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• W2029451118 abstract "Newly synthesized thyroglobulin (Tg), the secretory glycoprotein that serves as precursor in thyroid hormone synthesis, normally forms transient covalent protein complexes with oxidoreductases of the endoplasmic reticulum (ER). The Tg-G2320R mutation is responsible for congenital hypothyroidism in rdw/rdw rats, in which a lack of secondary thyroid enlargement (goiter) implicates death of thyrocytes as part of disease pathogenesis. We found that mutant Tg-G2320R was retained within the ER with no detectable synthesis of thyroxine, had persistent exposure of free cysteine thiols, and was associated with activated ER stress response but incomplete ER-associated degradation (ERAD). Tg-G2320R associated with multiple ER resident proteins, most notably ERp72, including covalent Tg-ERp72 interactions. In PC Cl3 thyrocytes, inducible overexpression of ERp72 increased the ability of cells to maintain Tg cysteines in a reduced state. Noncovalent interactions of several ER chaperones with newly synthesized Tg-G2320R diminished over time in parallel with ERAD of the mutant protein, yet a small ERAD-resistant Tg fraction remained engaged in covalent association with ERp72 even 2 days post-synthesis. Such covalent protein aggregates may set the stage for apoptotic thyrocyte cell death, preventing thyroid goiter formation in rdw/rdw rats. Newly synthesized thyroglobulin (Tg), the secretory glycoprotein that serves as precursor in thyroid hormone synthesis, normally forms transient covalent protein complexes with oxidoreductases of the endoplasmic reticulum (ER). The Tg-G2320R mutation is responsible for congenital hypothyroidism in rdw/rdw rats, in which a lack of secondary thyroid enlargement (goiter) implicates death of thyrocytes as part of disease pathogenesis. We found that mutant Tg-G2320R was retained within the ER with no detectable synthesis of thyroxine, had persistent exposure of free cysteine thiols, and was associated with activated ER stress response but incomplete ER-associated degradation (ERAD). Tg-G2320R associated with multiple ER resident proteins, most notably ERp72, including covalent Tg-ERp72 interactions. In PC Cl3 thyrocytes, inducible overexpression of ERp72 increased the ability of cells to maintain Tg cysteines in a reduced state. Noncovalent interactions of several ER chaperones with newly synthesized Tg-G2320R diminished over time in parallel with ERAD of the mutant protein, yet a small ERAD-resistant Tg fraction remained engaged in covalent association with ERp72 even 2 days post-synthesis. Such covalent protein aggregates may set the stage for apoptotic thyrocyte cell death, preventing thyroid goiter formation in rdw/rdw rats. The endoplasmic reticulum (ER) 2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; Tg, thyroglobulin; PERK, protein kinase-like ER-resident kinase; IP, immunoprecipitation; PDI, protein disulfide-isomerase; T4, l-thyroxine; TUNEL, deoxynucleotidyl transferase-mediated dUTP nick end labeling; PEG, polyethylene glycol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline.2The abbreviations used are: ER, endoplasmic reticulum; ERAD, ER-associated degradation; Tg, thyroglobulin; PERK, protein kinase-like ER-resident kinase; IP, immunoprecipitation; PDI, protein disulfide-isomerase; T4, l-thyroxine; TUNEL, deoxynucleotidyl transferase-mediated dUTP nick end labeling; PEG, polyethylene glycol; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. serves as the compartment that initiates secretory protein biosynthesis and provides both a more oxidizing environment than the cytosol for promotion of disulfide bond formation as well as ER molecular chaperones that facilitate folding of secretory pathway polypeptides (1van Anken E. Braakman I. