The molecular chaperone hsp40 regulates the activity of P58IPK, the cellular inhibitor of PKR

The interferon-induced double-stranded RNA-activated protein kinase, PKR, likely contributes to both the antiviral and the antiproliferative effects of interferon. We previously found that influenza virus avoids the translational inhibitory effects of activated PKR by activating a cellular inhibitory protein, termed P58^IPK, based on its M_r of 58,000. P58^IPK is a member of the tetratricopeptide family of proteins and possesses significant homology to the conserved J region of the DnaJ family of heat shock proteins. We earlier hypothesized that P58^IPK was kept in an inactive state with its own inhibitor (termed I-P58^IPK) in uninfected cells. We therefore attempted the purification and characterization of I-P58^IPK. The following data suggest that we have identified the molecular chaperone, hsp40, as I-P58^IPK. (i) The MonoP-purified I-P58^IPK protein reacted with hsp40 antibody. (ii) This preparation demonstrated high specific activity in an in vitro functional assay containing only purified recombinant and native components. (iii) Purified, recombinant hsp40 protein inhibited P58^IPK in an identical in vitro assay. (iv) Finally, we demonstrate that hsp40 directly complexes with P58^IPK, in vitro, suggesting the inhibition occurs through a direct interaction. Our data, taken together, provide evidence for a novel intersection between the heat shock and interferon pathways, and suggest that influenza virus regulates PKR activity through the recruitment of a cellular stress pathway.     

PERK eIF2alpha kinase regulates neonatal growth by controlling the expression of circulating insulin-like growth factor-I derived from the liver

Humans afflicted with the Wolcott-Rallison syndrome and mice deficient for PERK (pancreatic endoplasmic reticulum eIF2alpha kinase) show severe postnatal growth retardation. In mice, growth retardation in Perk-/- mutants is manifested within the first few days of neonatal development. Growth parameters of Perk-/- mice, including comparison of body weight to length and organ weights, are consistent with proportional dwarfism. Tibia growth plates exhibited a reduction in proliferative and hypertrophic chondrocytes underlying the longitudinal growth retardation. Neonatal Perk-/- deficient mice show a 75% reduction in liver IGF-I mRNA and serum IGF-I within the first week, whereas the expression of IGF-I mRNA in most other tissues is normal. Injections of IGF-I partially reversed the growth retardation of the Perk-/- mice, whereas GH had no effect. Transgenic rescue of PERK activity in the insulin- secreting beta-cells of the Perk-/- mice reversed the juvenile but not the neonatal growth retardation. We provide evidence that circulating IGF-I is derived from neonatal liver but is independent of GH at this stage. We propose that PERK is required to regulate the expression of IGF-I in the liver during the neonatal period, when IGF-I expression is GH-independent, and that the lack of this regulation results in severe neonatal growth retardation.     

内质网分子伴侣糖调节蛋白94在大鼠酒精性肝损伤中的高表达和意义

目的检测酒精性肝损伤大鼠肝组织内质网分子伴侣糖调节蛋白94的表达情况,探讨酒精性肝损伤的发病机制.方法24只SD雌性大鼠分成对照组和模型组.模型组大鼠给予乙醇加鱼油灌胃配合高脂饮食8周,建立酒精性肝损伤模型,应用半定量逆转录聚合酶链反应(RT-PCR)法和免疫组化法检测大鼠肝组织内质网分子伴侣糖调节蛋白94 mRNA和蛋白水平.结果与正常组比较,模型组大鼠肝组织糖调节蛋白94 mRNA和蛋白表达明显增强(P＜0.01).结论酒精性肝损伤大鼠存在糖调节蛋白94高表达,这可能是该病的发病机制之一.     

Tyrosine phosphorylation acts as a molecular switch to full-scale activation of the eIF2alpha RNA-dependent protein kinase

Phosphorylation of the alpha-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine/threonine RNA-dependent protein kinase (PKR) is an eIF2alpha kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2alpha phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2alpha kinase PKR.     

Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

The folding environment in the endoplasmic reticulum (ER) depends on multiple abundant chaperones that function together to accommodate a range of substrates. The ways in which substrate engagement shapes either specific chaperone dynamics or general ER attributes in vivo remain unknown. In this study, we have evaluated how changes in substrate flux through the ER influence the diffusion of both the lectin chaperone calreticulin and an inert reporter of ER crowdedness. During acute changes in substrate load, the inert probe revealed no changes in ER organization, despite significant changes in calreticulin dynamics. By contrast, inhibition of the lectin chaperone system caused rapid changes in the ER environment that could be reversed over time by easing new substrate burden. Our findings provide insight into the normal organization and dynamics of an ER chaperone and characterize the capacity of the ER to maintain homeostasis during acute changes in chaperone activity and availability.     

