Acute ablation of PERK results in ER dysfunctions followed by reduced insulin secretion and cell proliferation
© Feng et al; licensee BioMed Central Ltd. 2009
Received: 5 August 2008
Accepted: 4 September 2009
Published: 4 September 2009
A deficiency in Perk (EIF2AK3) causes multiple neonatal defects in humans known as the Wolcott Rallison syndrome. Perk KO mice exhibit the same array of defects including permanent neonatal diabetes (PND). PND in mice was previously shown by us to be due to a decrease in beta cell proliferation and insulin secretion. The aim of this study was to determine if acute ablation of PERK in the 832/13 beta cells recapitulates these defects and to identify the primary molecular basis for beta cell dysfunction.
The INS1 832/13 transformed rat beta cell line was transduced with a dominant-negative Perk transgene via an adenoviral vector. AdDNPerk-832/13 beta cells exhibited reduced expression of insulin and MafA mRNAs, reduced insulin secretion, and reduced cell proliferation. Although proinsulin content was reduced in AdDNPerk-832/13 beta cells, proinsulin was abnormally retained in the endoplasmic reticulum. A temporal study of the acute ablation of Perk revealed that the earliest defect seen was induced expression of two ER chaperone proteins, GRP78/BiP and ERp72. The oxidized states of ERp72 and ERp57 were also increased suggesting an imbalance in the redox state of the ER.
Acute ablation of Perk in INS 832/13 beta cells exhibited all of the major defects seen in Perk KO mice and revealed abnormal expression and redox state of key ER chaperone proteins. Dysregulation of ER chaperone/folding enzymes ERp72 and GRP78/BiP occurred early after ablation of PERK function suggesting that changes in ER secretory functions may give rise to the other defects including reduced insulin gene expression, secretion, and cell proliferation.
Monogenic forms of permanent neonatal diabetes have revealed key aspects of the development and function of the insulin secreting beta cells . Mutations in Perk (EIF2AK3) underlies the complex genetic disorder of the Wolcott Rallison syndrome, which includes permanent neonatal diabetes (PND), exocrine pancreas deficiency, growth retardation, hepatic dysfunctions, and skeletal dysplasias [2, 3]. All of the dysfunctions in WRS are mirrored in mice deficient for PERK [4, 5], and detailed genetic studies in mice have shown that the diabetes is caused by the loss of expression of PERK in the insulin secreting beta cells , whereas the exocrine pancreas deficiency is caused by the absence of PERK in the pancreatic acinar cells . The initial interpretation of the molecular basis of these dysfunctions in humans and mice was based upon studies performed in vitro with cultured fibroblasts . These studies showed that the catalytic domain of PERK resides in the cytoplasm where it phosphorylates the translation initiation factor eIF2 alpha, which results in either repression of global protein synthesis or activation of translation of specific mRNAs encoding gene regulatory proteins. PERK regulatory domain resides in the lumen of the ER and is controlled by the binding of the ER chaperone proteins, GRP78/BiP and GRP94, and calcium . Disturbances in the ER such as ER stress and accumulation of unfolded proteins, or normal physiological changes in calcium levels can activate PERK [9, 10]. Based upon these studies it was proposed that PND in humans and mice deficient in PERK was caused by uncontrolled ER stress and apoptotic cell death of the beta cells . However a recent comprehensive analysis of the islet and beta cell development revealed that the cause of diabetes in Perk deficient mice is due to failure to expand beta cell mass and defects in beta cell development and insulin secretion during the critical fetal and neonatal periods . Thus three distinct defects are seen in Perk-deficient beta cells, which raises questions about the causal connection and progression of beta cell dysfunction. The chronic loss of PERK expression over the four-week period between when defects in beta cell development are first seen during the fetal stage and the onset of overt diabetes three weeks after birth confounds resolving these questions and determining the molecular basis of the defects in Perk-deficient mice. To investigate the acute effects of PERK ablation we have expressed a dominant negative mutation of Perk in the transformed INS1 832/13 beta cell line. We found that acute ablation of PERK in 832/13 beta cells mimics all of the defects seen in the beta cells of Perk-deficient mice and detailed temporal studies suggest that defects in the function of the endoplasmic reticulum may give rise to the defects in beta cell developmental and proliferation.