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 191-228Crossref PubMed Scopus (169) Google Scholar). Secretory polypeptides that fold to a native conformation become eligible for anterograde transport, whereas those that fail to fold correctly are retained within the ER until they either achieve an acceptable conformation or are targeted for ER-associated degradation (ERAD) (2McCracken A.A. Brodsky J.L. Curr. Top. Microbiol. Immunol. 2005; 300: 17-40PubMed Google Scholar). These processes of ER quality control (3Ellgaard L. Helenius A. Nat. Rev. Mol. Cell. Biol. 2003; 4: 181-191Crossref PubMed Scopus (1672) Google Scholar) help to prevent secretory protein congestion of the ER lumen that can adversely affect secretory pathway efficiency. A growing list of genetic diseases have been accounted for by structural changes in secretory proteins that alter their folding and render them deficient for ER export (4Rutishauser J. Spiess M. Swiss Med. Wkly. 2002; 132: 211-222PubMed Google Scholar). Many of these ER storage diseases produce a simple deficiency of the secreted protein (5Kim P.S. Arvan P. Endocr. Rev. 1998; 19: 173-202PubMed Google Scholar), whereas others may be cytotoxic to cells that attempt to produce them (6Wu J. Kaufman R.J. Cell Death Differ. 2006; 13: 374-384Crossref PubMed Scopus (722) Google Scholar). Preventing toxic accumulation of misfolded secretory proteins, for example via ERAD, is vital for proper ER homeostasis and may be a strategy for therapies of ER storage diseases (7Sekijima Y. Wiseman R.L. Matteson J. Hammarstrom P. Miller S.R. Sawkar A.R. Balch W.E. Kelly J.W. Cell. 2005; 121: 73-85Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 8Chen Y. Bellamy W.P. Seabra M.C. Field M.C. Ali B.R. Hum. Mol. Genet. 2005; 14: 2559-2569Crossref PubMed Scopus (51) Google Scholar). ERAD is built around highly conserved mechanisms that target misfolded proteins for intracellular disposal (9Meusser B. Hirsch C. Jarosch E. Sommer T. Nat. Cell Biol. 2005; 7: 766-772Crossref PubMed Scopus (999) Google Scholar). Key steps in this pathway include recognition of the ERAD substrate for unfolding and targeting for retrotranslocation to the cytosol (10Nishikawa S. Brodsky J.L. Nakatsukasa K. J. Biochem. 2005; 137: 551-555Crossref PubMed Scopus (129) Google Scholar) followed by proteolysis via the ubiquitin-proteasome system (11Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (363) Google Scholar). An inability to effectively degrade mutant secretory (or membrane) proteins may result in a gain-of-toxic-function that leads to cell death. This can occur either in an autosomal dominant manner, such as occurs with familial neurohypophyseal diabetes insipidus (12Russell T.A. Ito M. Ito M. Yu R.N. Martinson F.A. Weiss J. Jameson J.L. J. Clin. Investig. 2003; 112: 1697-1706Crossref PubMed Scopus (86) Google Scholar) or in an autosomal recessive manner, such as in patients expressing the Z-variant of α1-antitrypsin (13Perlmutter D.H. Methods Mol. Biol. 2003; 232: 39-56PubMed Google Scholar). Thyroglobulin (Tg), the major secretory glycoprotein of thyrocytes that serves as the precursor protein for thyroid hormone synthesis, begins to fold in the ER (14Kim P.S. Arvan P. J. Biol. Chem. 1991; 266: 12412-12418Abstract Full Text PDF PubMed Google Scholar, 15Kim P. Bole D. Arvan P. J. Cell Biol. 1992; 118: 541-549Crossref PubMed Scopus (101) Google Scholar) with the assistance of multiple molecular chaperones (16Kim P.S. Arvan P. J. Cell Biol. 1995; 128: 29-38Crossref PubMed Scopus (168) Google Scholar, 17Di Jeso B. Ulianich L. Pacifico F. Leonardi A. Vito P. Consiglio E. Formisano S. Arvan P. Biochem. J. 2003; 370: 449-458Crossref PubMed Google Scholar) including endogenous oxidoreductases that transiently form covalent, mixed disulfides with nascent Tg (18Di Jeso B. Park Y.