Overexpression of ERp29 in the thyrocytes of FRTL-5 cells

It was previously reported that the up-regulation of ERp29 mRNA depends on the levels of thyroid stimulating hormone (TSH) in the thyrocytes of FRTL-5 cells. In order to investigate the putative new function of ERp29 as an endoplasmic molecular (ER) chaperone, an ERp29-overexpressing FRTL-5 cell line was established. This cell line had approximately three times the levels of ERp29 protein and an enhanced level of thyroglobulin (Tg) secretion. The results showed both enhanced ERp29 expression and an interaction with the other ER chaperones such as GRP94, BiP, ERp72 and calnexin. In addition, ERp29 enhanced the expression of PKR-like ER kinase (PERK), which is a transmembrane protein located in the ER membrane. These findings suggest that ERp29 assists in protein folding as well as in the secretion of the secretory/plasma membrane proteins under close co-operation with other ER chaperones and the ER stress signaler, PERK.     

Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells

The endoplasmic reticulum protein 29 (ERp29) has a critical role in regulating protein folding, maturation and secretion. However, its role in carcinogenesis remains elusive. Recently, we reported that ERp29 is a novel tumor suppressor and regulates mesenchymal-epithelial transition in MDA-MB-231 breast cancer cells. Here, we investigated whether ERp29 plays a role in the response of breast cancer cells to chemotherapeutic agents. We found that expression of ERp29 increased the resistance to doxorubicin, but not cisplatin and paclitaxel, and decreased the doxorubicin-induced cell apoptosis in MDA-MB-231 cells, whereas knockdown of ERp29 in MCF-7 cells increased the doxorubicin cytotoxicity. A proteomics study identified up-regulation of Hsp27 and down-regulation of stathmin-1, galectin and prohibitin in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells. Further, we demonstrated that ERp29 up-regulated expression of Hsp27 by down-regulating eukaryotic translational initiation factor 2α (eIF2α). When Hsp27 was knocked down by siRNA in the doxorubicin-resistant, ERp29 over-expressing MDA-MB-231 cells and parental MCF-7 cells, cell viability was significantly decreased and doxorubicin-induced cell apoptosis was enhanced. These results indicate that Hsp27 is involved in the ERp29-mediated resistance to doxorubicin. Therefore, targeting of Hsp27, with a combination of other chemotherapeutic agents, is a rational strategy in treating doxorubicin-resistant cancer cells.     

Identification of ERp29, an endoplasmic reticulum lumenal protein, as a new member of the thyroglobulin folding complex

Folding and post-translational modification of the thyroid hormone precursor, thyroglobulin (Tg), in the endoplasmic reticulum (ER) of the thyroid epithelial cells is facilitated by several molecular chaperones and folding enzymes, such as BiP, GRP94, calnexin, protein disulfide isomerase, ERp72, and others. They have been shown to associate simultaneously and/or sequentially with Tg in the course of its maturation, thus forming large heterocomplexes in the ER of thyrocytes. Here we present evidence that such complexes include a novel member, an ER-resident lumenal protein, ERp29, which is present in all mammalian tissues with exceptionally high levels of expression in the secretory cells. ERp29 was induced upon treatment of FRTL-5 rat thyrocytes with the thyroid-stimulating hormone, which is essential for the maintenance of thyroid cells and Tg biosynthesis. Chemical cross-linking followed by the cell lysis and immunoprecipitation of ERp29 or Tg revealed association of these proteins and additionally, immunocomplexes that also included major ER chaperones, BiP and GRP94. Sucrose density gradient analysis indicated co-localization of ERp29 with Tg and BiP in the fractions containing large macromolecular complexes. This was supported by immunofluorescent microscopy showing co-localization of ERp29 with Tg in the putative transport vesicular structures. Affinity chromatography using Tg as an affinity ligand demonstrated that ERp29 might be selectively isolated from the FRTL-5 cell lysate or purified lumenal fraction of rat liver microsomes along with the other ER chaperones. Preferential association with the urea-denatured Tg-Sepharose was indicative of either direct or circuitous ERp29/Tg interactions in a chaperone-like manner. Despite the presence of the C-terminal ER-retrieval signal, significant amounts of ERp29 were also recovered from the culture medium of stimulated thyrocytes, indicating ERp29 secretion. Based on these data, we suggest that the function of ERp29 in thyroid cells is connected with folding and/or secretion of Tg.     

