Quaternary structure of the HSC70 cochaperone HIP

HSC70 interacting protein (HIP) is an essential cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor. To determine the quaternary structure and the gross conformation of the protein in solution, a wide array of biochemical and biophysical techniques has been used. Size-exclusion chromatography and sedimentation velocity indicate the presence of a single species with a Stokes radius, R(s), of 55 A and a sedimentation coefficient, s degrees (20,w), of 4.34 S. The combination of these data gives a molecular mass of 101 000 Da, a value close to that of the theoretical molecular mass of a dimer (87 090 Da). Sedimentation equilibrium, performed at various protein concentrations and rotor speeds, gives a molecular mass of 88 284 Da, almost in exact agreement with the molecular mass of a dimer. On the basis of these data, a frictional ratio f/f(0) of 1.6 is obtained, suggesting an elongated shape for the HIP dimer. Secondary structure predictions, supported by circular dichroism experiments, indicate that HIP is an almost all alpha-protein, able to form extended coiled coils. Using threading and comparative model building methods, a structural model of a segment of HIP involved in HSC70 binding has been constructed and potential sites of interaction between HIP and HSC70 are proposed on the basis of electrostatic as well as shape complementarity. Altogether, these results indicate that HIP is an elongated dimer, able to bind two HSC70 molecules through its TPR regions, and suggest the existence of a versatile binding site on HSC70 that may be involved in the interaction of the chaperone with the cochaperones or other interacting proteins.     

Structure and activity of JAC1 J-domain implicate the involvement of the cochaperone activity with HSC70 in chloroplast photorelocation movement

Chloroplast photorelocation movement towards weak light and away from strong light is essential for plants to adapt to the fluctuation of ambient light conditions. In the previous study, we showed that blue light receptor phototropins mediated blue light-induced chloroplast movement in Arabidopsis by regulating short actin filaments localized at the chloroplast periphery (cp-actin filaments) rather than actin cables in the cytoplasm. However, the signaling pathway for the chloroplast photorelocation movement is still unclear. We also identified JAC1 (J-domain protein required for chloroplast accumulation response 1) as an essential component for the accumulation response and dark positioning in Arabidopsis. We recently determined the crystal structure of the J-domain of JAC1. The JAC1 J-domain has a positively charged surface, which forms a putative interface with the Hsc70 chaperone by analogy to that of bovine auxilin. Furthermore, the mutation of the highly conserved HPD motif in the JAC1 J-domain impaired the in vivo activity of JAC1. These data suggest that JAC1 cochaperone activity with HSC70 is essential for chloroplast photorelocation movement.Key words: Arabidopsis, auxilin, blue light, clathrin, endocytosis, J-domain, organelle movement, phototropin     

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.     

Stress-induced release of HSC70 from human tumors

In this study, we demonstrate that the pro-inflammatory cytokine interferon-gamma (IFN-gamma) induces the active release of the constitutive form of the 70-kDa heat shock protein (HSC70) from K562 erythroleukemic cells. Treatment of K562 cells with IFN-gamma induced the upregulation of the inducible form of the 70-kDa heat shock protein (HSP70), but not the constitutive form of HSC70 within the cytosol, in a proteasome-dependent manner. In addition, IFN-gamma induced the downregulation of surface-bound HSC70, but did not significantly alter surface-bound HSP70 expression. These findings indicate that HSC70 can be actively released from tumor cells and is indicative of a previously unknown mechanism by which immune modulators stimulate the release of intracellular HSC70. This mechanism may account for the potent chaperokine activity of heat shock proteins recently observed during heat shock protein-based immunotherapy against a variety of cancers.     

Identification of two molecular chaperons (HSX70, HSC70) in mature human erythrocytes

Two-dimensional gel electrophoresis of cytosolic proteins from mature human erythrocytes combined with immunoblotting revealed the presence of a group of heat shock proteins (HSPs) that included two molecular chaperons of the HSP70 family (HSX70, inducible; HSC70, constitutively expressed) and HSP90. As expected for cells devoid of organelles, erythrocytes do not contain stress proteins that are localized either in the mitochondria (HSP60, glucose-regulated protein (GRP 75) or in the endoplasmic reticulum (GRP78 or Ig heavy chain-binding protein, endoplasmin). Since red cells are unable to replace proteins whose structure has been damaged by environmental changes the results are taken to imply a role for chaperons in monitoring, protecting, and maintaining the structure and stability of erythrocyte proteins.     

Structural basis of J cochaperone binding and regulation of Hsp70

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.     