-n. Ulianich L. Treglia A.S. Urbanas M.L. High S. Arvan P. Mol. Cell. Biol. 2005; 25: 9793-9805Crossref PubMed Scopus (62) Google Scholar). Tg deficiency is a well recognized cause of congenital hypothyroidism (19Medeiros-Neto G. Kim P.S. Yoo S.E. Vono J. Targovnik H. Camargo R. Hossain S.A. Arvan P. J. Clin. Investig. 1996; 98: 2838-2844Crossref PubMed Google Scholar) in which disease-causing Tg mutants are blocked in ER export and then are routed for proteasomal degradation (20Tokunaga F. Brostrom C. Koide T. Arvan P. J. Biol. Chem. 2000; 275: 40757-40764Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Consequently, the thyroid gland develops hypertrophy and hyperplasia (goiter) secondary to chronic elevation of thyroid stimulating hormone (21Di Jeso B. Arvan P. Braverman L.E. Utiger R. The Thyroid. 9th Ed. Lippincott Williams & Wilkins, Philadelphia2004Google Scholar). However, recent genetic linkage analyses have found the thyroglobulin gene region as a major locus in human familial congenital hypothyroidism even in the absence of goiter formation (22Ahlbom B.E. Yaqoob M. Gustavsson P. Abbas H.G. Anneren G. Larsson A. Wadelius C. Hum. Genet. 2002; 110: 145-147Crossref PubMed Scopus (9) Google Scholar). Moreover, in the rdw/rdw rat dwarf (23Koto M. Sato T. Okamoto M. Adachi J. Exp. Anim. 1988; 37: 21-30Crossref Scopus (31) Google Scholar), homozygous expression of the Tg-G2320R mutant (24Kim P.S. Ding M. Menon S. Jung C.G. Cheng J.M. Miyamoto T. Li B. Furudate S. Agui T. Mol. Endocrinol. 2000; 14: 1944-1953Crossref PubMed Scopus (62) Google Scholar, 25Hishinuma A. Furudate S. Oh-Ishi M. Nagakubo N. Namatame T. Ieiri T. Endocrinology. 2000; 141: 4050-4055Crossref PubMed Google Scholar) does not lead to goiter but rather to a hypoplastic thyroid gland (26Umezu M. Kagabu S. Jiang J. Sato E. Lab. Anim. Sci. 1998; 48: 496-501PubMed Google Scholar), suggesting a possible gain-of-toxic-function in affected thyrocytes. In this study, we have investigated the Tg-G2320R mutant protein and found persistent cysteine thiol exposure consistent with thiol-mediated ER retention (27Carelli S. Ceriotti A. Cabibbo A. Fassina G. Ruvo M. Sitia R. Science. 1997; 277: 1681-1684Crossref PubMed Scopus (82) Google Scholar), activation of the ER stress response, and extensive but incomplete ERAD with a small degradation-resistant fraction remaining engaged in covalent complexes with ERp72 for days, if not longer. Evidence is provided to suggest that ERp72 might function as a thiol reductase in the ER, and because interaction with ERp72 has recently been shown to inhibit ERAD of mutant Tg (28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (100) Google Scholar), these interactions might potentially predispose to toxicity of thyrocytes in rdw/rdw rats. PEG-Maleimide Treatment—Incubation was performed in Cys/Met-free DMEM containing 10 mm mPEG 5000-maleimide (SunBio) for 75 min at 37 °C. The samples were then boiled for 1 min in a buffer containing 1% SDS, 2% 2-mercaptoethanol, 0.1 m NaCl, 25 mm Tris, pH 6.8. Silver Staining and Detection of ER Chaperones and T4—Thyroid homogenates were freshly prepared by boiling for 5 min in a buffer containing 4% SDS, 2% mercaptoethanol, 10 mm Tris, pH 6.8. After SDS-PAGE, samples were electrotransferred to nitrocellulose before immunoblotting with respective chaperone antibodies or anti-T4 antibody (Sigma) followed by horseradish peroxidase-conjugated secondary antibodies. Bands were visualized with enhanced chemiluminescence. Immunofluorescence of Phospho-PERK—Paraffin-embedded wild-type and rdw/rdw rat thyroid paraffin sections (10 μm) were placed on coated glass slides and deparaffinized by three successive immersions in xylene for 10 min. The sections were rehydrated for 5 min each in a decreasing