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)

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.
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PMID:The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR. 1555 31

The molecular mechanisms associated with reestablishment of renal epithelial polarity after injury remain incompletely delineated. Stress proteins may act as molecular chaperones, potentially modulating injury or enhancing recovery. We tested whether overexpression of heat shock protein 70 (HSP70) would stabilize Na-K-ATPase attachment to the cytoskeleton, under conditions of ATP depletion, and whether a direct association existed between Na-K-ATPase and HSP70 in cultured renal epithelial cells. LLC-PK1 cells were transfected with a tagged HSP70 (70FLAG) or vector alone (VA). Detachment of Na-K-ATPase was detected in Triton soluble lysate after ATP depletion. 70FLAG cells demonstrated a significant (P < 0.01) decrease in detachment of Na-K-ATPase after either 2 or 4 h of ATP depletion. Interactions between HSP70 and Na-K-ATPase were determined by coimmunoprecipitation of 70FLAG and Na-K-ATPase, by direct and competitive binding assays and by immunocytochemical localization. Binding of HSP70 and Na-K-ATPase increased dramatically following injury. Interactions were: 1) reversible; 2) reciprocal to changes in the HSP70 binding protein clathrin; and 3) present only when ATP turnover was inhibited in cell lysate, an established characteristic of HSP binding. These studies indicate that 1) overexpression of HSP70 is associated with decreased detachment of Na-K-ATPase from the cytoskeleton following injury; 2) HSP70 binds to Na-K-ATPase; and 3) binding of HSP70 to Na-K-ATPase is dynamic and specific, increasing in response to injury and decreasing during recovery. Interaction between the molecular chaperone HSP70 and damaged or displaced Na-K-ATPase may represent a fundamental cellular mechanism underlying maintenance and recovery of renal tubule polarity following energy deprivation.
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PMID:HSP70 binding modulates detachment of Na-K-ATPase following energy deprivation in renal epithelial cells. 1570 13

Heat shock protein 70 (Hsp 70) and heat shock protein 40 (Hsp 40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp 70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp 70 was able to suppress the thermosensitivity of an Escherichia coli dnaK 756 strain, but not that of the corresponding deletion strain (DeltadnaK 52) or dnaK 103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp 70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp 70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK 756 strain. We also identified two key amino acid residues (V 401 and Q 402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp 70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp 70 and PfHsp 70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp 40 chaperone machinery.
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PMID:Plasmodium falciparum heat shock protein 70 is able to suppress the thermosensitivity of an Escherichia coli DnaK mutant strain. 1597 16

This study was conducted to explore the relationship between two isolates of Neospora caninum (N. caninum) (KBA-2 and VMDL-1) using proteomics. To achieve the goal, proteins of N. caninum tachyzoite lysates of KBA-2 and VMDL-1 were separated by two-dimensional gel electrophoresis (2-DE), stained with silver-nitrate and analyzed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) to compare protein profiles. In addition, proteins separated by 2-DE were transferred to membranes, probed with bovine anti-N. caninum KBA-2 immunoglobulin G, and reactive proteins were visualized and compared between the two isolates. Most spots on 2-DE profiles and antigenic spots on 2-DE immunoblot profiles were located at similar locations in terms of isoelectric point and molecular weight. Proteins common to both isolates included the following: heat shock protein 70, subtilisin-like serine protease, nucleoside triphosphatase, heat shock protein 60, pyruvate kinase, tubulin alpha, tubulin beta, enolase, putative protein disulfide isomerase, actin, fructase-1,6-bisphosphatase, putative ribosomal protein S2, microneme protein Nc-P38, lactate dihydrogenase, fructose-1,6-bisphosphatase aldolase, serine threonine phosphatase 2C, 14-3-3 protein homologue, N. caninum dense granule-1 and NcGRA2. As a consequence, even though N. caninum KBA-2 and VMDL-1 isolates were isolated from geographically distinct locations there were significant homology in the proteome and antigenic proteome profiles. In addition, proteomic approach was verified as a useful tool for understanding of host immune response against different isolates of protozoa.
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PMID:Comparison of proteome and antigenic proteome between two Neospora caninum isolates. 1609 74

Import of mitochondrial matrix proteins involves the general translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated motor (PAM) drives the completion of preprotein translocation into the matrix. Five subunits of PAM are known: the preprotein-binding matrix heat shock protein 70 (mtHsp70), the nucleotide exchange factor Mge1, Tim44 that directs mtHsp70 to the inner membrane, and the membrane-bound complex of Pam16-Pam18 that regulates the ATPase activity of mtHsp70. We have identified a sixth motor subunit. Pam17 (encoded by the open reading frame YKR065c) is anchored in the inner membrane and exposed to the matrix. Mitochondria lacking Pam17 are selectively impaired in the import of matrix proteins and the generation of an import-driving activity of PAM. Pam17 is required for formation of a stable complex between the cochaperones Pam16 and Pam18 and promotes the association of Pam16-Pam18 with the presequence translocase. Our findings suggest that Pam17 is required for the correct organization of the Pam16-Pam18 complex and thus contributes to regulation of mtHsp70 activity at the inner membrane translocation site.
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PMID:Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor. 1610 94

