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 Escherichia coli Hsp40 DnaJ uses its J-domain to target substrate polypeptides for binding to the Hsp70 DnaK, but the mechanism of J-domain function has been obscured by a substrate-like interaction between DnaJ and DnaK. ATP hydrolysis in DnaK is associated with a conformational change that captures the substrate, and both DnaJ and substrate can stimulate ATP hydrolysis. However, substrates cannot trigger capture by DnaK in the presence of ATP, and substrates stimulate a DnaK conformational change that is uncoupled from ATP hydrolysis. The role of the J-domain was examined using the fluorescent derivative of a fusion protein composed of the J-domain and a DnaK-binding peptide. In the absence of ATP, DnaK-binding affinity of the fusion protein is similar to that of the unfused peptide. However, in the presence of ATP, the affinity of the fusion protein is dramatically increased, which is opposite to the decrease in DnaK affinity typically exhibited by peptides. Binding of a fusion protein that contains a defective J-domain is insensitive to ATP. According to results from isothermal titration calorimetry, the J-domain binds to the DnaK ATPase domain with weak affinity (K(D) = 23 microM at 20 degrees C). The interaction is characterized by a positive enthalpy, small heat capacity change (DeltaC(p)= -33 kcal mol(-1)), and increasing binding affinity for increasing temperatures in the physiological range. In conditions that support binding of the J-domain to the ATPase domain, the J-domain accelerates ATP hydrolysis and a simultaneous conformational change in DnaK that is associated with peptide capture. The defective J-domain is inactive, despite the fact that it binds to the DnaK ATPase domain with higher than wild-type affinity. The results are most consistent with an allosteric mechanism of J-domain action in which the J-domain couples ATP hydrolysis to peptide capture by accelerating ATP hydrolysis and delaying DnaK closure until ATP is hydrolyzed.
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PMID:The J-domain of Hsp40 couples ATP hydrolysis to substrate capture in Hsp70. 1271 35

The human DnaJ (Hsp40) proteins HSJ1a and HSJ1b are type II DnaJ proteins with different C termini generated by alternate splicing. Both protein isoforms can regulate the ATPase activity and substrate binding of Hsp70. In this study, we have confirmed the neuronal expression of HSJ1a and HSJ1b proteins and localized their expression in human neural retina using isoform-specific antisera. HSJ1a and HSJ1b were enriched in photoreceptors, particularly the inner segments, but had different intracellular localization due to the prenylation of HSJ1b by a geranylgeranyl moiety. Because of their enrichment at the site of rhodopsin production, we investigated the effect of HSJ1 isoforms on the cellular processing of wild-type and mutant rhodopsin apoprotein in SK-N-SH cells. The expression of HSJ1b, but not HSJ1a, inhibited the normal cellular processing of wild-type rhodopsin-GFP, which co-localized with HSJ1b at the ER. HSJ1b expression also increased the incidence of inclusion formation by the wild-type protein. Both isoforms were recruited to mutant P23H rhodopsin inclusions, but only HSJ1b enhanced inclusion formation. Investigation of a prenylation-null mutant showed that the modulation of rhodopsin processing and inclusion formation was dependent on the correct subcellular targeting of HSJ1b to the cytosolic face of the ER. An Hsp70 interaction-null mutant of HSJ1b had the same effect as HSJ1b, suggesting that these phenomena were independent of Hsp70 and, furthermore, overexpression of Hsp70 with HSJ1b did not modulate the HSJ1b effect on inclusion formation, showing that Hsp70 was not limiting. The data provide evidence that cytoplasmic chaperones when targeted to the ER can influence the folding and processing of a GPCR and show that DnaJ protein function can be further specialized by alternative splicing and post-translational modification.
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PMID:The chaperone environment at the cytoplasmic face of the endoplasmic reticulum can modulate rhodopsin processing and inclusion formation. 1275 72

CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the tetratricopeptide repeat motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70 dependent. Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these cofactors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP overexpression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains coimmunoprecipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells, however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.
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PMID:Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells. 1283 80

The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.
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PMID:Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD. 1284 7

ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.
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PMID:Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP. 1292 75