alcohol series (100, 90, 70% ethanol) and finally immersed in PBS for 5 min. The sections were then incubated in PBS-based blocking solution (5% goat serum) for 5 min at room temperature. Rabbit anti rat-phospho-PERK (Thr-980) from Cell Signaling Technology (catalog no. 3191) was incubated overnight in a humidity chamber at room temperature. The slides were then washed three times in PBS before incubating them with goat anti-rabbit Alexa 488 (Molecular Probes) for 1 h. The slides were then washed three more times in PBS, rinsed with water, and mounted with ProLong anti-fading medium on top of the sample. Site-directed Mutagenesis of Mouse Tg cDNA—Full-length mouse Tg cDNA was cloned into pcDNA3.1A. Mutations were introduced using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the presence of the mutations was confirmed by DNA sequencing. Transient Transfection and Stable Cell Lines—COS-7 cells were grown in complete DMEM containing 10% fetal bovine serum and 100 units/ml penicillin/streptomycin (Invitrogen). Cells were transfected with 0.8 μg of plasmid DNA per well using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s protocol. Pulse-chase experiments were performed 36 h after transfection. PC Cl3 rat thyroid cell lines were grown in complete Coons F12 medium supplemented with 5% fetal bovine serum and 100 units/ml penicillin/streptomycin plus a four-hormone mixture (10 milliunits/ml thyrotropin, 10 μg/ml insulin, 5 μg/ml apotransferrin, 10 nm hydrocortisone). G418-resistant PC Cl3 thyrocytes engineered for constitutive expression of the reverse tet transactivator, called rtTA-7 cells (35Knauf J.A. Kuroda H. Basu S. Fagin J.A. Oncogene. 2003; 22: 4406-4412Crossref PubMed Scopus (132) Google Scholar), were kindly provided by Dr. James Fagin (Memorial-Sloan Kettering Cancer Center, New York). These cells were maintained in PC Cl3 complete growth medium plus 100 μg of G418. The mouse ERp72 cDNA (from Dr. M. Green, Saint Louis University, MO) subcloned to the BamHI site of pTRE2 (Clontech, Palo Alto, CA) was transfected into rtTA-7, and stable expressors were selected for both hygromycin resistance and doxycycline-inducible expression of ERp72 protein by immunoblotting. rtTA-7-ERp72 cells were maintained in PC Cl3 complete growth medium plus 150 μg/ml and 100 μg/ml G418. Metabolic Labeling and Immunoprecipitation—Transfected COS-7 cells were starved for 30 min in Met/Cys-free DMEM and then labeled for 1 h at 37°C with 80 mCi/ml of [35S] Easytag™ express protein labeling mix (PerkinElmer Life Sciences). Following pulse labeling, the cells were washed twice with PBS containing Ca2+ and Mg2+ and chased with DMEM supplemented with an excess of cold methionine and cysteine. When indicated, 15 μm MG132, 100 μm kifunensine, 1 mm 1-deoxymannojirimycin, 1 mm castanospermine, or 10 μg/ml brefeldin A was included in pretreatment, pulse labeling, or chase incubations. At the end of each chase period, the medium was aspirated, and the labeled cells were treated with ice-cold PBS containing 50 mm iodoacetamide (Sigma) for 10 min to alkylate free cyteine thiols in situ. Labeled cells were then lysed for 1 h on ice in 1 ml of IP buffer (1% Triton X-100, 25 mm Tris-HCl, 0.1 m NaCl, pH 6.8) containing a protease inhibitor mixture (29Caldwell S.R. Hill K.J. Cooper A.A. J. Biol. Chem. 2001; 276: 23296-23303Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and 5 units/ml apyrase (Sigma). Nuclei and cell debris were pelleted at 4000 rpm for 10 min at 4 °C. The supernates were then preincubated with zysorbin-protein A (Zymed Laboratories, San Francisco, CA) for 1 h before immunoprecipitation or co-precipitation overnight with the respective antibodies and zysorbin at 4 °C. The immunoprecipitates were washed three times with immunoprecipitation (IP) buffer, boiled in SDS-gel sample buffer, and the supernates were subjected to SDS-4% PAGE. The gels were dried, and Tg bands were visualized by phosphorimaging with quantitation done using ImageQuant 5.0 software (GE Healthcare). Polyclonal rabbit antisera against BiP, calnexin, ERp72, PDI, and ERp57 were directed against 18–22 amino acid sequences at the C termini of the respective proteins. Antibodies to Tg were as described previously (30Kim P.S. Hossain S.A. Park Y.