X-100 on ice for 30 min. The reaction was terminated by addition of 2 mm PMSF. The samples were incubated with 10% trichloroacetic acid for 10 min on ice, and the proteins were recovered by centrifugation at 15,000 × g for 5 min at 4 °C. The precipitates were washed twice with cold acetone and were solubilized by sample buffer for SDS-PAGE. Endoglycosidase H Treatment—Microsomes were suspended in 1% SDS and 1% 2-mercaptoethanol and incubated at 94 °C for 5 min. The samples were diluted 5-fold with 50 mm sodium citrate, pH 4.5, 1% Triton X-100, 1 mm PMSF, and 10 mm pepstatin A. Endoglycosidase H (Seikagaku Corporation) was added to 10 units/ml, and the samples were incubated for 16 h at 37 °C. Extraction of Mnl1p—The microsomes were incubated in 20 mm Hepes-KOH pH 7.4, 100 mm KCl, 300 mm mannitol, 1 mm EGTA, and 1 mm PMSF containing 0.1 m Na2CO3, pH 11.5, or 1% Triton X-100 on ice for 30 min. The samples were centrifuged at 80,000 × g for 30 min at 4 °C to separate soluble and insoluble fractions. Cycloheximide Chase Experiments—The cells were grown to OD600 = 1.5–2, and cycloheximide was added directly to the cell culture at 0.2 mg/ml. 0, 30, 60, or 90 min after the addition of cycloheximide, an equal volume of cell culture was removed and was incubated further in the presence of 10 mm NaN3 for 10 min on ice. Cell extracts were prepared as described by Yaffe and Schatz (33Yaffe, M. P., and Schatz, G. (198) Proc. Natl. Acad. Sci. U. S. A. 81, 4819–4823Google Scholar). Modification of Cys with AMS and Maleimde-PEG5000—The cells were suspended in 10% trichloroacetic acid and disrupted by agitation with glass beads. Trichloroacetic acid-precipitated proteins were collected by centrifugation at 15,000 × g for 5 min at 4 °C followed by washing twice with cold acetone. Protein pellets were suspended in 80 mm Tris-HCl, pH 6.8, 2% SDS, 6 m urea, 1 mm PMSF in the presence of 25 mm AMS (Invitrogen) or 5 mm maleimide-PEG5000 (Laysan Bio, Inc). After incubation on ice for 15 min at 37 °C for 10 min, the samples were boiled for 2 min and analyzed by SDS-PAGE under reducing conditions. To generate a fully reduced form of PDI, the trichloroacetic acid-precipitated pellets were boiled in 80 mm Tris-HCl, pH 6.8, 2% SDS, 6 m urea, 1 mm PMSF for 5 min, and the soluble fraction was incubated with 100 mm DTT at 30 °C for 30 min. Then proteins were precipitated with 10% trichloroacetic acid again and subsequently solubilized in 80 mm Tris-HCl, pH 6.8, 2% SDS, 6 m urea, 1 mm PMSF in the presence of 25 mm AMS or 5 mm maleimide-PEG5000. After incubation on ice for 15 min, at 37 °C for 10 min, the samples were boiled for 2 min and analyzed by SDS-PAGE under reducing conditions. Mnl1p Is an ER Luminal Protein with a Cleavable Signal Sequence—Mnl1p was previously shown to reside in the yeast ER (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We first asked whether the hydrophobic segment at the N terminus of Mnl1p functions as a cleavable signal sequence or a transmembrane segment to anchor the protein to the ER membrane. Immunoblotting of microsomes prepared from cells expressing FLAG-tagged Mnl1p (Mnl1p-FLAG) with the anti-FLAG antibody showed a 102-kDa band, which shifted to 91 kDa after treatment with endoglycosidase H, indicating that Mnl1p contains N-linked carbohydrate chains (Fig. 1A). Carboxypeptidase Y (CPY), which has four N-glycan sites, was used as a positive control. Mnl1p was, like an ER luminal protein BiP, resistant against externally added proteinase K but became protease-susceptible after disruption of the membrane with Triton X-100 (Fig. 1B). When treated with alkaline sodium carbonate, Mnl1p and BiP were mainly recovered in the supernatant after centrifugation, whereas Sec63p, an integral ER membrane protein, was recovered in the pellet (Fig. 1C, lanes 2 and 3). Treatment of the microsomes with Triton X-100 solubilized all of these proteins (Fig. 1C, lanes 4 and 5). These results