Ablation of PERK in INS1 832/13 beta cells results in reduced insulin gene expression, insulin content, and insulin secretion
Islets isolated from neonatal Perk KO mice exhibited an almost complete ablation of glucose-stimulated insulin secretion (GSIS) . To determine if an acute deficiency of PERK impacts insulin secretion, 832/13 beta cells were transduced with AdDNPerk. Forty-eight hours post-transduction of AdDNPerk-832/13 cells showed a 3-fold reduction in insulin secretion in response to 15 mM glucose stimulation compared to 832/13 transduced with the AdLacZ control (Figure 2C). Treatment of AdDNPerk-832/13 cells with tolbutamide, a KATP channel blocker, also showed reduced enhancement of glucose stimulated insulin secretion compared to the control cells. Treatment with diazoxide, a KATP channel opener, blocked glucose-stimulated insulin secretion (Figure 2D) in both genotypes as expected. We conclude that the reduced GSIS is due to a combination of decreased insulin content as well from a partial reduction in KATP-dependent insulin secretion.
Proliferation of AdDNPerk 832/13 beta cells is reduced
Accumulation of proinsulin in the ER occurs in the absence of increased protein synthesis in Perk deficient beta cells
ERp72 and GRP78/BiP expression are induced first after initiating acute ablation of Perk followed by other changes in gene expression
Temporal Changes in gene expression following Perk ablation in 832/13 cells
ERp72 protein expression and oxidized state is increased in PERK-ablated 832/13 cells
The redox state of the PDI family of ER folding enzymes including ERp72 and ERp57 is known to vary according to changes in the oxidative and functional states of the ER. In addition to increased levels of ERp72 protein, AdDNPerk-832/13 beta cells exhibited an increase in the oxidized isoforms of ERp72 and ERp57 (Figure 8C) that were first seen at 24 hours post transduction and increased significantly by 36 hours.
Despite the vast differences between the milieu and ontogeny of cultured beta cells and native beta cells in the endocrine pancreas, we found that the array of defects in the beta cells of Perk KO mice  were remarkably present following acute ablation of Perk in the transformed rat INS1 832/13 beta cells. These defects include reduced cell proliferation (without changes in cell death), reduced expression of proinsulin and insulin, reduced glucose-stimulated insulin-secretion, and abnormal retention of proinsulin in the ER leading some cells to have a highly distended ER. That these defects underlie the cause of neonatal diabetes in Perk KO mice, and by inference in humans with the Wolcott Rallison syndrome, was shown by the study of tissue-specific Perk KO mice and the ability of a beta cell specific-Perk transgene to rescue diabetes [6, 7, 25]. Given the close correspondence of cellular pathology of the AdDNPerk-832/13 beta cells to the beta cells of Perk KO mice, we were encouraged to take advantage of cultured beta cells to elucidate the molecular function of PERK in regulating beta cell development and physiology.
Acute ablation of Perk, afforded by transduction of the dominant negative Perk transgene, allowed us to determine the progress and order of the ensuing defects over time. As we had seen in Perk KO mice, the expression of insulin synthesis genes Ins-1, Ins-2, and MafA) and cell cycle genes was reduced but the reduction in their expression lagged behind the induction of the ER chaperone genes ERp72 and GRP78/BiP thus suggesting that a perturbation in the ER may result in repression of genes involved in beta cell function and proliferation. Initial reports showing that loss of PERK function resulted in neonatal diabetes in humans and mice [2, 4, 5] suggested that the molecular defect was related to the function of PERK in the regulation of the ER stress - Unfolded Protein Response (UPR). Our extensive analyses of Perk-deficient beta cells in mice  and in AdDNPerk-832/13 beta cells in culture have not supported this hypothesis. The original hypothesis assumed that global protein synthesis would be derepressed as a direct consequence of the absence of PERK, and that this would result in uncontrolled protein synthesis and overloading of the ER culminating in apoptotic cell death . Although the ER in Perk-deficient beta cells show marked accumulation of proinsulin, we found that global protein synthesis was not elevated nor was there an elevation in the proinsulin content in beta cells. Cell death did not increase, but instead cell proliferation was ablated. Moreover, the expression of ERp72 and GRP78/BiP was still induced in AdDNPerk-832/13 beta cells after blocking protein synthesis with cyclohexamide suggesting that their induction was not due to protein overload.
ERp72 and GRP78/BiP are positively regulated and insulin gene expression is negatively regulated by ATF6 [18, 26], and we found that the processed nuclear form of ATF6 was substantially increased in PERK-ablated AD293 cells. Treatment of cells with Brefeldin-A also induces the processing of the ATF6 , which is caused by relocalization of the S2P protease from the Golgi to the ER. We found that the S2P protease is indeed mislocalized in PERK-ablated AD293 cells and is associated with ER markers (Gupta, et al. unpublished data), which suggests that the increased basal level of the nuclear form of ATF6 is due to mislocalization of the proteases that cleave precursor ATF6 rather than by ER stress. Further studies are required to confirm the importance of these findings in insulin-secreting beta cells.