-N. Lee I. Yoo S.-E. Arvan P. Proc. Natl. Acad. Sci. (U. S. A.). 1998; 95: 9909-9913Crossref PubMed Scopus (107) Google Scholar). Sequential Immunoprecipitation—Transiently transfected COS-7 cells expressing Tg-G2320R were pulse-labeled and chased as described in the text. First round immunoprecipitations were done using antibodies specific for Tg, BiP, ERp72, or PDI. After immunoprecipitation, the residual supernatants from the first round immunoprecipitation were split into three equal aliquots. A second round of immunoprecipitation was then done on these supernates using Tg-, ERp72-, or PDI-specific antibodies. Second round immunoprecipitations were carried out overnight in the presence of fresh protease inhibitors. Endoglycosidase H Treatment—At different chase times after pulse labeling, an aliquot of transfected COS-7 cell lysates was resuspended in 0.5% SDS and 1% 2-mercaptoethanol in 20 mm Tris-HCl, pH 7.4, and boiled for 5 min. The denatured lysates were then either mock-digested or digested in the presence of 250 units of endoglycosidase H (New England Biolabs, Beverly, MA) in 50 mm sodium citrate, pH 5.5, for 1 h at 37°C. The samples were then immunoprecipitated with an anti-Tg antibody followed by reducing SDS-PAGE and phosphorimaging. TUNEL Staining—Paraffin-embedded wild-type and rdw/rdw rat thyroid paraffin sections (10 μm) were placed on coated glass slides and deparaffinized by three successive immersions in xylene for 10 min. The sections were rehydrated for 5 min each in a decreasing alcohol series (100, 90, 70% ethanol) and finally immersed in PBS for 5 min. Staining for apoptosis was performed using the TACS-XL in situ apoptosis detection kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol except for the following modifications. Tissue sections were permeabilized with proteinase K for 1 h, the labeling reaction was allowed to proceed for 2 h, TACS Blue labeling was performed for 30 min, and nuclear fast red counterstaining was extended to 20 min. Tg-G2320R Exhibits a Disulfide Maturation Defect—The Tg monomer forms ∼60 intrachain disulfide bonds within its globular structure (17Di Jeso B. Ulianich L. Pacifico F. Leonardi A. Vito P. Consiglio E. Formisano S. Arvan P. Biochem. J. 2003; 370: 449-458Crossref PubMed Google Scholar). The Tg-G2320R mutation, responsible for the dwarf phenotype of the rdw/rdw rat, was examined for the steady state level of Tg disulfide maturation. The full-length wild-type mouse Tg cDNA was mutagenized to encode the G2320R mutation and this was transiently expressed in COS cells. Proteins of the transiently transfected cells were reacted with PEG-maleimide, a reagent that alkylates free thiols and adds ∼5 kDa of molecular mass to each reactive cysteine thiol (31Tsai B. Rodighiero C. Lencer W.I. Rapoport T.A. Cell. 2001; 104: 937-948Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar). If a sufficient number of thiols were to be alkylated, a change in the molecular mass of the 330-kDa Tg monomer would be detected by SDS-PAGE/immunoblotting as a slower migrating form. Well folded wild-type Tg migrated comparably under untreated versus treated conditions (Fig. 1A), and untreated Tg-G2320R migrated comparably to intracellular wild-type Tg after PEG-maleimide treatment (Fig. 1B, lanes 3 and 4), whereas PEG-maleimide treatment shifted Tg-G2320R mobility