ERp29 deficiency affects sensitivity to apoptosis via impairment of the ATF6–CHOP pathway of stress response

Endoplasmic reticulum protein 29 (ERp29) belongs to the redox-inactive PDI-Dβ-subfamily of PDI-proteins. ERp29 is expressed in all mammalian tissues examined. Especially high levels of expression were observed in secretory tissues and in some tumors. However, the biological role of ERp29 remains unclear. In the present study we show, by using thyrocytes and primary dermal fibroblasts from adult ERp29^?/? mice, that ERp29 deficiency affects the activation of the ATF6–CHOP-branch of unfolded protein response (UPR) without influencing the function of other UPR branches, like the ATF4-eIF2α-XBP1 signaling pathway. As a result of impaired ATF6 activation, dermal fibroblasts and adult thyrocytes from ERp29^?/? mice display significantly lower apoptosis sensitivities when treated with tunicamycin and hydrogen peroxide. However, in contrast to previous reports, we could demonstrate that ERp29 deficiency does not alter thyroglobulin expression levels. Therefore, our study suggests that ERp29 acts as an escort factor for ATF6 and promotes its transport from ER to Golgi apparatus under ER stress conditions.     

PDI family protein ERp29 forms 1:1 complex with lectin chaperone calreticulin

Lectin chaperone calreticulin is well known to interact with ERp57 which is one of PDI family proteins. The interaction of ERp57 with calreticulin is believed to assist disulfide bond formation of nascent glycoprotein in the ER. Various kinds of PDI family proteins are present in the ER, however, their precise roles have been unclear. In this study, interaction assay between PDI family proteins and calreticulin by SPR analysis was performed. Our analysis revealed for the first time formation of a 1:1 complex between ERp29 and calreticulin. The dissociation constant of interaction between ERp29 and calreticulin was shown to be almost identical to ERp57–calreticulin interaction. We speculate that the recognition site of ERp29 within calreticulin is different from that of ERp57.     

The C-Terminal Domain of ERp29 Mediates Polyomavirus Binding, Unfolding, and Infection

Penetration of the endoplasmic reticulum (ER) membrane by polyomavirus (PyV) is a decisive step in virus entry. We showed previously that the ER-resident factor ERp29 induces the local unfolding of PyV to initiate the ER membrane penetration process. ERp29 contains an N-terminal thioredoxin domain (NTD) that mediates its dimerization and a novel C-terminal all-helical domain (CTD) whose function is unclear. The NTD-mediated dimerization of ERp29 is critical for its unfolding activity; whether the CTD plays any role in PyV unfolding is unknown. We now show that three hydrophobic residues within the last helix of the ERp29 CTD that were individually mutated to either lysine or alanine abolished ERp29's ability to stimulate PyV unfolding and infection. This effect was not due to global misfolding of the mutant proteins, as they dimerize and do not form aggregates or display increased protease sensitivity. Moreover, the mutant proteins stimulated secretion of the secretory protein thyroglobulin with an efficiency similar to that of wild-type ERp29. Using a cross-linking coimmunoprecipitation assay, we found that the physical interaction of the ERp29 CTD mutants with PyV is inefficient. Our data thus demonstrate that the ERp29 CTD plays a crucial role in PyV unfolding and infection, likely by serving as part of a substrate-binding domain.     

Dimerization of ERp29, a PDI-like protein, is essential for its diverse functions

Protein disulfide isomerase (PDI)-like proteins act as oxido-reductases and chaperones in the endoplasmic reticulum (ER). How oligomerization of the PDI-like proteins control these activities is unknown. Here we show that dimerization of ERp29, a PDI-like protein, regulates its protein unfolding and escort activities. We have demonstrated previously that ERp29 induces the local unfolding of polyomavirus in the ER, a step required for viral infection. We now find that, in contrast to wild-type ERp29, a mutant ERp29 (D42A) that dimerizes inefficiently is unable to unfold polyomavirus or stimulate infection. A compensatory mutation that partially restores dimerization to the mutant ERp29 (G37D/D42A) rescues ERp29 activity. These results indicate that dimerization of ERp29 is crucial for its protein unfolding function. ERp29 was also suggested to act as an escort factor by binding to the secretory protein thyroglobulin (Tg) in the ER, thereby facilitating its secretion. We show that this escort function likewise depends on ERp29 dimerization. Thus our data demonstrate that dimerization of a PDI-like protein acts to regulate its diverse ER activities.     