collectively indicate that Mnl1p is an ER luminal protein, but not anchored to the ER membrane. Indeed, Mnl1p-FLAG and BiP in the yeast sec11 mutant, a temperature-sensitive mutant of the subunit of the ER signal peptidase (26Böhni P.C. Deshaies R.J. Schekman R.W. J. Cell Biol. 1988; 106: 1035-1042Crossref PubMed Scopus (104) Google Scholar), exhibited higher molecular weight bands (the bands from Mnl1p-FLAG with different extents of N-glycosylation became clearer after endonuclease H treatment) at restrictive temperature (37 °C) than at permissive temperature (23 °C) (Fig. 1D). Therefore the N-terminal hydrophobic segment of Mnl1p most likely functions as a cleavable signal sequence that guides the mature protein to the ER lumen. Mnl1p Interacts with PDI—Because Mnl1p facilitates ERAD of glycoproteins in the ER (9Nakatsukasa K. Nishikawa S. Hosokawa N. Nagata K. Endo T. J. Biol. Chem. 2001; 276: 8635-8638Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Jakob C.A. Bodmer D. Spirig U. Battig P. Marcil A. Dignard D. Bergeron J.J. Thomas D.Y. Aebi M. EMBO Rep. 2001; 2: 423-430Crossref PubMed Scopus (218) Google Scholar), we searched for its possible partner proteins cooperating with Mnl1p in the ERAD. When microsomes with Mnl1p-FLAG were solubilized with 1% Nonidet P-40 and subjected to immunoprecipitation with the immobilized anti-FLAG antibody, PDI was found to be retained on the beads in particular when Mnl1p-FLAG was expressed from a multicopy plasmid (Fig. 2, A and B). When solubilized microsomes were subjected to immunoprecipitation with the anti-PDI antibodies, Mnl1p-FLAG was in turn detected in the co-immunoprecipitated fractions (Fig. 2, C and D). Hemagglutinin-tagged Yos9p, hemagglutinin-tagged Mns1p (α1,2-mannosidase in yeast), or BiP was not detected by the anti-hemagglutinin antibody (for Yos9p and Mns1p) or anti-BiP antibodies (for BiP) in the co-immunoprecipitated fractions (data not shown). We next asked whether the interactions between Mnl1p and PDI involve intermolecular disulfide bonds. To minimize oxidation of Mnl1p after cell lysis, the proteins were precipitated by trichloroacetic acid from the spheroplasts expressing Mnl1p-FLAG and solubilized with SDS in the presence of iodoacetamide, which can alkylate thiol groups to prevent artificial disulfide shuffling. Then Mnl1p-FLAG was immunoprecipitated with the anti-FLAG antibody and analyzed by SDS-PAGE under reducing or nonreducing conditions followed by immunoblotting with anti-Mnl1p antibodies and anti-PDI antibodies (Fig. 2E). Mnl1p exhibited two high molecular weight bands of ∼250 kDa (denoted by circles) in addition to the band of 102 kDa corresponding to the Mnl1p monomer (denoted by a triangle) under nonreducing conditions (Fig. 2E, lanes 2 and 4) but not under reducing conditions (Fig. 2E, lanes 10 and 12). The 250–300-kDa bands were also detected by anti-PDI antibodies under nonreducing conditions (Fig. 2E, lanes 6 and 8), suggesting that the 250–300-kDa bands correspond to complexes formed by intermolecular disulfide bond(s) between Mnl1p and PDI. Quantification of the pulldown results under the conditions of Mnl1p being expressed from a multicopy plasmid suggests that about half the Mnl1p molecules interact with PDI and that ∼30% of PDI with Mnl1p through disulfide bonds (data not shown). Inter- and Intramolecular Disulfide Bond Formation of Mnl1p by PDI—Mnl1p contains eight cysteine residues in its mature domain, which are named C1–C8 (Fig. 3A); C1–C3 are in the MHD and C4–C8 in the C-terminal domain of Mnl1p. To identify the cysteine residues involved in the intermolecular disulfide bond with PDI, we made a series of Cys → Ser mutants of Mnl1p (C1S to C8S), in which each of the eight Cys was replaced by Ser, and analyzed their disulfide bond formation by nonreducing SDS-PAGE followed by immunoblotting with anti-Mnl1p antibodies and anti-PDI antibodies (Fig. 3B). The upper (filled circles) and lower (open