to a slower position (Fig. 1B, lane 5), indicating abnormal exposure of cysteine thiols in the mutant protein. What are the consequences of high level expression of maturation-defective Tg-G2320R in the thyroid of rdw/rdw rats? By silver staining, wild-type thyroid tissue contains a more intensely staining Tg band at ∼330 kDa, whereas rdw/rdw thyroid exhibits increased bands of 150 and 94 kDa and one or more proteins with molecular mass of ∼70 kDa (Fig. 2A, marked with asterisks). Immunoblotting showed that these reflect increased levels of ER chaperones (Fig. 2B, upper three panels) along with ER oxidoreductases ERp72, PDI, and ERp57 (next three panels) as well as the lectin-like chaperone calreticulin (next panel), whereas calnexin was only modestly affected (fibronectin levels served as a loading control (last panel)). Coordinate up-regulation of ER luminal chaperones in thyrocytes expressing Tg-G2320R (32Oh-Ishi M. Omori A. Kwon J.Y. Agui T. Maeda T. Furudate S.I. Endocrinology. 1998; 139: 1288-1299Crossref PubMed Scopus (26) Google Scholar, 33Baryshev M. Sargsyan E. Wallin G. Lejnieks A. Furudate S. Hishinuma A. Mkrtchian S. J. Mol. Endocrinol. 2004; 32: 903-920Crossref PubMed Scopus (52) Google Scholar) is consistent with activation of the ATF6 and Ire1 stress-signaling proteins of the tripartite ER stress response (34Schroder M. Kaufman R.J. Annu. Rev. Biochem. 2005; 74: 739-789Crossref PubMed Scopus (2432) Google Scholar) that have been reported to be activated in rdw/rdw thyroid tissue (33Baryshev M. Sargsyan E. Wallin G. Lejnieks A. Furudate S. Hishinuma A. Mkrtchian S. J. Mol. Endocrinol. 2004; 32: 903-920Crossref PubMed Scopus (52) Google Scholar). To examine activation of the third ER stress-signaling protein, PERK, rdw/rdw rat thyroid tissue sections were immunostained with anti-phospho-PERK, which reacted in excess of that found in wild-type rat thyroid tissue (Fig. 2C) indicating generalized ER stress response activation. At the same time, formation of l-thyroxine (T4) was undetectable by anti-T4 immunoblotting within polypeptides contained in thyroid tissue extracts of rdw/rdw rats (Fig. 2D). Altogether, in conjunction with earlier work, the data in Fig. 2 demonstrate a loss of thyroid hormonogenesis accompanied by full-blown activation of ER stress response pathways in the rdw/rdw rat thyroid gland. Tg-G2320R (rdw) Mutant Thyroglobulin Is Retained Intracellularly and Degraded by ERAD—In transiently transfected COS cells, within 2 h after wild-type Tg synthesis, Tg glycans began to acquire resistance to digestion with endoglycosidase H. By contrast, the Tg-G2320R mutant did not acquire endoglycosidase H resistance even at 4 h, indicating its failure to arrive at the Golgi complex (Fig. 3A), and no rdw Tg was secreted (not shown). Instead, cells disposed of the mutant protein, primarily between 6 and 24 h after synthesis (Fig. 3B) when ≤10% of molecules still remained (Fig. 3C). The presence of MG132 (15 μm) greatly inhibited the intracellular degradation (Fig. 3, B and C), indicating that ERAD of Tg-G2320R involves proteasomal disposal. ERAD of another mutant, Tg-L2263P (encoded by cog), is known to involve an ER mannosidase/EDEM (ER degradation-enhancing α-mannoside-like protein)-sensitive pathway inhibited by kifunensine and 1-deoxymannojirimicin (20Tokunaga F. Brostrom C. Koide T. Arvan P. J. Biol. Chem. 2000; 275: 40757-40764Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Pretreatment of COS cells with these glycan-processing inhibitors produced similar effects on the ERAD of Tg-G2320R at 16 h of chase; kifunensine (KF) and 1-deoxymannojirimicin (DMM) clearly inhibited ERAD of Tg-G2320R, whereas castanospermine (CAS) was largely without effect (Fig. 3D). Brefeldin A (BFA), an inhibitor of ARF1-GEF (ADP-ribosylation factor 