Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DnaJ family of molecular chaperones is characterized by the presence of a highly conserved 70-amino-acid J domain. Escherichia coli DnaJ interacts with the 70-kDa heat-shock protein (DnaK), in vitro, to stimulate the 70-kDa heat-shock protein ATPase activity and modify substrate binding. The conservation of the interaction of DnaJ-like proteins with the 70-kDa heat-shock proteins has been demonstrated for the yeast protein YDJ1, a protein that shows full domain conservation with E. coli DnaJ. Human neurone-specific DnaJ-like proteins, HSJ1a and HSJ1b, possess a J domain and a glycine/phenylalanine-rich region in common with E. coli DnaJ, although the overall amino acid identity is less than 23%. We have investigated, in vitro, the interaction of HSJ1a and HSJ1b with the mammalian brain constitutive 70-kDa heat-shock protein (hsc70). The weak intrinsic ATPase activity of the constitutive 70-kDa heat-shock protein is enhanced more than fivefold by stoichiometric amounts of both HSJ1a and HSJ1b. This enhancement is mediated by an increase in the rate of bound ATP hydrolysis, whereas the rate of ADP release is unaffected. HSJ1 proteins appear to regulate the affinity of the 70-kDa constitutive heat-shock protein for the permanently unfolded substrate, carboxymethylated alpha-lactalbumin. A recent report [Palleros, D. R., Reid, K. L., Shi, L., Welch, W. J. & Fink, A. L. (1993) Nature 365, 664-666] has suggested that substrate release by 70-kDa heat-shock proteins requires a conformational change in these proteins induced by K+ in concert with ATP binding. In the presence of ATP, HSJ1 proteins reduce 70-kDa constitutive heat-shock protein/carboxymethylated alpha-lactalbumin complex formation both in the presence and absence of K+. This suggests that HSJ1 proteins induce a conformational change in the 70-kDa constitutive heat-shock protein that can mimic the effect mediated by K+ and therefore modulate 70-kDa heat-shock protein substrate release by another mechanism rather than merely stimulating the 70-kDa heat-shock protein ATPase activity. As HSJ1 proteins have limited similarity to DnaJ, we suggest that this action is being mediated by the J domain alone, and that this modulation of 70-kDa heat-shock-protein substrate binding will be common to all proteins that contain a J domain.
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PMID:Regulation of 70-kDa heat-shock-protein ATPase activity and substrate binding by human DnaJ-like proteins, HSJ1a and HSJ1b. 795 63

The DnaK and DnaJ heat shock proteins function as the primary Hsp70 and Hsp40 homologues, respectively, of Escherichia coli. Intensive studies of various Hsp70 and DnaJ-like proteins over the past decade have led to the suggestion that interactions between specific pairs of these two types of proteins permit them to serve as molecular chaperones in a diverse array of protein metabolic events, including protein folding, protein trafficking, and assembly and disassembly of multisubunit protein complexes. To further our understanding of the nature of Hsp70-DnaJ interactions, we have sought to define the minimal sequence elements of DnaJ required for stimulation of the intrinsic ATPase activity of DnaK. As judged by proteolysis sensitivity, DnaJ is composed of three separate regions, a 9-kDa NH2-terminal domain, a 30-kDa COOH-terminal domain, and a protease-sensitive glycine- and phenylalanine-rich (G/F-rich) segment of 30 amino acids that serves as a flexible linker between the two domains. The stable 9-kDa proteolytic fragment was identified as the highly conserved J-region found in all DnaJ homologues. Using this structural information as a guide, we constructed, expressed, purified, and characterized several mutant DnaJ proteins that contained either NH2-terminal or COOH-terminal deletions. At variance with current models of DnaJ action, DnaJ1-75, a polypeptide containing an intact J-region, was found to be incapable of stimulating ATP hydrolysis by DnaK protein. We found, instead, that two sequence elements of DnaJ, the J-region and the G/F-rich linker segment, are each required for activation of DnaK-mediated ATP hydrolysis and for minimal DnaJ function in the initiation of bacteriophage lambda DNA replication. Further analysis indicated that maximal activation of ATP hydrolysis by DnaK requires two independent but simultaneous protein-protein interactions: (i) interaction of DnaK with the J-region of DnaJ and (ii) binding of a peptide or polypeptide to the polypeptide-binding site associated with the COOH-terminal domain of DnaK. This dual signaling process required for activation of DnaK function has mechanistic implications for those protein metabolic events, such as polypeptide translocation into the endoplasmic reticulum in eukaryotic cells, that are dependent on interactions between Hsp70-like and DnaJ-like proteins.
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PMID:A bipartite signaling mechanism involved in DnaJ-mediated activation of the Escherichia coli DnaK protein. 862 73

The uncoating of clathrin-coated vesicles can be mediated in vitro by the 'uncoating ATPase' that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Although we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.
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PMID:Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins. 887 Jun 55

DnaJ proteins are characterized by a 'J' domain which is homologous to a region of the Escherichia coli protein DnaJ. DnaJ has been shown to interact with the chaperone protein DnaK, and a number of eukaryotic DnaJ-like proteins have been found to interact with the 70 kDa heat-shock protein/70 kDa heat-shock cognate protein (Hsp70/Hsc70), the eukaryotic homologues of DnaK. Cysteine-string proteins (Csps) are believed to function in calcium-stimulated exocytosis and in this paper we describe a specific ATP-dependent interaction between a Csp (Csp1) and Hsc70/Hsp70. We also show that Csp1 can stimulate the ATPase activity of both Hsc70 and Hsp70 several-fold. Furthermore, we demonstrate that Csp2, a Csp variant found in adrenal chromaffin cells, can enhance the ATPase activity of Hsc70 to a similar extent as Csp1, whereas Csp(137-198), a truncated protein lacking the 'J' domain of Csp1 is unable to stimulate the ATPase activity of Hsc70. This suggests that the functions of Csp1 and Csp2 must differ in some aspect other than interaction with Hsc70. This study is also important from a general view of DnaJ/Hsc70 interactions, as Csps lack a G/F-rich region which has been suggested to be essential for activation of the ATPase activity of DnaK by DnaJ. Thus, this work would imply that a G/F-rich region is not an essential feature of DnaJ proteins for stimulation of the ATPase activity of Hsp70 proteins.
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PMID:Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein. 914 60