Regulation of protein synthesis by the heme-regulated eIF2alpha kinase: relevance to anemias

During erythroid differentiation and maturation, it is critical that the 3 components of hemoglobin, alpha-globin, beta-globin, and heme, are made in proper stoichiometry to form stable hemoglobin. Heme-regulated translation mediated by the heme-regulated inhibitor kinase (HRI) provides one major mechanism that ensures balanced synthesis of globins and heme. HRI phosphorylates the alpha-subunit of eukaryotic translational initiation factor 2 (eLF2alpha) in heme deficiency, thereby inhibiting protein synthesis globally. In this manner, HRI serves as a feedback inhibitor of globin synthesis by sensing the intracellular concentration of heme through its heme-binding domains. HRI is essential not only for the translational regulation of globins, but also for the survival of erythroid precursors in iron deficiency. Recently, the protective function of HRI has also been demonstrated in murine models of erythropoietic protoporphyria and beta-thalassemia. In these 3 anemias, HRI is essential in determining red blood cell size, number, and hemoglobin content per cell. Translational regulation by HRI is critical to reduce excess synthesis of globin proteins or heme under nonoptimal disease states, and thus reduces the severity of these diseases. The protective role of HRI may be more common among red cell disorders.     

内质网分子伴侣糖调节蛋白94在非酒精性脂肪肝中的表达及意义

目的:检测非酒精性脂肪肝患者肝组织中糖调节蛋白94的表达情况,探讨非酒精性脂肪肝病(nonalcoholic fattyliver disease,NAFLD)的发病机制.方法:应用半定量逆转录聚合酶链反应(RTPCR)法和免疫组化法检测脂肪肝组和正常对照组肝组织中糖调节蛋白94基因和蛋白表达,HE染色观察肝脏脂肪变性、炎性活动和坏死,并进行相关分析.结果:非酒精性脂肪肝组脂肪变性、炎症评分及坏死评分较正常对照组明显增加,差异具有统计学意义(3.89±0.32 VS 0.20±0.4;3.67±0.49 VS 0:0.44±0.51 VS 0,P＜0.01或0.05).与正常组比较,脂肪肝患者肝组织糖调节蛋白94mRNA和蛋白表达明显增强(0.86±0.02 VS 0.37±0.03;0.34±0.01 VS 0.31±0.01,均P＜0.01).糖调节蛋白94 mRNA和蛋白表达与脂肪变性、炎症评分及坏死评分明显呈正相关(P＜0.01).结论:非酒精脂肪肝引起的肝脏损害可能与糖调节蛋白94表达有关.     

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.The 90-kDa heat shock protein (Hsp90) is an abundant chaperone in the cytosol of eukaryotes (1). Together with its cochaperones, it functions in the conformational control of many regulatory proteins (2–4). Kinases constitute the largest group of Hsp90 client proteins with more than 60% of the human kinases that depend on Hsp90 in terms of their activity (5, 6).Hsp90 forms V-shaped homodimers connected via a C-terminal domain. The middle domain (M-domain) is involved in client binding (7, 8), and the N-terminal domain binds ATP. Upon ATP binding, the N-terminal domains dimerize, leading to the closed state (9–13), whereas the open state is regained upon ATP-hydrolysis (14). Both conformation and ATPase activity are affected by interaction with a cohort of cochaperones (15). Given the large number and diversity of client proteins, cochaperones are believed to deliver specificity in this context.The Hsp90-mediated maturation of kinases is strictly dependent on the cochaperone Cdc37 (cell division control protein 37) (16, 17) and phosphorylation of this cofactor is important for its function (18, 19). Binding of Cdc37 to Hsp90 causes inhibition of the ATPase activity of Hsp90 and has therefore been proposed to facilitate client kinase loading onto the Hsp90 machinery (20).The viral Src kinase (v-Src) is one of the most stringent known Hsp90 clients (5, 21). v-Src belongs to the family of nonreceptor tyrosine kinases, which play important roles in many cellular pathways. v-Src kinase is constitutively active and leads to the formation of sarcomas in chicken (22). It shows 98% sequence identity with its cellular counterpart c-Src (cellular Src kinase), the first identified protooncogene (23). Hsp90 binds to and stabilizes c-Src in its nascent state, but it dissociates after the kinase folding is achieved (24). Due to this complete loss of interaction, c-Src has been defined as a nonclient (5). Src consists of a unique domain followed by the SH3 and SH2 domains and a flexible linker, which connects the SH2 domain with the highly conserved kinase domain. c-Src contains an additional stretch at its C terminus that includes a tyrosine at the position 527, whose phosphorylation status regulates kinase activity (25, 26). In addition, v-Src differs from c-Src by several point mutations (27–30). Some of these were shown to increase c-Src activity in vivo and have been linked to cancer progression and metastasis in humans (31–33). Due to these differences, v-Src cannot be down-regulated and is permanently active, even in the absence of activating stimuli (26, 30). The analysis of proteins nearly identical in sequence but highly different in chaperone dependence offers an excellent model system for understanding the features that render a protein Hsp90-dependent. We used these kinases to reconstitute and dissect the chaperoning effect of Hsp90 on v-Src kinase in vitro. The analysis of chimeras comprising elements of c-Src and v-Src allowed us to determine the molecular basis of the stringent Hsp90 dependence of v-Src.     