1-guanine nucleotide exchange factor) that blocks ER-to-Golgi anterograde transport, was similarly without effect on ERAD of Tg-G2320R (Fig. 3D). Based on these results, inhibitors of glycan processing and intracellular protein trafficking cannot be used to distinguish significant differences between ERAD of Tg-G2320R and Tg-L2263P mutants. Binding of ER Chaperones and Oxidoreductases to Mutant Tg-G2320R—Following a 60-min pulse labeling, co-immunoprecipitation of wild-type Tg or Tg-G2320R mutant with ER molecular chaperones was examined at various chase times. A fraction of newly synthesized wild-type Tg was associated with BiP at 0 h chase time, and this declined precipitously even during the first hour of chase (Fig. 4A) at a time when total labeled intracellular Tg was still maintained (upper panel). By contrast, Tg-G2320R association with BiP did not dramatically decline over 4 h (Fig. 4A), although it did decline over 24 h (Fig. 4B) in parallel with the kinetics of ERAD (upper panel). In addition to BiP, an obvious increase in the early association of both ERp72 and PDI with Tg-G2320R was evident (Fig. 4A), and substantial association continued with the remaining intracellular fraction throughout the entire 24-h post-synthesis (Fig. 4B). A similar but far less dramatic increase in association of mutant Tg was also observed with ERp57 (Fig. 4A, bottom), but because of the relatively low Tg co-precipitation (see below), this was not pursued extensively. Similarly, small amounts of wild-type and mutant Tg-G2320R were co-immunoprecipitated with calnexin during the first 4 h after Tg synthesis (Fig. 4A); although at much later chase times, the fraction of the small amount of residual labeled intracellular mutant Tg (upper panel) that was bound by calnexin became proportionately larger (Fig. 4B). As a control for these studies, when Tg was intentionally unfolded in situ with increasing doses of dithiothreitol prior to co-immunoprecipitation, the pattern of chaperone/oxidoreductase co-precipitation looked quite similar for wild-type Tg, Tg-G2320R, and Tg-L2263P (with all ER chaperones and oxidoreductases becoming significantly engaged as the Tg protein was increasingly denatured, Fig. 5). These data suggest that the differences shown in Fig. 4 reflect distinct chaperone preferences for Tg-G2320R rather than technical difficulties with antibody-mediated co-immunoprecipitation. Moreover, under conditions of mannosidase inhibition when mutant Tg-G2320R persists, more mutant Tg could also be found associated with BiP, ERp72, and PDI binding partners (Fig. 4C). Indeed, the final amount of labeled Tg-G2320R at 24 h of chase (Fig. 4B, upper panel) appeared to be near quantitatively co-precipitated by BiP, calnexin, ERp72, or PDI (Fig. 4B, lower panels). Multiple Chaperones Interact with the Same Population of Mutant Tg Molecules—Based on the foregoing results, a sequential IP protocol (see “Experimental Procedures”) was employed to examine whether a fraction of Tg-G2320R was simultaneously associated with more than one ER oxidoreductase. A first IP with anti-Tg recovered virtually all labeled Tg at either 0 or 6 h of chase, such that essentially no additional labeled Tg could be recovered from the remaining IP-supernatant during a subsequent round of IP with any antibody (Fig. 6A, lanes 2–4). Similarly, the supernatant after a first IP with anti-ERp72 yielded virtually no additional Tg that could be co-precipitated in a second IP with the same antibody (Fig. 6A, lane 7); just as a first round anti-PDI IP yielded little additional Tg co-precipitation with a second round of anti-PDI (lane 12). By contrast, a second round IP with anti-Tg recovered significant additional labeled Tg that was not associated with either of these ER oxidoreductases (Fig. 6A, lanes 6 