Association of Hsp47, Grp78, and Grp94 with procollagen supports the successive or coupled action of molecular chaperones

Hps47, Grp78, have been implicated with procellagen maturation events. In particular Hps47 has been shown to blind to nascent procellagen α1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collgen has previsously been suggested to mechanism. The early association of Hps47 with procollagen and its relatively late relese suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hps47. Herein, we examined how these events occurs in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the releses of Hps47, Grp78, and Grp94 from maturing procollange is delayed. Thus, in cell experiencing metabolic stress, newly synthesized procollagen unable to property fold became stable bound to a complex of molecular chaperones. In that Hps47, Grp78, and Grp98 could be recovered with nascent procollagen and as oligomer in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.     

Grp78, Grp94, and Grp170 interact with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum

In alpha1-antitrypsin (alpha1-AT) deficiency, a mutant form of alpha1-AT polymerizes in the endoplasmic reticulum (ER) of liver cells resulting in chronic hepatitis and hepatocellular carcinoma by a gain of toxic function mechanism. Although some aspects of the cellular response to mutant alpha1-AT Z have been partially characterized, including the involvement of several proteasomal and nonproteasomal mechanisms for disposal, other parts of the cellular response pathways, particularly the chaperones with which it interacts and the signal transduction pathways that are activated, are still not completely elucidated. The alpha1-AT Z molecule is known to interact with calnexin, but, according to one study, it does not interact with Grp78. To carry out a systematic search for the chaperones with which alpha1-AT Z interacts in the ER, we used chemical cross-linking of several different genetically engineered cell systems. Mutant alpha1-AT Z was cross-linked with Grp78, Grp94, calnexin, Grp170, UDP-glucose glycoprotein:glucosyltransferase, and two unknown proteins of approximately 110-130 kDa. Sequential immunoprecipitation/immunoblot analysis and coimmunoprecipitation techniques demonstrated each of these interactions without chemical cross-linking. The same chaperones were found to interact with two nonpolymerogenic alpha1-AT mutants that are retained in the ER, indicating that these interactions are not specific for the alpha1-AT Z mutant. Moreover, sucrose density gradient centrifugation studies suggest that approximately 85% of alpha1-AT Z exists in heterogeneous soluble complexes with multiple chaperones and approximately 15% in extremely large polymers/aggregates devoid of chaperones. Agents that perturb the synthesis and/or activity of ER chaperones such as tunicamycin and calcium ionophore A23187, have different effects on the solubility and degradation of alpha1-AT Z as well as on its residual secretion.     

The hsp110 and Grp1 70 stress proteins: newly recognized relatives of the Hsp70s

Both the Grp170 and Hsp110 families represent relatively conserved and distinct sets of stress proteins, within a more diverse category that also includes the Hsp70s. All of these families are found in a wide variety of organisms from yeasts to humans. Although Hsp110s or Grp170s are not Hsp70s any more than Hsp70s are Hsp110s or Grp170s, it is still reasonable to refer to this combination of related families as the Hsp70 superfamily based on arguments discussed above and since no obvious prokaryotic Hsp110 or Grp170 has yet been identified. These proteins are related to their counterparts in the Hsp70/Grp78 family of eukaryotic stress proteins but are characterized by significantly larger molecular weights. The members of the Grp170 family are characterized by C-terminal ER retention sequences and are ER localized in yeasts and mammals. As a Grp, Grp170 is recognized to be coregulated with other major Grps by a well-known set of stress conditions, sometimes referred to as the unfolded protein response (Kozutsumi et al 1988; Nakaki et al 1989). The Hsp110 family members are localized in the nucleus and cytoplasm and, with other major Hsps, are also coregulated by a specific set of stress conditions, most notably including hyperthermic exposures. Hsp110 is sometimes called Hsp105, although it would be preferable to have a uniform term. The large Hsp70-like proteins are structurally similar to the Hsp70s but differ from them in important ways. In both the Grp170 and Hspl10 families, there is a long loop structure that is interposed between the peptide-binding ,-domain and the alpha-helical lid. In the Hsp110 family and Grp170, there are differing degrees of expansion in the alpha-helical domain and the addition of a C-terminal loop. This gives the appearance of much larger lid domains for Hsp110 and Grp170 compared with Hsp70. Both Hsp110 and Grp170 families have relatively conserved short sequences in the alpha-helical domain in the lid, which are conserved motifs in numerous proteins (we termed these motifs Magic and TedWylee as discussed earlier). The structural differences detailed in this review result in functional differences between the large (Grp170 and Hspl10) members of the Hsp70 superfamily, the most distinctive being an increased ability of these proteins to bind (hold) denatured polypeptides compared with Hsc70, perhaps related to the enlarged C-terminal helical domain. However, there is also a major difference between these large stress proteins; Hsp110 does not bind ATP in vitro, whereas Grp170 binds ATP avidly. The role of the Grp170 and Hsp110 stress proteins in cellular physiology is not well understood. Overexpression of Hsp110 in cultured mammalian cells increases thermal tolerance. Grp170 binds to secreted proteins in the ER and may be cooperatively involved in folding these proteins appropriately. These roles are similar to those of the Hsp70 family members, and, therefore, the question arises as to the differential roles played by the larger members of the superfamily. We have discussed evidence that the large members of the superfamily cooperate with members of the Hsp70 family, and these chaperones probably interact with a large number of chaperones and cochaperones in their functional activities. The fundamental point is that Hsp110 is found in conjunction with Hsp70 in the cytoplasm (and nucleus) and Grp170 is found in conjunction with78 in tha ER in every eucaryotic cell examined from yeast to humans. This would strongly argue that Hsp110 Grp170 exhibit functions in eucaryotes not effectively performed by Hsp70s or Grp78, respectively. Of interest in this respect is the observation that all Hsp110s loss of function or deletion mutants listed in the Drosophila deletion project database are lethal. The important task for the future is to determine the roles these conserved molecular chaperones play in normal and physiologically stressed cells.     

