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Glucose-regulated protein 78 is essential for cardiac myocyte survival.

ABSTRACT: Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.

SUBMITTER: Wang X

PROVIDER: S-EPMC6261960 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Glucose-regulated protein 78 is essential for cardiac myocyte survival.

Wang Xiaoding X Bi Xukun X Zhang Guangyu G Deng Yingfeng Y Luo Xiang X Xu Lin L Scherer Philipp E PE Ferdous Anwarul A Fu Guosheng G Gillette Thomas G TG Lee Amy S AS Jiang Xuejun X Wang Zhao V ZV

Cell death and differentiation 20180417 12

Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all ...[more]

PMID: 29666470

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Project description:RationaleEmphysema and osteoporosis are epidemiologically associated diseases of cigarette smokers. The causal mechanism(s) linking these illnesses is unknown. We hypothesized autoimmune responses may be involved in both disorders.ObjectivesTo discover an antigen-specific autoimmune response associated with both emphysema and osteoporosis among smokers.MethodsReplicate nonbiased discovery assays indicated that autoimmunity to glucose regulated protein 78 (GRP78), an endoplasmic reticulum chaperone and cell surface signaling receptor, is present in many smokers. Subject assessments included spirometry, chest CT scans, dual x-ray absorptiometry, and immunoblots for anti-GRP78 IgG. Anti-GRP78 autoantibodies were isolated from patient plasma by affinity chromatography, leukocyte functions assessed by flow cytometry, and soluble metabolites and mediators measured by immunoassays.Measurements and main resultsCirculating anti-GRP78 IgG autoantibodies were detected in plasma specimens from 86 (32%) of the 265 smoking subjects. Anti-GRP78 autoantibodies were singularly prevalent among subjects with radiographic emphysema (OR 3.1, 95%CI 1.7-5.7, p?=?0.003). Anti-GRP78 autoantibodies were also associated with osteoporosis (OR 4.7, 95%CI 1.7-13.3, p?=?0.002), and increased circulating bone metabolites (p?=?0.006). Among emphysematous subjects, GRP78 protein was an autoantigen of CD4 T-cells, stimulating lymphocyte proliferation (p?=?0.0002) and IFN-gamma production (p?=?0.03). Patient-derived anti-GRP78 autoantibodies had avidities for osteoclasts and macrophages, and increased macrophage NFkB phosphorylation (p?=?0.005) and productions of IL-8, CCL-2, and MMP9 (p?=?0.005, 0.007, 0.03, respectively).ConclusionsHumoral and cellular GRP78 autoimmune responses in smokers have numerous biologically-relevant pro-inflammatory and other deleterious actions, and are associated with emphysema and osteoporosis. These findings may have relevance for the pathogenesis of smoking-associated diseases, and development of biomarker immunoassays and/or novel treatments for these disorders.

Project description:Post translational modification of proteins plays a significant role in immune recognition. In particular the modification of arginine to citrulline which is mediated by PAD enzymes is increased during cellular stress (autophagy) which permits the presentation of modified epitopes upon MHC class II molecules for recognition by CD4 T cells. Citrullination also occurs in tumour cells as a result of continuous environmental stresses and increased autophagy. We have shown in animal models the efficient stimulation of citrullinated epitope specific CD4 T cells resulting in dramatic elimination/regression of tumours. The ER chaperone glucose-regulated protein 78 (GRP78) is known to also be required for stress-induced autophagy and is directly linked to autophagosome formation. GRP78 is known to be highly expressed by many tumour types. In this study we investigate the potential of targeting citrullinated GRP78 for cancer therapy. A citrullinated GRP78 specific antibody was used to assess citrullinated GRP78 expression in murine and human tumour cells by flow cytometry. Five peptides were selected and used to vaccinate HLA transgenic mice and immune responses were characterised by ex vivo cytokine ELISpot assay. T cell repertoire in humans was assessed through proliferation assays and cytokine ELISpot assay. Citrullinated peptide was identified in murine B16 melanoma by mass spectrometry and the peptide vaccine was assessed for tumour therapy in a mouse melanoma model. We show the identification CD4 T cell responses to one citrullinated GRP78 epitope that are restricted through HLA DP*0401 and HLA-DR*0101 alleles. This peptide is detected by mass spectrometry in B16 melanoma grown in vivo and citrulline specific CD4 responses to two peptides spanning this epitope mediate efficient therapy of established B16 melanoma tumours in HHDII/DP4 (p<0.0001) transgenic mouse model. Finally, we demonstrate the existence of a repertoire of responses to the citrullinated GRP78 peptide in healthy individuals (p=0.0023) with 13/17 (76%) individuals showing a response to this peptide. We propose that citrullinated GRP78 is a candidate tumour antigen and vaccination against citrullinated GRP78 may provide a promising tumour therapy approach.

Project description:GRP78, a resident endoplasmic reticulum (ER) chaperone involved in protein transport, folding and assembly, has been reported in sperm. It is shown to be localized in the neck region of human sperm. We have previously reported GRP78 to be less phosphorylated in asthenozoosperm.The present study aimed to determine whether sperm GRP78 undergoes phosphorylation changes during epididymal maturation and whether there are any differences in GRP78 phosphoforms in asthenozoosperm vis-à-vis normozoosperm. Testicular- and cauda epididymal- sperm from adult male Holtzman rats, and semen ejaculates collected from normal and asthenozoospermic individuals were investigated. DIGE carried out to determine phosphorylation of GRP78 in asthenozoosperm and normal sperm reveals a shift in the location of GRP78 of asthenozoosperm towards the alkaline pH, indicative of reduced GRP78 phosphorylation. Immunoprecipitation studies using antibodies specific to GRP78, serine-, threonine-, and tyrosine phosphorylation and Pan phospho antibody demonstrates GRP78 to be phosphorylated at all three residues in rat spermatozoa. Phosphatase assays using Calf intestinal alkaline phosphatase and Lambda protein phosphatase followed by nanofluidic proteomic immunoassay (NIA) show that in rat, GP4.96, GP4.94 and GP4.85 are the three phosphoforms in mature (caudal) sperm as against two phosphoforms GP4.96and GP4.94in immature (testicular) sperm. In mature human sperm GP5.04, GP4.96, and GP4.94were the 3 phosphoforms observed. GP4.94[P = 0.014]andGP5.04 [P = 0.02] are significantly reduced in asthenozoosperm. Ours is the first report indicating GRP78 in sperm to be phosphorylated at serine, threonine and tyrosine residues contrary to published literature reporting GRP78 not to be tyrosine phosphorylated. We report the presence of GRP78 phosphoforms in rat- and human- sperm and our data suggest that GRP78 phosphorylation in sperm undergoes spatial reorganization during epididymal maturation. Significant differences observed in 2 out of 3 phosphoforms in asthenozoosperm suggest that GRP78 phosphorylation may have functional relevance in sperm with consequent clinical implications.

Project description:Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-beta1 (IFN-beta1). In this connection, the IFN-beta1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-beta1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.

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Glucose-regulated protein 78 is essential for cardiac myocyte survival.

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Glucose-regulated protein 78 is essential for cardiac myocyte survival.

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