In addition to its role in repressing global protein synthesis during ER stress, PERK positively regulates the translation of ATF4, which in turn upregulates GADD153/CHOP [8, 28, 29]. Mutations in these downstream genes, however, do not lead to diabetes in mice, [6, 7] thus arguing against the hypothesis that diabetes associated with Perk deficiency is related to the role of PERK in regulating the unfolded protein response. Considerable evidence suggests that PERK is important for mediating the ER stress response in adult beta cells [11, 30–32], but we argue that the primary function of PERK in beta cells is to regulate beta cell development and function during the fetal-neonatal period. Interestingly IRE1, another mediator of the ER stress response, has also been shown to have an important beta cell function by positively regulating insulin biosynthesis .
We speculate that the Impacted-ER phenotype, characterized by gross distension of the endoplasmic reticulum and abnormal retention of proinsulin observed in Perk-deficient beta cells, represents the extreme manifestation of ER dysfunction. The induction of ERp72 and GRP/78 mRNA expression is the first hint of ER dysfunction, and is followed by increased levels of these proteins and the oxidized isoform of ERp72 and ERp57. The increased oxidized isoforms of ERp72 and ERp57 suggests that redox state of the ER is abnormal or that the oxidation and reduction of protein disulfides is imbalanced. The formation of disulfide bonds is essential for the proper folding proinsulin and subsequent maturation of insulin . The protein disulfide isomerase family including ERp72, PDI, and ERp57, mediate their oxidative protein folding functions by forming transient mixed disulfides with ER client proteins [34, 35] such as proinsulin. In addition to catalyzing the proper folding of proteins in preparation for transport from the ER to the Golgi, the PDI enzymes participate in ER associated protein degradation (ERAD) by reducing thiols of misfolded proteins in preparation for retrotranslocation to the cytoplasm for proteosomal degradation [20, 34]. Recently Forster and colleagues  have shown that ERp72 and PDI have opposing functions in the ER. ERp72 assists proper protein folding whereas PDI facilitates unfolding of misfolded proteins in preparation for proteosomal degradation. We propose that PERK regulates the expression of key genes that encode ER redox and protein folding functions in the insulin-secreting beta cells during the dynamic metabolic changes that occur during the developmental transitions through embryonic, fetal, neonatal, and juvenile stages. PERK enzyme activity is regulated through its ER luminal domain via binding of GRP78/BiP complexed with calcium [36, 37]. Physiological changes in ER calcium modulate PERK activity  as well as insulin secretion. The regulation of ER calcium is coupled to the redox state of specific ER proteins including SERCA, the major ER calcium pump . We speculate that PERK acts as an ER calcium sensor that couples ER functions of folding, quality control, and protein trafficking that are intimately tied to the redox state of the ER as a function of the dynamic changes in the physiological environment of the beta cell during the early developmental transitions.
We found that acute ablation of PERK in the transformed 832/13 beta cell line leads to reduced proliferation and reduced insulin gene expression, insulin content, and insulin secretion, demonstrating that acute ablation of PERK recapitulates the major cellular and molecular defects seen in Perk KO mice. In addition proinsulin accumulates abnormally in the ER, which occurs after the elevation of two key chaperone proteins GRP78/BiP and ERp72. ERp72 accumulates in a sub compartment of the ER and its oxidized isoform is substantially increased. Because the changes in the expression of GRP78/BiP and ERp72 are the earliest changes observed after ablation of Perk, we suggest that ER dysfunctions give rise to defects in proinsulin trafficking, insulin secretion, and cell proliferation.
INS-1 832/13 insulin-secreting beta cells were obtained from Dr. Christopher Newgard (Duke University). The 832/13 cells were cultured in RPMI-1640 (Mediatech Cellgro) supplemented with 11 mM glucose, 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol and Antibiotic Antimycotic Solution (Sigma) at 37°C in 5% CO2, 95% air. The cells were sub-cultured twice weekly. For determining relative proliferation, BrdU (1 mM) was added to the cell for 1 hr followed by permeabilization and fixation for 10 minutes with 4% formaldehyde, 0.1% Triton x-100 in PBS. The cells were then denatured with 1N HCL for 30 mins, and anti-BrdU (1:50, DAKO) applied for 1 hour. For estimating cell death the DeadEndTM Fluorometric TUNEL System (Promega, Inc.) was used. Cells were fixed in 4% methanol-free formaldehyde solution for 10 min, permeabilized with 0.2% Triton X-100 solution for 5 minutes, and labeled according to instructions of the kit.