Hepatic lipase maturation: a partial proteome of interacting factors

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.     

Retention of mutant low density lipoprotein receptor in endoplasmic reticulum (ER) leads to ER stress

Familial hypercholesterolemia is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and retention of mutant LDLR are poorly understood. In the present study we show that the G544V mutant LDLR associates with the chaperones Grp78, Grp94, ERp72, and calnexin in the ER of transfected Chinese hamster ovary cells. Retention of the mutant LDLR was shown to cause ER stress and activation of the unfolded protein response. We observed a marked increase in the activity of two ER stress sensors, IRE1 and PERK. These results show that retention of mutant LDLR in ER induces cellular responses, which might be important for the clinical outcome of familial hypercholesterolemia.     

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.     

T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78 kDa), phospho-eIF2α (phosphorylated eukaryotic initiation factor 2α) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2α, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis.     

Endoplasmic reticulum stress contributed to inflammatory bowel disease by activating p38 MAPK pathway

Recent evidence suggests that endoplasmic reticulum (ER) stress plays a vital role in inflammatory bowel disease (IBD). Therefore, the aim of this study was to investigate the mechanism by which ER stress promotes inflammatory response in IBD. The expression of Gro-α, IL-8 and ER stress indicator Grp78 in colon tissues from patients with Crohn’s disease (CD) and colonic carcinoma was analyzed by immunohistochemistry staining. Colitis mouse model was established by the induction of trinitrobenzene sulphonic acid (TNBS), and the mice were treated with ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Then the body weight, colon length and colon inflammation were evaluated, and Grp78 and Gro-α in colon tissues were detected by immunohistochemistry. Epithelial cells of colon cancer HCT116 cells were treated with tunicamycin to induce ER stress. Grp78 was detected by Western blot, and chemokines were measured by PCR and ELISA. The expression levels of Grp78, Gro-α and IL-8 were significantly upregulated in intestinal tissues of CD patients. Mice with TNBS induced colitis had increased expression of Grp78 and Gro-α in colonic epithelia. TUDCA reduced the severity of TNBS-induced colitis. In HCT116 cells, tunicamycin increased the expression of Grp78, Gro-α and IL-8 in a concentration-dependent manner. Furthermore, p38 MAPK inhibitor significantly inhibited the upregulation of Gro-α and IL-8 induced by tunicamycin. In conclusion, ER stress promotes inflammatory response in IBD, and the effects may be mediated by the activation of p38 MAPK signaling pathway.Key words: Inflammatory bowel disease, endoplasmic reticulum stress, IL-8, Gro-α, p38 MAPK     

Diabetes- and angiotensin II-induced cardiac endoplasmic reticulum stress and cell death: metallothionein protection

We have shown cardiac protection by metallothionein (MT) in the development of diabetic cardiomyopathy (DCM) via suppression of cardiac cell death in cardiac-specific MT-overexpressing transgenic (MT-TG) mice. The present study was undertaken to define whether diabetes can induce cardiac endoplasmic reticulum (ER) stress and whether MT can prevent cardiac cell death via attenuating ER stress. Diabetes was induced by streptozotocin in both MT-TG and wild-type (WT) mice. Two weeks, and 2 and 5 months after diabetes onset, cardiac ER stress was detected by expression of ER chaperones, and apoptosis was detected by CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and cleaved caspase-3 and caspase-12. Cardiac apoptosis in the WT diabetic mice, but not in MT-TG diabetic mice, was significantly increased 2 weeks after diabetes onset. In parallel with apoptotic effect, significant up-regulation of the ER chaperones, including glucose-regulated protein (GRP)78 and GRP94, cleaved ATF6 and phosporylated eIF2α, in the hearts of WT, but not MT-TG diabetic mice. Infusion of angiotensin II (Ang II) also significantly induced ER stress and apoptosis in the hearts of WT, but not in MT-TG mice. Direct administration of chemical ER stress activator tunicamycin significantly increased cardiac cell death only in WT mice. Pre-treatment with antioxidants completely prevented Ang II-induced ER stress and apoptosis in the cultured cardiac cells. These results suggest that ER stress exists in the diabetic heart, which may cause the cardiac cell death. MT prevents both diabetes- and Ang II-induced cardiac ER stress and associated cell death most likely via its antioxidant action, which may be responsible for MT's prevention of DCM.     