Three mRNAs from the murine polyomavirus early region encode the three well-characterized tumor antigens. We report the existence of a fourth alternatively spliced mRNA which encodes a fourth tumor antigen, tiny T antigen, which comprises the amino-terminal domain common to all of the T antigens but is extended by six unique amino acid residues. The amount of tiny T antigen in infected cells is small because of its short half-life. Tiny T antigen stimulates the ATPase activity of Hsc70, most likely because of its DnaJ-like motif. The common amino-terminal domain may interface with chaperone complexes to assist the T antigens in carrying out their diverse functions of replication, transcription, and transformation in the appropriate cellular compartments.
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PMID:Tiny T antigen: an autonomous polyomavirus T antigen amino-terminal domain. 922

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four- to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.
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PMID:The amino-terminal transforming region of simian virus 40 large T and small t antigens functions as a J domain. 923 32

The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s. The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs. We have characterized mutations in the HPD motif of the synaptic vesicle protein cysteine-string protein (Csp). Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70. The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent. The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70. Thus, the HPD motif of Csp is required for binding to Hsc70. We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase. Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation. In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins. Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein. These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones. This suggests that Csps may have a general chaperone function in regulated exocytosis.
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PMID:The molecular chaperone function of the secretory vesicle cysteine string proteins. 939 74

DnaJ-like proteins are molecular chaperones that regulate Hsp70 ATPase activity both in protein folding, assembly and disassembly of protein complexes. Here we report the isolation of MsJ1, an alfalfa gene encoding a protein homologous to cytosolic DnaJ-like proteins. MsJ1 was induced under heat-shock treatment in both leaves and stems of adult plants. In the absence of heat shock MsJ1 expression was tissue-specific with the highest levels of mRNA in roots and in embryonal structures. High levels of transcript were also detected in cotyledons where active degradation of storage protein occurs. In synchronized alfalfa suspension-cultured cells the MsJ1 transcript was actively expressed and showed a phase-specific modulation during cell cycle with a 2-fold induction in G2/M. These findings suggest that DnaJ-like proteins play an active role in regulating normal cellular events like protein degradation, morphogenesis and cell cycle progression.
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PMID:MsJ1, an alfalfa DnaJ-like gene, is tissue-specific and transcriptionally regulated during cell cycle. 1043 24

The activity of Hsp70 proteins is regulated by accessory proteins, among which the most studied are the members of the DnaJ-like protein family. BiP/GRP78 chaperones the translocation and maturation of secreted and membrane proteins in the endoplasmic reticulum. No DnaJ-like partner has been described so far to regulate the function of mammalian BiP/GRP78. We show here that murine BiP/GRP78 interacts with the lumenal J domain of the murine transmembrane protein MTJ1 (J-MTJ1). J-MTJ1 stimulates the ATPase activity of BiP/GRP78 at stoichiometric concentrations. The C-terminal tail of BiP/GRP78 is not required for the interaction with J-MTJ1, leaving the function of this portion of the molecule still unclear. Physical interactions between J-MTJ1 and BiP/GRP78 are stable and can be abolished by a single histidine --> glutamine substitution in the highly conserved HPD motif shared by all DnaJ-like proteins. The J-MTJ1 fragment, but not the mutant J-MTJ1:H89Q fragment, stimulates the ATPase activity of Escherichia coli DnaK, although at a higher concentration than its genuine partner DnaJ. Full-length DnaJ does not stimulate BiP over the range of concentrations investigated. These results indicate that the J domain of MTJ1 is sufficient for its interaction with BiP/GRP78 and cannot be substituted by E. coli DnaJ.
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PMID:Interaction of murine BiP/GRP78 with the DnaJ homologue MTJ1. 1077 98

The nature of kinesin interactions with membrane-bound organelles and mechanisms for regulation of kinesin-based motility have both been surprisingly difficult to define. Most kinesin is recovered in supernatants with standard protocols for purification of motor proteins, but kinesin recovered on membrane-bound organelles is tightly bound. Partitioning of kinesin between vesicle and cytosolic fractions is highly sensitive to buffer composition. Addition of either N-ethylmaleimide or EDTA to homogenization buffers significantly increased the fraction of kinesin bound to organelles. Given that an antibody against kinesin light chain tandem repeats also releases kinesin from vesicles, these observations indicated that specific cytoplasmic factors may regulate kinesin release from membranes. Kinesin light tandem repeats contain DnaJ-like motifs, so the effects of hsp70 chaperones were evaluated. Hsc70 released kinesin from vesicles in an MgATP-dependent and N-ethylmaleimide-sensitive manner. Recombinant kinesin light chains inhibited kinesin release by hsc70 and stimulated the hsc70 ATPase. Hsc70 actions may provide a mechanism to regulate kinesin function by releasing kinesin from cargo in specific subcellular domains, thereby effecting delivery of axonally transported materials.
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PMID:Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport. 1084 36


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