Proinsulin content, insulin content and insulin secretion
After 48 hr post-transduction, 832/13 cell pellets were sonicated in 1 mol/l acetic acid containing 0.1% bovine serum albumin. Aliquots of cell extracts were assayed for proinsulin and insulin content using proinsulin EIA (ALPCO) and Mouse Insulin Ultrasensitive EIA (ALPCO), respectively. The data were normalized to DNA content. Insulin secretion was assayed at 3 mmol and 15 mmol/l glucose. For KATP channel dependent secretion assay, 200 μmol/l tolbutamide or 250 μmol/l diazoxide were added.
Plasmids and adenovirus production
The c-myc tagged dominant-negative mouse Perk transgene (PERKΔC) was kindly provided by Dr. David Ron (New York University). PERKΔC was generated by deleting the kinase domain of Perk residing in the carboxy terminal coding sequence, retaining the amino terminal ER luminal domain (Harding, 1999). The PERKΔC transgene was excised by us from the parent vector by digestion with Spe 1 and Xho 1 and then inserted to the Adenovirus vector pShuttle-IERS-hrGFP-1 (Stratagene). The resulting construct, which we denote as AdDNPerk, was transfected into AD-293 cells using the MBS Mammalian Transfection kit (Stratagene) for amplification. The adenovirus was then purified by Adeno-X™ Virus Purification kit (Clontech) and the titer was determined by the QuickTiter™ Adenovirus Titer Immunoasssay Kit (Cell Biolabs, Inc.). The pShuttle-CMV-lacZ transgene, denoted herein as AdLacZ, served as a control. Transduction of 832/13 cells was carried out at a cell confluency of about 70%, at different multiplicities of infection (MOI) from 5 to 20. A MOI of 20 was used in most experiments unless otherwise stated. After 2 hours of incubation with the virus at 37°C, 5% CO2, cells were washed once in RPMI and cultured for an additional 6-48 h before assay.
To assess ATF6 processing the expression plasmid pCMVshort-EYFP-ATF6-alpha, provided by Kazutoshi Mori, was transfected into AD293 cells.
The pancreata from E18.5 to p2 mice were inflated in situ by injecting 3 mg/ml Collagenase P (Invitrogen) in HBSS (Sigma) and digestion was carried out at 37°C for about 12 mins at which time 1 ml of ice-cold HBSS was added to stop the enzyme reactions. The dispersed pancreatic tissue was pelleted and washed once with HBSS. The pellets were then suspended in 1 ml Histopaque-1077, overlaid with 0.2 ml RPMI 1640 medium and centrifuged at 890 g for 12 min to separate the islet from the other cell types and residual debris. Islets were manually picked under a dissection microscope and washed once with ice-cold HBSS.
Gene expression levels
Quantification of gene expression was carried out by using qPCR Core Kit for SYBR Green I (Eurogentec) amplifying cDNA with the ABI Prism 7000 Sequence Detection System. The following cycling conditions were used for all primer pairs: 50°C (2 minutes), 95°C (10 minutes), 40 cycles of (95°C [15 seconds], 60°C [1 minute]). Levels of Xbp1-s (spliced form) were normalized to Xbp1-t (total) levels. All other mRNAs were normalized to the levels of actin and/or GAPDH. The mouse primer sequences were