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 "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

Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.
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PMID:Mutational analysis of cysteine-string protein function in insulin exocytosis. 1019 13

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 activity of Hsp70 proteins is regulated by accessory proteins, which include members of the DnaJ-like protein family. Characterized by the presence of a highly conserved 70-amino acid J domain, DnaJ homologues activate the ATPase activity of Hsp70 proteins and stabilize their interaction with unfolded substrates. DnaJ homologues have been identified in most organelles where they are involved in nearly all aspects of protein synthesis and folding. Within the endoplasmic reticulum (ER), DnaJ homologues have also been shown to assist in the translocation, secretion, retro-translocation, and ER-associated degradation (ERAD) of secretory pathway proteins. By using bioinformatic methods, we identified a novel mammalian DnaJ homologue, ERdj4. It is the first ER-localized type II DnaJ homologue to be reported. The signal sequence of ERdj4 remains uncleaved and serves as a membrane anchor, orienting its J domain into the ER lumen. ERdj4 co-localized with GRP94 in the ER and associated with BiP in vivo when they were co-expressed in COS-1 cells. In vitro experiments demonstrated that the J domain of ERdj4 stimulated the ATPase activity of BiP in a concentration-dependent manner. However, mutation of the hallmark tripeptide HPD (His --> Gln) in the J domain totally abolished this activation. ERdj4 mRNA expression was detected in all human tissues examined but showed the highest level of the expression in the liver, kidney, and placenta. We found that ERdj4 was highly induced at both the mRNA and protein level in response to ER stress, indicating that this protein might be involved in either protein folding or ER-associated degradation.
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PMID:Identification and characterization of a novel endoplasmic reticulum (ER) DnaJ homologue, which stimulates ATPase activity of BiP in vitro and is induced by ER stress. 1183 48

P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha). We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain. Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70. However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.
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PMID:Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function. 1193 89

The DnaJ (Hsp40) cochaperone regulates the DnaK (Hsp70) chaperone by accelerating ATP hydrolysis in a cycle closely linked to substrate binding and release. The J-domain, the signature motif of the Hsp40 family, orchestrates interaction with the DnaK ATPase domain. We studied the J-domain by creating 42 mutant E. coli DnaJ variants and examining their phenotypes in various separate in vivo assays, namely, bacterial growth at low and high temperatures, motility, and propagation of bacteriophage lambda. Most mutants studied behaved like wild type in all assays. In addition to the (33)HisProAsp(35) (HPD) tripeptide found in all known functional J-domains, our study uncovered three new single substitution mutations (Y25A, K26A, and F47A) that totally abolish J-domain function. Furthermore, two glycine substitution mutants in an exposed flexible loop (R36G, N37G) showed partial loss of J-domain function alone and complete loss of function as a triple (RNQ-GGG) mutant coupled with the phenotypically silent Q38G. Interestingly, all the essential residues map to a small region on the same solvent-exposed face of the J-domain. Engineered mutations in the corresponding residues of the human Hdj1 J-domain grafted in E. coli DnaJ also resulted in loss of function, suggesting an evolutionarily conserved interaction surface. We propose that these clustered residues impart critical sequence determinants necessary for J-domain catalytic activity and reversible contact interface with the DnaK ATPase domain.
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PMID:Scanning mutagenesis identifies amino acid residues essential for the in vivo activity of the Escherichia coli DnaJ (Hsp40) J-domain. 1245 54