circles) bands of the 250–300-kDa Mnl1p-PDI complexes disappeared in C5S and C6S mutant cells, respectively (Fig. 3B, lanes 30, 31, 41, and 42). Combination of the C5S and C6S mutations (C5S,C6S) resulted in the complete disappearance of the two bands (Fig. 3B, lanes 25 and 36). Therefore C5 and/or C6 of Mnl1p form intermolecular disulfide bonds with PDI, and the upper and lower bands of 250–300 kDa correspond to Mnl1p-PDI complexes with disulfide bonds involving C5 and C6, respectively. During the analyses of the Mnl1p-PDI complex, we noted that the migration rates of the Mnl1p monomer varied for different Cys mutants under nonreducing conditions, but not under reducing conditions (Fig. 3B). The C1S and C3S mutants showed faster migration rates than the wild type (WT) Mnl1p, suggesting that the upper band corresponds to the Mnl1p monomer with fully oxidized C1 and C3 (denoted as ox) and the lower band to the one without a disulfide bond between C1 and C3 (denoted as red). The S1S2 (C61S,C64S) and S5S6 (C406S,C409S) mutants of PDI have Cys → Ser mutations in the CGHC motif in one of the two active site thioredoxin domains (see Fig. 7A) and still have disulfide forming activity (34Holst B. Tachibana C. Winther J.R. J. Cell Biol. 1997; 138: 1229-1238Crossref PubMed Scopus (71) Google Scholar, 35Kulp M.S. Frickel E.M. Ellgaard L. Weissman J.S. J. Biol. Chem. 2006; 281: 876-884Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Mnl1p in cells expressing the S1S2 or S5S6 PDI mutants showed only the lower bands for the reduced Mnl1p monomer under nonreducing conditions (Fig. 3C), suggesting that oxidation of C1 and C3 requires both of the two active thioredoxin domains in PDI. Interestingly, the
Endoplasmic-reticulum-associated protein degradation
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Abstract Unfolded protein response (UPR) is a conserved signaling pathway that is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER) and stimulates production of ER chaperones to restore ER proteostasis. However, little is known how UPR-induced proteins return to their pre-stress levels upon removal of ER stress. TUNICAMYCIN-INDUCED1 (TIN1) is an Arabidopsis protein that is normally expressed in pollen but is rapidly induced by ER stresses in vegetative tissues. Here we show that the ER stress-induced TIN1 is rapidly degraded in the UPR recovery phase. We found that TIN1 degradation depends on its asparagine-linked glycans and requires both EMS-mutagenized bri1 suppressor 5 (EBS5) and EBS6 for its recruitment to the ER-associated degradation (ERAD) complex. Loss-of-function mutations in Arabidopsis ERAD components greatly stabilize TIN1. Interestingly, two other UPR-induced proteins that are coexpressed with TIN1 remained stable upon removal of ER stress, suggesting that rapid degradation during the stress-recovery phase likely applies to a subset of UPR-induced proteins. Further investigation should uncover the mechanisms by which the ERAD machinery differentially recognizes UPR-induced ER proteins.
Endoplasmic-reticulum-associated protein degradation
Tunicamycin
Proteostasis
Protein Degradation
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Endoplasmic-reticulum-associated protein degradation
Protein Degradation
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Abstract Protein S -nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S -nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S -nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S -nitrosylate the ER stress sensors IRE1α and PERK. While S -nitrosylation of IRE1α inhibited its ribonuclease activity, S -nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S -nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S -nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death.
S-Nitrosylation
Nitrosylation
Endoplasmic-reticulum-associated protein degradation
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