Mechanisms of pre‐apoptotic calreticulin exposure in immunogenic cell death

Dying tumour cells can elicit a potent anticancer immune response by exposing the calreticulin (CRT)/ERp57 complex on the cell surface before the cells manifest any signs of apoptosis. Here, we enumerate elements of the pathway that mediates pre‐apoptotic CRT/ERp57 exposure in response to several immunogenic anticancer agents. Early activation of the endoplasmic reticulum (ER)‐sessile kinase PERK leads to phosphorylation of the translation initiation factor eIF2α, followed by partial activation of caspase‐8 (but not caspase‐3), caspase‐8‐mediated cleavage of the ER protein BAP31 and conformational activation of Bax and Bak. Finally, a pool of CRT that has transited the Golgi apparatus is secreted by SNARE‐dependent exocytosis. Knock‐in mutation of eIF2α (to make it non‐phosphorylatable) or BAP31 (to render it uncleavable), depletion of PERK, caspase‐8, BAP31, Bax, Bak or SNAREs abolished CRT/ERp57 exposure induced by anthracyclines, oxaliplatin and ultraviolet C light. Depletion of PERK, caspase‐8 or SNAREs had no effect on cell death induced by anthracyclines, yet abolished the immunogenicity of cell death, which could be restored by absorbing recombinant CRT to the cell surface.     

Isolation of ERp29, a novel endoplasmic reticulum protein, from rat enamel cells evidence for a unique role in secretory-protein synthesis.

Recently we cloned and described ERp29, a novel 29-kDa endoplasmic reticulum (ER) protein that is widely expressed in rat tissues. Here we report our original isolation of ERp29 from dental enamel cells, and the comprehensive sequence analysis that correlated ERp29 with its cognate cDNA, both in enamel cells and liver. Fractionation of enamel cells using a new freeze-thaw procedure showed that ERp29 partitioned with known reticuloplasmins, and not with soluble proteins from mitochondria or cytosol. The absence of ERp29 in secreted enamel matrix indicated that the C-terminal tetrapeptide (KEEL motif) confers effective ER-retention in enamel cells. ERp29 behaved as a single species (approximately 40 kDa) during size-exclusion chromatography of liver reticuloplasm, suggesting that most ERp29 is not stably associated with other proteins. Immunoblot analysis showed that ERp29 was up-regulated during enamel secretion and expressed most highly in secretory tissues, indicative of a role in secretory-protein synthesis. Unlike other reticuloplasmins, ERp29 was down-regulated during enamel mineralization and thereby dissociated from a calcium-handling role. Tissue-specific variations in ERp29 molecular abundance were revealed by quantification of reticuloplasmin mole ratios. In conclusion: (a) ERp29 is a novel reticuloplasmin of general functional importance; (b) a unique role in protein processing is implicit from the distinctive expression patterns and molecular structure; (c) ERp29 is primarily involved in normal protein secretory events, not the ER stress response; (d) a major role is likely in tissues where ERp29 was equimolar with established molecular chaperones and foldases. This study implicates ERp29 as a new member of the ER protein-processing machinery, and identifies tissues where the physiological role of ERp29 is most likely to be clearly manifested.     

