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 lin
e 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.
Mkrtchian S, etal., Eur J Biochem 1998 Jan 15;251(1-2):304-13.
We have isolated, cDNA cloned and characterised a 29-kDa protein (ERp29), containing a C-terminal endoplasmic reticulum(ER)-retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of
proteins in the ER [(e.g. culture in presence of the Ca2+ ionophore A23187, inhibitors of Ca2+-ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N-glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1% of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress-response machinery of the ER.
Mkrtchian S, etal., FEBS Lett. 1998 Jul 24;431(3):322-6.
ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution
as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunoprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.
INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor of the pancreas with poor prognosis. The lack of understanding of the molecular mechanisms of PDAC and biomarkers for early diagnosis might be two of the reasons for the poor prognosis of PDAC. MATERIA
LS AND METHODS: ILK and ERP29 protein expressions in PDAC, peritumoral tissues, benign pancreatic lesions, and normal pancreatic tissues were measured by immunohistochemistry and the clinical and pathological significances of ILK and ERP29 in PDAC were analyzed. RESULTS: The percentages of positive ILK and negative ERP29 expressions were significantly higher in PDAC tumors than in peritumoral tissues, benign pancreatic tissues, and normal pancreatic tissues (P < 0.01). Benign pancreatic lesions with positive ILK and negative ERP29 expressions exhibited dysplasia or intraepithelial neoplasia. The percentage of cases with positive ILK and negative ERP29 expressions was significantly lower in PDAC patients without lymph node metastasis and invasion, and having TNM stage I/II disease than in patients with lymph node metastasis, invasion, and TNM stage III/IV disease (P < 0.05 or P < 0.01). Kaplan-Meier survival analysis showed that positive ILK and negative ERP29 expressions were significantly associated with survival in PDAC patients (P < 0.001). Cox multivariate analysis revealed that positive ILK and negative ERP29 expressions were independent poor prognosis factors in PDAC patients. CONCLUSIONS: Positive ILK and negative ERP29 expressions are associated with the progression of PDAC and poor prognosis in patients with PDAC.
Resistance of cancer cells to radiotherapy is a major clinical problem in cancer treatment. Therefore, understanding the molecular basis of cellular resistance to radiotherapy and identification of novel targets are essential for improving treatment efficacy for cancer patients. Our previous studie
s have demonstrated a significant role of ERp29 in breast cancer cell survival against doxorubicin-induced genotoxic stress. We here reported that ERp29 expression in the triple negative MDA-MB-231 breast cancer cells significantly increased cell survival against ionizing radiation. Methylation PCR array analysis identified that ERp29 expression increased promoter hypomethylation of the DNA repair gene, O(6)-methylguanine DNA-methyltransferase (MGMT), by downregulating DNA methyltransferase 1. Knockdown of MGMT in the ERp29-transfected cancer cells increased radiosensitivity, leading to a decreased post-irradiation survival. In addition, radiation treatment in the MGMT-knockdown cells elevated phosphorylation of gamma-H2AX and cleavage of caspase 3, indicating that depletion of MGMT facilitates DNA double strands breaks and increases cell apoptosis. Hence, our studies prove a novel function of ERp29\MGMT in cancer cell survival against radiation. Targeting ERp29\MGMT axis may be useful for providing better treatment efficacy in combination with radiotherapy in breast cancer.
Sargsyan E, etal., Gene 2002 Feb 20;285(1-2):127-39.
ERp29 is a soluble protein localized in the endoplasmic reticulum (ER) of eukaryotic cells, which is conserved in all mammalian species. The N-terminal domain of ERp29 displays sequence and structural similarity to the protein disulfide isomerase despite the lack of the characteristic double cystein
e motif. Although the exact function of ERp29 is not yet known, it was hypothesized that it may facilitate folding and/or export of secretory proteins in/from the ER. ERp29 is induced by ER stress, i.e. accumulation of unfolded proteins in the ER. To gain an insight into the mechanisms regulating ERp29 expression we have cloned and characterized the rat ERp29 gene and studied in details its distribution in human tissues. Comparison with the murine and human genes and phylogenetic analysis demonstrated common origin and close ortholog relationships of these genes. Additionally, we have cloned approximately 3 kb of the 5'-flanking region of the ERp29 gene and functionally characterized its promoter. Such characteristics of the promoter as GC-rich sequence, absence of TATA-box, multiple transcription start sites taken together with the ubiquitous gene expression, reaching maximum levels in the specialized secretory tissues, indicate that ERp29 belongs to the group of the constitutively expressed housekeeping genes. A 337 bp fragment of the 5' flank was identified as a core promoter sufficient for the transcriptional activation of the gene. Gel mobility shift assay indicated interaction of the predicted GC and E box elements within the core promoter with Sp1/Sp3 and USF1/USF2 transcription factors, respectively, suggesting their key role in the basal expression of the gene.
Sargsyan E, etal., J Biol Chem 2002 May 10;277(19):17009-15.
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, an
d 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.
