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)

Thyroid hormone significantly affects molecular and neuroanatomical properties of the developing nervous system. Altered connectivity in hypothyroidism may reflect reductions in process growth, alterations in process maintenance, or changes in synaptogenesis or synaptic maintenance. These events are dependent on microtubules, neurofilaments, microfilaments, and associated molecular components. Reductions in delivery of microtubules and neurofilaments to the distal axon by slow component a (SCa) of axonal transport may contribute to the neuroanatomical abnormalities of hypothyroidism (Stein et al., J Neurosci Res 28:121-133, 1991). However, hypothyroidism might also affect the axon and synaptic connections by altering slow component b (SCb), which includes actin microfilaments and proteins that contribute to synaptic function, i.e., clathrin, HSC70 (clathrin uncoating ATPase), spectrin, and calmodulin. To determine the effect of hypothyroidism on SCb proteins, slow axonal transport was analyzed in optic nerves of hyt/hyt hypothyroid mice, which have severe primary hypothyroidism, and euthyroid control mice. Clathrin, spectrin, HSC70, and actin showed significant reductions in transport velocity in hyt/hyt optic nerves relative to euthyroid nerves, but the transport rate for calmodulin was less affected. However, the amount of calmodulin was significantly elevated in hyt/hyt nerve over euthyroid nerves. Hypothyroidism selectively reduces transport of SCb proteins, which are thought to play significant roles in synaptic function and in the growth cone. The effects of hypothyroidism on microtubules and neurofilaments combined with actions on SCb suggest that changes in neuronal function associated with reduced thyroid hormone during development and maturity (i.e., alterations in neuronal connectivity, nerve conduction, and synaptic function) may be mediated in part by effects on slow axonal transport.
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PMID:Hypothyroidism selectively reduces the rate and amount of transport for specific SCb proteins in the hyt/hyt mouse optic nerve. 172 71

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.
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PMID:Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. 182 89

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome-associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP-binding domains and truncates the carboxyl terminal peptide-binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane-bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium-inducible, microsome-associated ATPase activity with properties similar to a proteolytically cleaved N-terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.
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PMID:Stch encodes the 'ATPase core' of a microsomal stress 70 protein. 813 51

The 70-kDa heat-shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin-coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two-cistron expression vector, and purified to homogeneity in a two-step procedure involving ion-exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 11 of cell culture. Amino-terminal sequencing of the recombinant protein gives the expected sequence, and non-denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self-associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42 degrees C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.
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PMID:Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70. 816 1

Immunoglobulin-binding protein (BiP), a 70-kDa heat shock protein in the endoplasmic reticulum, binds transiently to nascent proteins, releasing them upon folding and assembly. The in vitro release of bound proteins from BiP requires ATP hydrolysis. Recently, the three-dimensional structure was solved for an ATP-hydrolyzing proteolytic 44-kDa fragment of a 71-kDa heat shock cognate protein, HSC71. Because of the high degree of homology in this region, BiP presumably forms a similar ATP binding structure. Amino-terminal deletions in BiP eliminated ATP-agarose binding. Alteration of a second potential ATP binding site had no effect, suggesting that only the HSC71-like site was capable of ATP binding. Crystallographic data from HSC71 implicated certain amino acids in interactions with the beta-phosphate, gamma-phosphate, and divalent cation of ATP. Mutation of each corresponding residue in BiP (Thr-37, Thr-229, and Glu-201) severely inhibited its ATPase activity. These BiP mutants were still capable of binding ATP and immunoglobulin heavy chains, suggesting that these mutations did not drastically alter the structure of BiP. They did however block the ATP-mediated release of heavy chains from BiP. Our results demonstrate that the structure of BiP in this region must be extremely similar to that elucidated for HSC71 and that mutations of residues proposed to interact with ATP block the ATP-mediated release of bound protein by inhibiting ATP hydrolysis.
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PMID:Mutations within the nucleotide binding site of immunoglobulin-binding protein inhibit ATPase activity and interfere with release of immunoglobulin heavy chain. 846 60

In vitro incubation of immunoprecipitated immunoglobulin-binding protein (BiP) complexes with calcium and [gamma-32P]ATP resulted in the phosphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same pH optimum as reported for its magnesium-dependent ATPase activity. This suggested the possibility that both activities could occur through ATP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation and ATPase activities, and mutation of either residue to a serine significantly reduced both activities. Glutamic acid 175 in HSC71 has been hypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activity but still supported some autophosphorylation. The in vitro phosphorylation site was mapped to Thr-229 by mutational analysis. This threonine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imply that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines which activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphorylated BiP demonstrated that Thr-229 is not a detectable site of phosphorylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of Thr-229.
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PMID:The immunoglobulin-binding protein in vitro autophosphorylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation. 850 3

We have previously shown that the molecular chaperone HSC70 self-associates in solution into dimers, trimers, and probably high order oligomers, according to a slow temperature- and concentration-dependent equilibrium that is shifted toward the monomer upon binding of ATP peptides or unfolded proteins. To determine the structural basis of HSC70 self-association, the oligomerization properties of the isolated amino- and carboxyl-terminal domains of this protein have been analyzed by gel electrophoresis, size exclusion chromatography, and analytical ultracentrifugation. Whereas the amino-terminal ATPase domain (residues 1-384) was found to be monomeric in solution even at high concentrations, the carboxyl-terminal peptide binding domain (residues 385-646) exists as a slow temperature- and concentration-dependent equilibrium involving monomers, dimers, and trimers. The association equilibrium constant obtained for this domain alone is on the order of 10(5) M-1, very close to that determined previously for the entire protein, suggesting that self-association of HSC70 is determined solely by its carboxyl-terminal domain. Furthermore, oligomerization of the isolated carboxyl-terminal peptide binding domain is, like that of the entire protein, reversed by peptide binding, indicating that self-association of the protein may be mediated by the peptide binding site and, as such, should play a role in the regulation of HSC70 chaperone function. A general model for self-association of HSP70 is proposed in which the protein is in equilibrium between two states differing by the conformation of their carboxyl-terminal domain and their self-association properties.
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PMID:The COOH-terminal peptide binding domain is essential for self-association of the molecular chaperone HSC70. 907 9

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.
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PMID:Cysteine string protein is required for calcium secretion coupling of evoked neurotransmission in drosophila but not for vesicle recycling. 943 17

Rats were subjected to transient cerebral ischemia by four-vessel occlusion of 30 min duration, followed by 2, 4, 8 or 24 h of recovery. Total RNA was isolated from the cerebral cortex and hippocampus, and reverse transcribed into cDNA. Hsp40 mRNA levels of samples were evaluated by quantitative PCR. Transient cerebral ischemia caused a marked increase in hsp40 mRNA levels to about 250% and 500% of control in the cortex and hippocampus respectively. Since hsp40 exerts a critical regulatory function in the HSC70/HSP70 ATPase cycle, an ischemia-induced rise of hsp40 mRNA levels could mark the onset of the recovery process after transient ischemia. On the other hand, the inhibitory action of hsp40 on P58 (a protein that activates protein synthesis by blocking the interferon-induced double-stranded RNA-activated protein kinase PKR) implies that the rise in hsp40 expression may equally well contribute to the post-ischemic suppression of protein synthesis.
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PMID:Effects of transient cerebral ischemia on hsp40 mRNA levels in rat brain. 958 51

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.
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PMID:Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70. 991 17


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