Role of ERp46 in β-cell lipoapoptosis through endoplasmic reticulum stress pathway as well as the protective effect of exendin-4

Endoplasmic reticulum (ER) stress is considered as a key factor in free fatty acid (FFA)-induced apoptosis. ERp46, a new member of the thioredoxin family, is highly expressed in pancreatic β-cells and plays an important role in glucose toxicity. In this study we examined the potential role of ERp46 in palmitic acid (PA)-induced cell apoptosis and the protective role of exendin-4, a long-acting agonist of the hormone glucagon-like peptide-1 (GLP-1) receptor. The glucose-sensitive mouse β-pancreatic cell line, βTC6, was used to investigate the mechanisms of PA-induced apoptosis. Our results showed that ERp46 expression was reduced in a dose- and time-dependent manner after PA treatment. Furthermore, inhibition of ERp46 expression by small interfering (si)RNA-mediated silencing enhanced the ER stress response via three separate pathways and increased βTC6 cell apoptosis rates. Moreover, exendin-4 reduced the ER stress response and levels of apoptosis in NC transfected cells after PA treatment, but not in cells transfected with ERp46siRNA. In conclusion, ERp46 plays a protective role in PA-induced cell apoptosis by decreasing the ER stress response and might be a novel target for anti-diabetic drugs. Exendin-4 might protect against βTC6 cell lipoapoptosis in part by activating ERp46 signaling pathway.     

ERp29 forms a feedback regulation loop with microRNA-135a-5p and promotes progression of colorectal cancer

Expression of endoplasmic reticulum (ER) stress-associated genes is often dysregulated in cancer progression. ER protein 29 (ERp29) is abnormally expressed in many neoplasms and plays an important role in tumorigenesis. Here, we showed ERp29 is a novel target for microRNA-135a-5p (miR-135a-5p) to inhibit the progression of colorectal cancer (CRC); correspondingly, ERp29 acts as an oncoprotein in CRC by promoting proliferation and metastasis of CRC cells, and suppressing apoptosis of the cells. More importantly, we found that miR-135a-5p expression is reversely upregulated by ERp29 through suppressing IL-1β-elicited methylation of miR-135a-5p promoter region, a process for enterocyte to maintain a balance between miR-135a-5p and ERp29 but dysregulated in CRC. Our study reveals a novel feedback regulation loop between miR-135a-5p and ERp29 that is critical for maintaining appropriate level of each of them, but partially imbalanced in CRC, resulting in abnormal expression of miR-135a-5p and ERp29, which further accelerates CRC progression. We provide supporting evidence for ERp29 and miR-135a-5p as potential biomarkers for diagnosis and treatment of CRC.Subject terms: Cell death, Oncogenes     

Diabetes mellitus and exocrine pancreatic dysfunction in perk-/- mice reveals a role for translational control in secretory cell survival.

The protein kinase PERK couples protein folding in the endoplasmic reticulum (ER) to polypeptide biosynthesis by phosphorylating the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha), attenuating translation initiation in response to ER stress. PERK is highly expressed in mouse pancreas, an organ active in protein secretion. Under physiological conditions, PERK was partially activated, accounting for much of the phosphorylated eIF2alpha in the pancreas. The exocrine and endocrine pancreas developed normally in Perk-/- mice. Postnatally, ER distention and activation of the ER stress transducer IRE1alpha accompanied increased cell death and led to progressive diabetes mellitus and exocrine pancreatic insufficiency. These findings suggest a special role for translational control in protecting secretory cells from ER stress.     

ERp29 is a ubiquitous resident of the endoplasmic reticulum with a distinct role in secretory protein production.

ERp29 was recently characterized biochemically as a novel protein that resides in mammalian endoplasmic reticulum (ER). Here we applied immunochemical procedures at the cellular level to investigate the hypothesized role of ERp29 in secretory protein production. ERp29 was localized exclusively to the ER/nuclear envelope of MDCK cells using confocal immunocytochemistry and comparative markers of the ER lumen, ER/Golgi membrane, nuclei, and mitochondria. A predominant association with rough ER was revealed by sucrose-gradient analysis of rat liver microsomes. Immunohistochemistry showed ERp29 expression in 35 functionally distinct cell types of rat, establishing ERp29 as a general ER marker. The ERp29 expression profile largely paralleled that of protein disulfide isomerase (PDI), the closest relative of ERp29, consistent with a role in secretory protein production. However strikingly different ERp29/PDI ratios were observed in various cell types, suggesting independent regulation and functional roles. Together, these findings associate ERp29 primarily with the early stages of secretory protein production and implicate ERp29 in a distinct functional role that is utilized in most cells. Our identification of several ERp29-enriched cell types suggests a potential selectivity of ERp29 for non-collagenous substrates and provides a physiological foundation for future investigations.