HspBP1, an Hsp70 cochaperone,has two structural domains and is capable of altering the conformation of the Hsp70 ATPase domain

We present here the first structural information for HspBP1, an Hsp70 cochaperone. Using circular dichroism, HspBP1 was determined to be 35% helical. Although HspBP1 is encoded by seven exons, limited proteolysis shows that it has only two structural domains. Domain I, amino acids 1-83, is largely unstructured. Domain II, amino acids 84-359, is predicted to be 43% helical using circular dichroism. Using limited proteolysis we have also shown that HspBP1 association changes the conformation of the ATPase domain of Hsp70. Only domain II of HspBP1 is required to bring about this conformational change. Truncation mutants of HspBP1 were tested for their ability to inhibit the renaturation of luciferase and bind to Hsp70 in reticulocyte lysate. A carboxyl terminal truncation mutant that was slightly longer than domain I was inactive in these assays, but domain II was sufficient to perform both functions. Domain II was less active than full-length HspBP1 in these assays, and addition of amino acids from domain I improved both functions. These studies show that HspBP1 domain II can bind Hsp70, change the conformation of the ATPase domain, and inhibit Hsp70-associated protein folding.     

Structural features required for the interaction of the Hsp70 molecular chaperone DnaK with its cochaperone DnaJ.

Hsp70 family members together with their Hsp40 cochaperones function as molecular chaperones, using an ATP-controlled cycle of polypeptide binding and release to mediate protein folding. Hsp40 plays a key role in the chaperone reaction by stimulating the ATPase activity and activating the substrate binding of Hsp70. We have explored the interaction between the Escherichia coli Hsp70 family member, DnaK, and its cochaperone partner DnaJ. Our data show that the binding of ATP, subsequent conformational changes in DnaK, and DnaJ-stimulated ATP hydrolysis are all required for the formation of a DnaK-DnaJ complex as monitored by Biacore analysis. In addition, our data imply that the interaction of the J-domain with DnaK depends on the substrate binding state of DnaK.     

The effect of the hexahistidine-tag in the oligomerization of HSC70 constructs

The hexahistidine is a fusion tag used for the isolation of proteins via an immobilized metal-ion affinity chromatography (IMAC). In the present study, we have purified and analyzed two constructs of the heat shock protein HSC70 in the presence or the absence of the His-tag (C30WT-His(+)/C30WT and C30DeltaL-His(+)/C30DeltaL). The oligomerization properties of the constructs were analyzed by size exclusion chromatography (SEC) and analytical ultracentrifugation (AU). Results from SEC analysis indicated that the His-tag promotes the dimerization of C30DeltaL-His(+) but has no effect on the elution profile of C30WT-His(+), compared to their respective untagged forms C30DeltaL and C30WT. These observations were also confirmed by AU analysis which indicates that C30DeltaL is stabilized in the dimeric form in the presence of the His-tag. These results emphasize the need to remove the His-tag before structural characterization of some recombinant proteins.     

Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells

CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the tetratricopeptide repeat motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70 dependent. Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these cofactors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP overexpression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains coimmunoprecipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells, however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.     

Expression of the cochaperone HspBP1 is not coordinately regulated with Hsp70 expression

Intracellular levels of the heat stress protein Hsp70 are elevated following exposure to elevated temperature. The cochaperone HspBP1 is an intracellular protein that is known to bind to and regulate Hsp70 activity. The purpose of this study was to determine if HspBP1 levels changed when Hsp70 levels were altered. Heat stress resulted in an increase in Hsp70 levels but no change in HspBP1 levels. Treatment of cells with the apoptosis inducing drug camptothecin lowered Hsp70 levels but again had no effect on HspBP1 levels. Cells treated with camptothecin plus heat stress did not exhibit an increase in Hsp70 levels. Over-expression in cells stably transfected with HspBP1 cDNA resulted in a 290% increase in HspBP1 levels without a similar change in Hsp70 levels. These results demonstrate that Hsp70 and HspBP1 are not coordinately regulated but provide evidence that an increase in the ratio of HspBP1 to Hsp70 correlates with apoptosis, in a similar way to reducing the amount of Hsp70.     

玉米细胞质HSC70的分离纯化及其抗体的制备

依据某些热激蛋白对ATP具有高度亲和性的特性,介绍了用ATP-琼脂糖亲和柱结合电洗脱分离纯化玉米幼苗细胞质HSC70及制备兔抗HSC70多抗的方法.先采用ATP-琼脂糖亲和柱初步分离几种能与ATP结合的热激蛋白,后用SDS-PAGE,切下分子量为70 kD的电泳谱带,电洗脱后用以免疫新西兰兔,以ELISA法和Western blot检测抗体效价和特异性.     

