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Binding of ATP and ATP analogues to the uncoating ATPase Hsc70 (70 kDa heat-shock cognate protein).

ABSTRACT: Nucleotide binding to the 70 kDa heat-shock cognate protein (Hsc70) from mung bean seeds and pig brain was investigated, as well as the clathrin uncoating activity of Hsc70 in the presence of these nucleotides. The two enzymes were found to behave identically. ATP bound to two different forms of Hsc70, with dissociation constants of 1.1 +/- 0.1 microM and 1.4 +/- 0.7 mM respectively at 25 degrees C. This corresponds to delta G0' = -34 and -16 kJ/mol respectively. From the temperature-dependence of the dissociation constant of the high-affinity site, delta H0' was calculated to -36 +/- 2 kJ/mol. This gives delta S0' = 6.7 J/mol per K. Adenosine 5'-[gamma-thio]triphosphate, ADP, adenosine 5'-[beta, gamma-imino]triphosphate and adenosine 5'-[beta, gamma-methylene]triphosphate showed dissociation constants of 2.3, 11, 31 and 284 microM respectively. The order of affinities corresponded to the order of effectiveness in uncoating of pig brain coated vesicles. The implications of these findings for the mechanism of Hsc70 action are discussed.

SUBMITTER: Buxbaum E

PROVIDER: S-EPMC1217706 | biostudies-other | 1996 Sep

REPOSITORIES: biostudies-other

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A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin.

Project description:An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.

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70-kDa heat shock cognate protein hsc70 mediates calmodulin-dependent nuclear import of the sex-determining factor SRY.

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The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus.

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A plastid-targeted heat shock cognate 70-kDa protein confers osmotic stress tolerance by enhancing ROS scavenging capability.

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Project description:Although there is very little sequence identity between the two proteins, the structures of rabbit skeletal muscle actin (375-amino acid residues) and the 44-kDa ATPase fragment of the bovine 70-kDa heat shock cognate protein (HSC70; 386 residues) are very similar. The alpha-carbon positions of 241 pairs of amino acid residues that are structurally equivalent within the two proteins can be superimposed with a root-mean-square difference in distance of 2.3 A; of these, 39 residues are identical, and 56 are conservative substitutions. In addition, the conformations of ADP are very similar in both proteins. A local sequence "fingerprint," which may be diagnostic of the adenine nucleotide beta-phosphate-binding pocket, has been derived. The fingerprint identifies members of the glycerol kinase family as candidates likely to have a similar structure in their nucleotide-binding domains. The structural differences between the two molecules mainly occur in loop regions of actin known to be involved in interactions with other monomers in the actin filament or in the binding of myosin; the corresponding regions in heat shock proteins may have functions that are as yet undetermined. Placing the Ca2+ ATP of actin on the ATPase fragment structure suggests Asp-206 (corresponding to His-161 of actin) as a candidate proton acceptor for the ATPase reaction.

| S-EPMC51803 | biostudies-other

Heat Shock Cognate 70 kDa Protein Is the Target of Tetradecyl 2,3-Dihydroxybenzoate for Neuritogenic Effect in PC12 Cells.

Project description:Tetradecyl 2,3-dihydroxybenzoate (ABG-001) is a lead compound derived from gentisides with a remarkable neuritogenic activity. However, the target of ABG-001 is yet to be defined to date. In this study, the potential target of ABG-001 was investigated via an activity-based protein profiling (ABPP) analysis, which is a chemical proteomic method for target identification by using chemical probes. Results indicated that the potential target proteins of ABG-001 were heat shock cognate 70 kDa protein (Hsc70), 78 kDa glucose-regulated protein (GRP78), and 14-3-3 theta protein. Then, the potential target of ABG-001 was confirmed by using inhibitors, the cellular thermal shift assay (CETSA) and small-interfering RNA (siRNA) analysis. The inhibitor of Hsc70 and siRNA significantly decreased the neurite outgrowth induced by ABG-001. Furthermore, ABG-001 induced neurite outgrowth was reduced by siRNA against Hsc70, and the results of CETSA suggested that Hsc70 showed a significant thermal stability-shifted effect upon ABG-001 treatment. These results indicated that Hsc70 is the target protein of ABG-001 in PC12 cells.

| S-EPMC8533567 | biostudies-literature

Non-canonical Interactions between Heat Shock Cognate Protein 70 (Hsc70) and Bcl2-associated Anthanogene (BAG) Co-Chaperones Are Important for Client Release.

Project description:Heat shock cognate protein 70 (Hsc70) regulates protein homeostasis through its reversible interactions with client proteins. Hsc70 has two major domains: a nucleotide-binding domain (NBD), that hydrolyzes ATP, and a substrate-binding domain (SBD), where clients are bound. Members of the BAG family of co-chaperones, including Bag1 and Bag3, are known to accelerate release of both ADP and client from Hsc70. The release of nucleotide is known to be mediated by interactions between the conserved BAG domain and the Hsc70 NBD. However, less is known about the regions required for client release, and it is often assumed that this activity also requires the BAG domain. It is important to better understand this step because it determines how long clients remain in the inactive, bound state. Here, we report the surprising observation that truncated versions of either human Bag1 or Bag3, comprised only the BAG domain, promoted rapid release of nucleotide, but not client, in vitro Rather, we found that a non-canonical interaction between Bag1/3 and the Hsc70 SBD is sufficient for accelerating this step. Moreover, client release did not seem to require the BAG domain or Hsc70 NBD. These results suggest that Bag1 and Bag3 control the stability of the Hsc70-client complex using at least two distinct protein-protein contacts, providing a previously under-appreciated layer of molecular regulation in the human Hsc70 system.

| S-EPMC5025674 | biostudies-literature

Heat-shock cognate 70 is required for the activation of heat-shock factor 1 in mammalian cells.

Project description:HSF1 (heat-shock factor 1) plays an essential role in mediating the appropriate cellular response to diverse forms of physiological stresses. However, it is not clear how HSF1 is regulated by interacting proteins under normal and stressful conditions. In the present study, Hsc70 (heat-shock cognate 70) was identified as a HSF1-interacting protein using the TAP (tandem affinity purification) system and MS. HSF1 can interact with Hsc70 in vivo and directly in vitro. Interestingly, Hsc70 is required for the regulation of HSF1 during heat stress and subsequent target gene expression in mammalian cells. Moreover, cells transfected with siRNAs (small interfering RNAs) targeted to Hsc70 showed greatly decreased HSF1 activation with expression of HSF1 target genes being dramatically reduced. Finally, loss of Hsc70 expression in cells resulted in an increase in stress-induced apoptosis. These results indicate that Hsc70 is a necessary and critical regulator of HSF1 activities.

| S-EPMC1317673 | biostudies-literature

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