All type I DnaJ (Hsp40) homologues share the presence of two highly conserved zinc centers. To elucidate their function, we constructed DnaJ mutants that separately replaced cysteines of either zinc center I or zinc center II with serine residues. We found that in the absence of zinc center I, the autonomous, DnaK-independent chaperone activity of DnaJ is dramatically reduced. Surprisingly, this only slightly impaired the in vivo function of DnaJ, and its ability to function as a co-chaperone in the DnaK/DnaJ/GrpE foldase machine. The DnaJ zinc center II, on the other hand, was found to be absolutely essential for the in vivo and in vitro function of DnaJ. This did not seem to be caused by a lack of substrate binding affinity or an inability to work as an ATPase-stimulating factor. Rather it appears that zinc center II mutant proteins lack a necessary additional interaction site with DnaK, which seems to be crucial for locking-in substrate proteins onto DnaK. These findings led us to a model in which ATP hydrolysis in DnaK is only the first step in converting DnaK into its high affinity binding state. Additional interactions between DnaK and DnaJ are required to make the DnaK/DnaJ/GrpE foldase machinery catalytically active.
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PMID:The roles of the two zinc binding sites in DnaJ. 1294 35

The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.
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PMID:Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli. 1297 Apr 59

We reported that the endoplasmic reticulum (ER) stress pathway involving CHOP, a member of the C/EBP transcription factor family, plays a key role in nitric oxide (NO)-mediated apoptosis of macrophages and pancreatic beta cells. We also showed that the cytosolic chaperone pair of hsp70 and dj1 (hsp40/hdj-1) or dj2 (HSDJ/hdj-2) prevents NO-mediated apoptosis upstream of cytochrome c release from mitochondria. To analyze roles of the chaperone pair in preventing apoptosis, RAW 264.7 macrophages stably expressing hsp70 and dj1 or dj2 were established. The chaperone pair prevented LPS/IFN-gamma-induced and NO-mediated apoptosis downstream of CHOP induction. hsp70 mutant protein lacking the ATPase domain or the C-terminal EEVD sequence were not effective in preventing CHOP-induced apoptosis. A mutant dj2 lacking the C-terminal prenylation CaaX motif, was also not effective. When wild-type RAW 264.7 cells were treated with LPS/IFN-gamma, NO-mediated apoptosis was induced, and proapoptotic Bcl-2 family protein Bax was translocated from cytosol to mitochondria. This translocation was prevented in cells stably expressing hsp70/dj2, and in CHOP knockout cells. Overexpression of CHOP in wild-type cells also induced translocation of Bax and this translocation was prevented in cells expressing hsp70/dj2. CHOP-induced apoptosis was prevented by Bax knock-down. Coimmunoprecipitation experiments showed that Bax interacts with both hsp70 and dj1/dj2. ATPase domain of hsp70 was necessary for the binding with Bax. These findings indicate that CHOP-induced apoptosis is mediated by translocation of Bax from the cytosol to the mitochondria, and hsp70/dj1 or dj2 chaperone pair prevents apoptosis by interacting with Bax and preventing translocation to the mitochondria.
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PMID:hsp70-DnaJ chaperone pair prevents nitric oxide- and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. 1475 10

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.
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PMID:Propagation of Saccharomyces cerevisiae [PSI+] prion is impaired by factors that regulate Hsp70 substrate binding. 1508 86

The folding and assembly of proteins in the endoplasmic reticulum (ER) lumen and membrane are monitored by ER quality control. Misfolded or unassembled proteins are retained in the ER and, if they cannot fold or assemble correctly, ultimately undergo ER-associated degradation (ERAD) mediated by the ubiquitin-proteasome system. Whereas luminal and integral membrane ERAD substrates both require the proteasome for their degradation, the ER quality control machinery for these two classes of proteins likely differs because of their distinct topologies. Here we establish the requirements for the ERAD of Ste6p*, a multispanning membrane protein with a cytosolic mutation, and compare them with those for mutant form of carboxypeptidase Y (CPY*), a soluble luminal protein. We show that turnover of Ste6p* is dependent on the ubiquitin-protein isopeptide ligase Doa10p and is largely independent of the ubiquitin-protein isopeptide ligase Hrd1p/Der3p, whereas the opposite is true for CPY*. Furthermore, the cytosolic Hsp70 chaperone Ssa1p and the Hsp40 co-chaperones Ydj1p and Hlj1p are important in ERAD of Ste6p*, whereas the ER luminal chaperone Kar2p is dispensable, again opposite their roles in CPY* turnover. Finally, degradation of Ste6p*, unlike CPY*, does not appear to require the Sec61p translocon pore but, like CPY*, could depend on the Sec61p homologue Ssh1p. The ERAD pathways for Ste6p* and CPY* converge at a post-ubiquitination, pre-proteasome step, as both require the ATPase Cdc48p. Our results demonstrate that ERAD of Ste6p* employs distinct machinery from that of the soluble luminal substrate CPY* and that Ste6p* is a valuable model substrate to dissect the cellular machinery required for the ERAD of multispanning membrane proteins with a cytosolic mutation.
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PMID:Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. 1525 59


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