Effects of chronic heat stress on the expressions of heat shock proteins 60, 70, 90, A2, and HSC70 in the rabbit testis

Few studies have focused on the expression of heat shock proteins (HSPs) after chronic heat stress. The objective of this study was to investigate the effect of chronic high temperature–humidity index treatment on the expressions of HSP60, HSP70, HSP90, HSPA2 and HSC70, in the Rex rabbit testis and the expressions of these proteins after recovery from the chronic heat shock. Thirty mature male rabbits of the same age were randomly divided into three groups: control, heat stress, and recovery. The western blot results showed that the expressional levels of HSP60, HSP90, and HSC70 increased significantly and HSPA2 was elevated slightly after a 9-week heat treatment. HSP70 was absent in the control testis and had a high level of expression after heat stress. All of these proteins partially reverted back to normal levels after a 9-week recovery. The immunohistochemical results indicated that the expression patterns of HSP60, HSP90, HSPA2, and HSC70 did not change.     

The two mitochondrial heat shock proteins 70, Ssc1 and Ssq1, compete for the cochaperone Mge1

Two members of the heat shock protein 70 kDa (Hsp70) family, Ssc1 and Ssq1, perform important functions in the mitochondrial matrix. The essential Ssc1 is an abundant ATP-binding protein required for both import and folding of mitochondrial proteins. The function of Ssc1 is supported by an interaction with the preprotein translocase subunit Tim44, the cochaperone Mdj1, and the nucleotide exchange factor Mge1. In contrast, only limited information is available on Ssq1. So far, a basic characterization of Ssq1 has demonstrated its involvement in the maintenance of mitochondrial DNA, the maturation of the yeast frataxin (Yfh1) after import, and assembly of the mitochondrial Fe/S cluster. Here, we analyzed the biochemical properties and the interaction partners of Ssq1 in detail. Ssq1 showed typical chaperone properties by binding to unfolded substrate proteins in an ATP-regulated manner. Ssq1 was able to form a specific complex with the nucleotide exchange factor Mge1. In particular, complex formation in organello was enhanced significantly when Ssc1 was inactivated selectively. However, even under these conditions, no interaction of Ssq1 with the two other mitochondrial Hsp70-cochaperones, Tim44 and Mdj1, was observed. The Ssq1-Mge1 interaction showed a lower overall stability but the same characteristic nucleotide-dependence as the Ssc1-Mge1 interaction. A quantitative analysis of the interaction properties indicated a competition of Ssq1 with Ssc1 for binding to Mge1. Perturbation of Mge1 function or amounts resulted in direct effects on Ssq1 activity in intact mitochondria. We conclude that mitochondria represent the unique case where two Hsp70s compete for the interaction with one nucleotide exchange factor.     

Methionine sulfoxide reductase 2 reversibly regulates Mge1, a cochaperone of mitochondrial Hsp70, during oxidative stress

Peptide methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in protein(s). Although these reductases have been implicated in several human diseases, there is a dearth of information on the identity of their physiological substrates. By using Saccharomyces cerevisiae as a model, we show that of the two methionine sulfoxide reductases (MXR1, MXR2), deletion of mitochondrial MXR2 renders yeast cells more sensitive to oxidative stress than the cytosolic MXR1. Our earlier studies showed that Mge1, an evolutionarily conserved nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70. In the present study, we show that Mxr2 regulates Mge1 by selectively reducing MetO at position 155 and restores the activity of Mge1 both in vitro and in vivo. Mge1 M155L mutant rescues the slow-growth phenotype and aggregation of proteins of mxr2Δ strain during oxidative stress. By identifying the first mitochondrial substrate for Mxrs, we add a new paradigm to the regulation of the oxidative stress response pathway.     

Domain structure of riboflavin synthase.

Riboflavin synthase of Escherichia coli is a homotrimer of 23.4 kDa subunits catalyzing the formation of the carbocyclic ring of the vitamin, riboflavin, by dismutation of 6,7-dimethyl-8-ribityllumazine. Intramolecular sequence similarity suggested that each subunit folds into two topologically similar domains. In order to test this hypothesis, sequence segments comprising amino-acid residues 1-97 or 101-213 were expressed in recombinant E. coli strains. The recombinant N-terminal domain forms a homodimer that can bind riboflavin, 6,7-dimethyl-8-ribityllumazine and trifluoromethyl-substituted 8-ribityllumazine derivatives as shown by absorbance, circular dichroism, and NMR spectroscopy. Most notably, the recombinant domain dimer displays the same diastereoselectivity for ligands as the full length protein. The minimum N-terminal peptide segment required for ligand binding comprises amino-acid residues 1-87. The recombinant C-terminal domain comprising amino-acid residues 101-213 is relatively unstable and was shown not to bind riboflavin but to differentiate between certain diastereomeric trifluoromethyl-8-ribityllumazine derivatives. The data show that a single domain comprises the intact binding site for one substrate molecule. The enzyme-catalyzed dismutation requires two substrate molecules to be bound in close proximity, and each active site of the enzyme appears to be located at the interface of an N-terminal and C-terminal domain.