Mobility of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 × 10⁻¹⁰ cm²/s. A coefficient only slightly larger (7.1 × 10⁻¹⁰ cm²/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.     

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.     

Sarcalumenin alleviates stress-induced cardiac dysfunction by improving Ca2+ handling of the sarcoplasmic reticulum

AIMS: Sarcalumenin (SAR) is a Ca(2+)-binding protein expressed in the longitudinal sarcoplasmic reticulum (SR) of striated muscle cells. Although its Ca(2+)-binding property is similar to that of calsequestrin, its role in the regulation of Ca(2+) cycling remains unclear. METHODS AND RESULTS: To investigate whether SAR plays an important role in maintaining cardiac function under pressure overload stress, SAR-knockout (SAR-KO) mice were subjected to transverse aortic constriction (TAC). To examine the relation of SAR with cardiac type of SR Ca(2+) pump, SERCA2a, we designed cDNA expression using cultured cells. We found that SAR expression was significantly downregulated in hypertrophic hearts from three independent animal models. SAR-KO mice experienced higher mortality than did wild-type (WT) mice after TAC. TAC significantly downregulated SERCA2a protein but not mRNA in the SAR-KO hearts, whereas it minimally did so in hearts from WT mice. Accordingly, SR Ca(2+) uptake and cardiac function were significantly reduced in SAR-KO mice after TAC. Then we found that SAR was co-immunoprecipitated with SERCA2a in cDNA-transfected HEK293T cells and mouse ventricular muscles, and that SERCA2a-mediated Ca(2+) uptake was augmented when SAR was co-expressed in HEK293T cells. Furthermore, SAR significantly prolonged the half-life of SERCA2a protein in HEK293T cells. CONCLUSION: These findings suggest that functional interaction between SAR and SERCA2a enhances protein stability of SERCA2a and facilitates Ca(2+) sequestration into the SR. Thus the SAR-SERCA2a interaction plays an essential role in preserving cardiac function under biomechanical stresses such as pressure overload.     

Syntaxin 5 regulates the endoplasmic reticulum channel-release properties of polycystin-2

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca²⁺) release channel. We performed a yeast two-hybrid screen by using the NH₂ terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca²⁺ levels, decreased endoplasmic reticulum (ER) Ca²⁺ stores, and reduced Ca²⁺ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca²⁺ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca²⁺ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca²⁺ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca²⁺ signaling associated with polycystic kidney disease.     

Deficiency of heme-regulated eIF2alpha kinase decreases hepcidin expression and splenic iron in HFE-/- mice

Heme-regulated eIF2alpha kinase (HRI) is essential for regulating globin translation in iron deficiency and in beta-thalassemia. We investigated the role of heme-regulated eIF2alpha kinase in hemoglobin and red blood cell production as well as in iron homeostasis in a mouse model of iron overload. We show that HRI deficiency does not significantly affect red cell parameters of hemochromatosis (HFE(-)(/)(-)) mice. Importantly, heme-regulated eIF2alpha kinase deficiency exacerbates decreases in hepcidin expression and splenic macrophage iron in HFE(-)(/)(-) mice. Furthermore, the serum level of bone morphogenic protein 2, which positively regulates hepcidin, is reduced in heme-regulated eIF2alpha kinase deficiency, but not in HFE deficiency.     

Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency

Haem-regulated eIF2alpha kinase (HRI) is essential for the regulation of globin gene translation and the survival of erythroid precursors in iron/haem deficiency. This study found that that in iron deficiency, fetal definitive erythropoiesis is inhibited at the basophilic erythroblast stage with increased proliferation and elevated apoptosis. This hallmark of ineffective erythropoiesis is more severe in HRI deficiency. Microarray gene profiling analysis showed that HRI was required for adaptive gene expression in erythroid precursors during chronic iron deficiency. The number of genes with expression affected more than twofold increased, from 213 in iron deficiency and 73 in HRI deficiency, to 3135 in combined iron and HRI deficiencies. Many of these genes are regulated by Gata1 and Fog1. We demonstrate for the first time that Gata1 expression in developing erythroid precursors is decreased in iron deficiency, and is decreased further in combined iron and HRI deficiencies. Additionally, Fog1 expression is decreased in combined deficiencies, but not in iron or HRI deficiency alone. Our results indicate that HRI confers adaptive gene expression in developing erythroblasts during iron deficiency through maintaining Gata1/Fog1 expression.