as follows: actin 5'-GCCCTGAGGCTCTTTTCC-3', 5'-TGCCACAGGATTCCATACCC-3'; GAPDH 5'-GGAGCGAGACCCCACTAACA-3', 5'-ACATACTCAGCACCGGCCTC-3'; Insulin I 5'-AGCATCTTTGTGGTCCCCAC-3', 5'-CCCCACACACCAGGTAA-3'; Insulin II 5'-CAGAAGCGTGGCATTGTAGA-3', 5'-TTGCAGTAGTTCTCCAGCTGG-3'; MafA, 5'-GCTGGTATCCATGTCCGTGC-3', 5'-GTCGGATGACCTCCTCCTTG-3'; Pdx-1, 5'-GAGCGTTCCAATACGGACCA-3',5'-TCAGCCGTTCTGTTTCTGGG-3'; ERP72, 5'-TTCCACGTGATGGATGTTCAG-3', 5'-AGTCTTACGATGGCCCACCA-3'; ERP57, 5'-GGCGGATGCAACATATCACC-3', 5'-TGTGGTTCGTACTGTCCCCC-3'; ERP58, 5'-CAAGAGGCTTGCCCCTGAG-3', 5'-GGTGTTTGTGTTGGCAGTGC-3'; Hrd1, 5'-TGGCTTTGAGTACGCCATTCT-3', 5'-CCACGGAGTGCAGCACATAC-3'; Ero1 L, 5'-AAACCCTGCCATTCTGATGAA-3', 5'-ACTCATCCACGGCTCCAA GT-3'; Ero1 beta, 5'-TGATTCGCAGGACCACTTTTG-3', 5'-TAGCCAGTGTACCGTTCCGG-3'; Herpud1, 5'-CCCACCTGAGCCGAGTCTAC-3', 5'-CTTGGAGACACTGGTGATCCAA-3'; ATF3, 5'-CCT ATGCAAAGCAGGATCCC-3', 5'-GCGTTGTCAGCCACAGTGG-3'; GRP94, 5'-CTGGGTCAAGCAGAAAGGAG-3' 5'-TCTCTGTTGCTTCCCGACTT-3'; BiP, 5'-GCTTCGTGTCTCCTCCTGAC-3', 5'-TAGGAGTCCAGCAACAGGCT-3'; Chop, 5'-CCAACAGAGGTCACACGCAC-3', 5'-TGACTGGAATCTGGAGAGCGA-3'; Xbp1-spliced, 5'-GAGTCCGCAGCAGGTG-3', 5'-GTGTCAGAGTCCATGGGA-3'; Xbp1-total, 5'-CACCTTCTTGCCTGCTGGAC-3', 5'-GGGAGCCCTCATATCCACAGT-3'. The rat primer sequences were as follows: Actin, 5'-ATC CTG GCC TCA CTG TCC AC-3', 5'-CTA GAA GCA TTT GCG GTG CA-3'; GAPDH, 5'-CACCACCAACTGCTTAGCCC-3', 5'-TGGCATGGACTGTGGTCATG-3'; Insulin I, 5'-CAGCACCTTTGTGGTCCTCA-3', 5'-CCCACACACCAGGTACAGAGC-3'; Insulin II, 5'-CTGCCCAGGCTTTTGTCAAA-3', 5'-CTTCCACCAAGTGAGAACCACA-3'; MafA, 5'-GGCACATTCTGGAGAGCGA-3', 5'-CCCGCCAACTTCTCGTATTTC-3'; Pdx-1, 5'-CCACCAAAGCTCACGCGT-3', 5'-CTGCGTATGCACCTCCTGC-3'; NeuroD, 5'-GCTTGAAGCCATGAATGCAG-3', 5'-TCCTCTCCCCCATTTCTCAGA-3'; ERp72, 5'-TCTAACCAATCACCGGGCTG-3',5'-TCATGGTAAGGTGCCGAGG-3'; BiP, 5'-ACCCTTACTCGGGCCAAATT-3', 5'-AGAGCGGAACAGGTCCATGT-3'; ERp58, 5'-CGAAAACTTCGAGAGTCGCG-3', 5'-GCAAGCCTCTTGCAATGTCC-3'; ERp57,, 5'-GGACTCAAG CGAAGTGACGG-3'; 5'-TCTGCTGCCAGCAAGAACTG-3'; Ero1 L, 5'-TTCACTGAGGAGGGCGGTT-3', 5'-CAGGACGGTCACTGCAATCA-3'; Ero1 beta, 5'-GTGCTTTGTCAAAGGTGG CC3'; 5'-GCAGAAGGGTCTTGGTGTCAG-3'; Crebpp1, 5'-ACGCCGGAGTCAATTCCTATC-3', 5'-GAGCTGATGTTGCGG GAAGA-3'; Psma5, 5'-TCACACCCCTGTCGTACTCG-3', 5'-TTTCAGTCGTGTGGCCTTTG-3'; Sec61a, 5'-GCTTCTGAATTT CCGGCAAG-3', 5'-AGGCAGGGAGTGTAGTCGGAC-3'; Sec63, 5'-TGGGTGAGTGAGACCTTCCC-3', 5'-AACCCCGGATCTTCCCAGTA-3'; Der1, 5'-CACCAGCCATGCTAAGCAGA-3', 5'-TCAGTGTGGGTCAGGTCCAAG-3'; Herpud1, 5'-GTGCTCTGTTGCTGGAGGCT-3', 5'-AGCACATCGTCATCCTGTGG-3'; Xbp-1 spliced, 5'-CTGAGTCCGAATCAGGTGCAG-3', 5'-ATCCATGGGAAGATGTTCTGG-3'; Xbp-1 total, 5'-CCCTTCTCCCTTCAGCGAC-3', 5'-CGTTGGCAAAAGTGTCCTCC-3'; Rat cyclin D2, 5'-TGCTGACCAAGATCACCCAC-3', 5'-CCTGGCAGGCTTTGAGACAA-3'; Rat cyclin B2, 5'-GCCCCTGAGGATGTCTCCAT-3', 5'-AGAGAAAGCTTGGCAGAGGCT-3'; Rat cyclin A2, 5'-AGTGTGAAGATGCCCTGGCT-3', 5'-TGGCTCCGGGTAAAGAGACA-3'; Rat cyclin E, 5'-GTCGCAGGGTTGCTGTTGAT-3', 5'-CATGCTTGCTCACGACCACT-3'