and 10). Most interesting, however, was that the supernatant after a first IP with anti-ERp72 did not yield additional labeled Tg that could be co-precipitated with anti-PDI (Fig. 6A, lane 8), and the supernatant after a first IP with anti-PDI IP did not yield appreciable labeled Tg in a second round co-precipitation with anti-ERp72 (lane 11). These data suggest that the fraction of labeled Tg bound to ERp72 was the same as that associated with PDI. This complex also included BiP (data not shown), indicating that multiple ER resident proteins interact with the same fraction of mutant Tg molecules. Covalent and Noncovalent Associations between Tg-G2320R and ER Oxidoreductases, Especially ERp72—Based on the observation that Tg-G2320R contains exposed free thiols (Fig. 1) and that both PDI and ERp72 interact with Tg-G2320R (Figs. 4, 5, and 6A), we tested the extent to which mixed disulfide bonds might form between the mutant Tg and ER-resident proteins. Lysates of transfected COS cells that had been pulse-labeled and chased for up to 1 day were untreated or treated with 2% SDS to disrupt all noncovalent associations before SDS dilution followed by co-IP of labeled Tg-G2320R with anti-BiP, anti-ERp72, or anti-PDI. Essentially no Tg-G2320R could be co-precipitated with BiP after disruption of noncovalent interactions (Fig. 6B). By contrast, co-IP of a fraction of mutant Tg with ERp72 and PDI persisted even after SDS treatment (Fig. 6B). Control experiments (not shown) established that co-IP of labeled Tg was prevented by prior reduction of the cell lysate, strongly suggesting that mutant Tg was disulfide-linked to the oxidoreductases. Although representing only a modest fraction of total Tg at the zero chase time, this fraction of mutant Tg covalently associated with ERp72 (or PDI) did not decline rapidly, as occurs in rat thyrocytes during resolution of covalent adducts of wild-type Tg with ERp57 and PDI (18Di Jeso B. Park Y.-n. Ulianich L. Treglia A.S. Urbanas M.L. High S. Arvan P. Mol. Cell. Biol. 2005; 25: 9793-9805Crossref PubMed Scopus (62) Google Scholar). Thus, by 24 h after synthesis, the mutant Tg fraction covalently associated with ERp72 closely approximated all residual Tg-G2320R that was refractory to ERAD (Fig. 6B). Given recent studies suggesting that ERp72 association can inhibit ERAD of mutant Tg (28Forster M.L. Sivick K. Park Y.N. Arvan P. Lencer W.I. Tsai B. J. Cell Biol. 2006; 173: 853-859Crossref PubMed Scopus (100) Google Scholar), the present data seem to direct special attention to potential function(s) of ERp72 within the ER. Demonstration of Potential ERp72 Reductase Activity in Thyrocytes—We found that when PDI and ERp72 from the ER of PC Cl3 thyrocytes (under native conditions) were incubated with PEG-maleimide (not shown), there was partitioning of PDI into discrete bands implying distinct PDI forms, each oxidized to a different extent, whereas ERp72 was more completely and uniformly alkylated. Because ERp72 Cys residues, on average, may exist in a more reduced state than do those o" @default.
• W2029451118 created "2016-06-24" @default.
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• W2029451118 creator A5007645545 @default.
• W2029451118 creator A5014890153 @default.
• W2029451118 creator A5029330427 @default.
• W2029451118 creator A5032586157 @default.
• W2029451118 creator A5037589415 @default.
• W2029451118 creator A5056527741 @default.
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• W2029451118 date "2007-03-01" @default.
• W2029451118 modified "2023-10-16" @default.
• W2029451118 title "Oxidoreductase Interactions Include a Role for ERp72 Engagement with Mutant Thyroglobulin from the rdw/rdw Rat Dwarf" @default.
• W2029451118 cites W1483092865 @default.
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