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.
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PMID:Message in a bottle: role of the 70-kDa heat shock protein family in anti-tumor immunity. 1614 35

We hypothesized that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca(2+) at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), and Na(+)/Ca(2+) exchanger (NCX) handled Ca(2+) in rat ventricular myocytes preconditioned with a kappa-opioid receptor agonist, U50488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotide and subsequently subjected to simulated ischemia. We determined the following: 1) the Ca(2+) transients induced by electrical stimulation and caffeine, which provide the overall picture of Ca(2+) homeostasis; 2) expression of RyR, SERCA, and NCX; and 3) Ca(2+) fluxes via NCX by the use of (45)Ca(2+) in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA, and NCX and the expression of RyR and SERCA. These effects led to increases in the release of Ca(2+) from the sarcoplasmic reticulum via RyR and in the removal of Ca(2+) from the cytoplasm by reuptake of Ca(2+) to the SR via SERCA and by extrusion of Ca(2+) out of the cell via NCX. UP also reduced mitochondrial Ca(2+) accumulation. All of the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotide. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca(2+) homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca(2+) overload, improved recovery in contractile function, and cardioprotection.
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PMID:Further study on the role of HSP70 on Ca2+ homeostasis in rat ventricular myocytes subjected to simulated ischemia. 1620 97

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651bp in length, having a 5' untranslated region (UTR) of 96bp, a 3' UTR of 575bp, and an open reading frame (ORF) of 1980bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8h and lasted to 16h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop.
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PMID:The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress. 1653 Apr 26

HSP70s are a family of ATP-dependent chaperones of relative molecular masses around 70kDa. Immunization of mice with HSP70 isolated from tumor tissues has been proved to elicit specific protective immunity against the original tumor. Recent researches have demonstrated that the ATPase domain of HSP70 and the tumor antigenic peptide that binds to Hsp70 were the crucial parts eliciting tumor-specific immunity. These findings suggested that a recombinant protein expressed in Escherichia coli, comprising a covalently fused fragment of tumor rejection antigen to ATPase domain of HSP70, could be used as a tumor vaccine. However, high-level expressions of heterologous recombinant proteins in E. coli often lead to the formation of inclusion bodies, resulting in defects in solubility and bioactivity. In the present work, we found an approach to resolve these problems, focusing on a refolding procedure via gel-filtration chromatography for denatured inclusion body proteins. Here, we expressed, purified and refolded a fusion protein comprising murine heat shock cognate protein 70 (Hsc70) N-terminal ATPase domain (Hsc70NTD) and a portion of TRP2 (aa153-417) as a model protein. The refolding effectivities were assessed according to their ATPase activities, the vaccine function was assessed according to immunization effect in inducing antigen-specific CTLs and to in vivo tumor protection. The results showed that the fusion protein refolded via gel-filtration chromatography exhibited ATPase activity, succeeded in eliciting antigen-specific CTL in vivo and delayed tumor growth on tumor-bearing mice.
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PMID:Fusion protein of ATPase domain of Hsc70 with TRP2 acting as a tumor vaccine against B16 melanoma. 1658 Jul 37

Inducible heat shock protein 70 (Hsp70) is one of the most important HSPs for maintenance of cell integrity during normal cellular growth as well as pathophysiological conditions. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial signaling transducer that regulates a diverse array of physiological and pathological processes and is essential for activating NF-kappaB signaling pathway in response to bacterial lipopolysaccharide (LPS). Here we report a novel mechanism of Hsp70 for preventing LPS-induced NF-kappaB activation in RAW264.7 macrophage-like cells. Our results show that Hsp70 can associate with TRAF6 physically in the TRAF-C domain and prevent TRAF6 ubiquitination. The stimulation of LPS dissociates the binding of Hsp70 and TRAF6 in a time-dependent manner. Hsp70 inhibits LPS-induced NF-kappaB signaling cascade activation in heat-shock treated as well as Hsp70 stable transfected RAW264.7 cells and subsequently decreases iNOS and COX-2 expression. Two Hsp70 mutants, Hsp70DeltaC(1-428aa) with N-terminal ATPase domain and Hsp70C(428-642aa) with C-terminal domain, lack the ability to influence TRAF6 ubiquitination and TRAF6-triggered NF-kappaB activation. Taken together, these findings indicate that Hsp70 inhibits LPS-induced NF-kappaB activation by binding TRAF6 and preventing its ubiquitination, and results in inhibition of inflammatory mediator production, which provides a new insight for analyzing the effects of Hsp70 on LPS-triggered inflammatory signal transduction pathways.
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PMID:Hsp70 inhibits lipopolysaccharide-induced NF-kappaB activation by interacting with TRAF6 and inhibiting its ubiquitination. 1669 80


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