Total 832/13 cellular protein was extracted with RIPA buffer (1% Nonidet P40, 0.5% sodium doxycholate, 0.1% SDS, 1 × PBS, pH 8.0) containing 1× protease and phosphatase inhibitor cocktails (Sigma, Inc.) Protein expression was assayed by Western blots. Primary antibodies used in the analysis were: ERp72 (1:2500, Stressgen, Inc), GRP78/BiP (1:500, Santa Cruz, Inc), ERp57 (1:300, Santa Cruz), and anti-GFP (Sigma, Inc.) to detect EFYP-ATF6. For Western blots performed on islets from single mice, 60 islets from each mouse were dissolved in 2× SDS sample buffer and then loaded onto a 4-15% gradient gel.
After 16 or 24 hr post-transduction, 832/13 cells were deprived of methionine and cysteine (Met/Cys) for 30 minutes at 37°C in Met/Cys-free DMEM, 10% dialyzed FBS. The cells were then labeled with [35S] Met/Cys (500 μCi/ml) at 37°C for 0, 15, 30 minutes, and the reactions were stopped by the addition of concentrated non-radioactive Met/Cys solution (0.1 M each). After two washes with PBS, the cells were lysed in RIPA buffer and then total cellular protein was precipitated with 10% trichloroacetic acid (TCA). The precipitates were washed with 20% ice-cold acetone, air-dried for 20 minutes and then dissolved in 30 mM Tris, 7 M urea, 2 M thiourea, and 4% CHAPS. The resident radioactivity was measured by scintillation counting and normalized to total protein content.
Paraffin-embedded sections or cryo-sections were subjected to immunohistochemistry using the following primary antibodies: ERp72 (1:1000, Stressgen), insulin (1:500, Linco Research); proinsulin (1:1000, Ole D. Madsen, Beta Cell Biology Consortium); GRP78/BiP (1:500, Santa Cruz). Appropriate secondary antibodies conjugated with Alexa Fluor350, 488 or 555 dye (Molecular Probes) were used to visualize the labeled cells. Fluorescence images were captured and analyzed with a Nikon Eclipse E1000 and Image-Pro Plus (Phase 3 Imaging Systems, GE Healthcare, Inc.).
Determination of the oxidative state of ERp72 and ERp57
At 24 hours post-transfection, the medium was removed, and the cells were briefly washed with ice cold PBS and lysed at 0°C for 5 min in 20% formic acid/2% SDS. Cell lysate proteins were precipitated with 10% TCA. Precipitates were washed twice with ice cold 70% acetone. The TCA precipitates were resuspended in 80 mM Tris-HCl pH 6.8, 2% SDS, supplemented with a cocktail of protease inhibitors with or without 10 mM 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS, Molecular Probes, Inc.). After incubating for 30 min at room temperature and then for 10 min at 37°C, samples were subjected to non-reducing SDS-PAGE followed by western blot analysis to detect ERp72 and ERp57. Non-transfected cells were treated with 10 mM DTT or 5 mM diamide for 10 min at 37°C as reduced and oxidized controls.
List of Abbreviations
endoplasmic reticulum associated protein degradation
eukaryotic translation initiation factor-2 alpha.
This study was supported by grants from the National Institutes of Health (R01-DK062049 and R01-GM056957) and the Pennsylvania Department of Health TSF (D.R.C.). We are grateful to Christopher Newgard, Kazutoshi Mori, and David Ron who provided important reagents for this study. We thank Sarah Kosak for excellent technical assistance.
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