Transport of preproteins into the mitochondrial matrix requires the presequence translocase of the inner membrane (TIM23 complex) and the presequence translocase-associated motor (PAM). The motor consists of five essential subunits, the mitochondrial heat shock protein 70 (mtHsp70) and four cochaperones, the nucleotide exchange-factor Mge1, the translocase-associated fulcrum Tim44, the J-protein Pam18, and Pam16. Pam16 forms a complex with Pam18 and displays similarity to J-proteins but lacks the canonical tripeptide motif His-Pro-Asp (HPD). We report that Pam16 does not function as a typical J-domain protein but, rather, antagonizes the function of Pam18. Pam16 specifically inhibits the Pam18-mediated stimulation of the ATPase activity of mtHsp70. The inclusion of the HPD motif in Pam16 does not confer the ability to stimulate mtHsp70 activity. Pam16-HPD fully substitutes for wild-type Pam16 in vitro and in vivo but is not able to replace Pam18. Pam16 represents a new type of cochaperone that controls the stimulatory effect of the J-protein Pam18 and regulates the interaction of mtHsp70 with precursor proteins during import into mitochondria.
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PMID:The presequence translocase-associated protein import motor of mitochondria. Pam16 functions in an antagonistic manner to Pam18. 1521 29

A model structure of the Hsc70/auxilin complex has been constructed to gain insight into interprotein substrate transfer and ATP hydrolysis induced conformational changes in the multidomain Hsc70 structure. The Hsc70/auxilin system, which is a member of the Hsp70/Hsp40 chaperone system family, uncoats clathrin-coated vesicles in an ATP hydrolysis-driven process. Incorporating previous results from NMR and mutant binding studies, the auxilin J-domain was docked into the Hsc70 ATPase domain lower cleft using rigid backbone/flexible side chain molecular dynamics, and the Hsc70 substrate binding domain was docked by a similar procedure. For comparison, J-domain and substrate binding domain docking sites were obtained by the rigid-body docking programs DOT and ZDOCK, filtered and ranked by the program ClusPro, and relaxed using the same rigid backbone/flexible side chain dynamics. The substrate binding domain sites were assessed in terms of conserved surface complementarity and feasibility in the context of substrate transfer, both for auxilin and another Hsp40 protein, Hsc20. This assessment favors placement of the substrate binding domain near D152 on the ATPase domain surface adjacent to the J-domain invariant HPD segment, with the Hsc70 interdomain linker in the lower cleft. Examining Hsc70 interdomain energetics, we propose that long-range electrostatic interactions, perhaps due to a difference in the pKa values of bound ATP and ADP, could play a major role in the structural change induced by ATP hydrolysis. Interdomain electrostatic interactions also appear to play a role in stimulation of ATPase activity due to J-domain binding and substrate binding by Hsc70.
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PMID:Experimentally biased model structure of the Hsc70/auxilin complex: substrate transfer and interdomain structural change. 1527 4

Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60(c-src) and in transformation.
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PMID:Genetic analysis of the polyomavirus DnaJ domain. 1601 58

The auxilin family of J-domain proteins load Hsp70 onto clathrin-coated vesicles (CCVs) to drive uncoating. In vitro, auxilin function requires its ability to bind clathrin and stimulate Hsp70 ATPase activity via its J-domain. To test these requirements in vivo, we performed a mutational analysis of Swa2p, the yeast auxilin ortholog. Swa2p is a modular protein with three N-terminal clathrin-binding (CB) motifs, a ubiquitin association (UBA) domain, a tetratricopeptide repeat (TPR) domain, and a C-terminal J-domain. In vitro, clathrin binding is mediated by multiple weak interactions, but a Swa2p truncation lacking two CB motifs and the UBA domain retains nearly full function in vivo. Deletion of all CB motifs strongly abrogates clathrin disassembly but does not eliminate Swa2p function in vivo. Surprisingly, mutation of the invariant HPD motif within the J-domain to AAA only partially affects Swa2p function. Similarly, a TPR point mutation (G388R) causes a modest phenotype. However, Swa2p function is abolished when these TPR and J mutations are combined. The TPR and J-domains are not functionally redundant because deletion of either domain renders Swa2p nonfunctional. These data suggest that the TPR and J-domains collaborate in a bipartite interaction with Hsp70 to regulate its activity in clathrin disassembly.
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PMID:Dissection of Swa2p/auxilin domain requirements for cochaperoning Hsp70 clathrin-uncoating activity in vivo. 1668 70


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