The altered expression of glucose-regulated proteins 78 in different phase of streptozotocin-affected pancreatic beta-cells

Endoplasmic reticulum (ER) stress-mediated apoptosis plays an important role in the destruction of pancreatic beta-cells and contributes to the development of type 1 diabetes. The chaperone molecule, glucose-regulated proteins 78 (Grp78), is required to maintain ER function during toxic insults. In this study, we investigated the changes of Grp78 expression in different phases of streptozotocin (STZ)-affected beta-cells to explore the relationship between Grp78 and the response of beta-cells to ER stress. An insulinoma cell line (NIT-1) treated with STZ for different time periods and STZ-induced diabetic Balb/C mice at different time points were used as the model system. The level of Grp78 and C/EBP homologous protein (CHOP) mRNA were detected by real-time polymerase chain reaction and their protein by immunoblot. Apoptosis and necrosis was measured by flow cytometry. In addition, the changes of Grp78 protein in STZ-treated nondiabetic mice were also detected by immunoblot. Grp78 expression significantly increased in the early phase but decreased in the later phase of affected beta-cells, while CHOP was induced and apoptosis occurred along with the decrease of Grp78. Interestingly, the Grp78 protein of STZ-treated nondiabetic mice increased stably compared with that of the control. From the results, we can conclude that Grp78 may contribute to the response of beta-cells to ER stress, and more attention should be paid to Grp78 in the improvement of diabetes.     

Endoplasmic reticulum stress-induced cysteine protease activation in cortical neurons: effect of an Alzheimer's disease-linked presenilin-1 knock-in mutation

Endoplasmic reticulum (ER) stress elicits protective responses of chaperone induction and translational suppression and, when unimpeded, leads to caspase-mediated apoptosis. Alzheimer's disease-linked mutations in presenilin-1 (PS-1) reportedly impair ER stress-mediated protective responses and enhance vulnerability to degeneration. We used cleavage site-specific antibodies to characterize the cysteine protease activation responses of primary mouse cortical neurons to ER stress and evaluate the influence of a PS-1 knock-in mutation on these and other stress responses. Two different ER stressors lead to processing of the ER-resident protease procaspase-12, activation of calpain, caspase-3, and caspase-6, and degradation of ER and non-ER protein substrates. Immunocytochemical localization of activated caspase-3 and a cleaved substrate of caspase-6 confirms that caspase activation extends into the cytosol and nucleus. ER stress-induced proteolysis is unchanged in cortical neurons derived from the PS-1 P264L knock-in mouse. Furthermore, the PS-1 genotype does not influence stress-induced increases in chaperones Grp78/BiP and Grp94 or apoptotic neurodegeneration. A similar lack of effect of the PS-1 P264L mutation on the activation of caspases and induction of chaperones is observed in fibroblasts. Finally, the PS-1 knock-in mutation does not alter activation of the protein kinase PKR-like ER kinase (PERK), a trigger for stress-induced translational suppression. These data demonstrate that ER stress in cortical neurons leads to activation of several cysteine proteases within diverse neuronal compartments and indicate that Alzheimer's disease-linked PS-1 mutations do not invariably alter the proteolytic, chaperone induction, translational suppression, and apoptotic responses to ER stress.     

The therapeutic mavericks: Potent immunomodulating chaperones capable of treating human diseases

Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an ‘eat-me’ signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.     

Insulin induces chaperone and CHOP gene expressions in adipocytes

Adipocyte secretes bioactive proteins called adipocytokines, and biosynthesis of secretory proteins requires molecular chaperones and folding enzymes in endoplasmic reticulum (ER). ER chaperones are known to be induced by unfolded protein response (UPR) and growth factors, however, it has not been determined how ER chaperones expression is regulated in adipocytes. Here we show that insulin treatment induced GRP78 and ERO1L mRNA levels in 3T3-L1 adipocytes. Insulin also upregulated CHOP mRNA levels, but did not induce phosphorylation of eIF2α. Pretreatment with insulin protected 3T3-L1 adipocytes against thapsigargin-mediated phosphorylation of eIF2α but did not against DTT-mediated one. In vivo mice study showed that GRP78 and CHOP expressions were regulated by feeding conditions. These results suggest that insulin signaling is important to induce mRNA expressions of GRP78 and CHOP, and may have a protective role against UPR.