Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain

Sriram, Muthu Irulappan; Osipiuk, J.; Freeman, BC; Morimoto, R I; Joachimiak, Andrzej

doi:10.1016/s0969-2126(97)00197-4

Cited by 137 publications

(131 citation statements)

References 47 publications

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“…by stabilizing the structure of the proteins or by protecting essential residues against environmental stresses (42)(43)(44)(45)(46)(47)(48)(49)(50)(51).…”

Section: Discussionmentioning

confidence: 99%

Primase Activity of Human DNA Polymerase α-Primase

Schneider¹,

Smith²,

Kautz³

et al. 1998

Journal of Biological Chemistry

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DNA polymerase ␣-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Coexpression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg 2؉ and Mn 2؉ but not Zn 2؉ . On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase ␣-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.

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“…by stabilizing the structure of the proteins or by protecting essential residues against environmental stresses (42)(43)(44)(45)(46)(47)(48)(49)(50)(51).…”

Section: Discussionmentioning

confidence: 99%

Primase Activity of Human DNA Polymerase α-Primase

Schneider¹,

Smith²,

Kautz³

et al. 1998

Journal of Biological Chemistry

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“…In many cancers, this differential expression pattern is lost, with increased basal expression of Hsp70 documented in multiple tumor types [6]. Hsc70 and Hsp70 are members of the actin class of ATPases and exhibit high structural homology, especially within their ATPase domain [7,8]. The Hsp70 family of chaperones exist in large multi-protein complexes with various co-chaperones including Bag-1, Hip, Hop, Hsp40, HspBP1 and Chip.…”

Section: Introductionmentioning

confidence: 99%

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells

Massey¹,

Williamson²,

Browne³

et al. 2009

Cancer Chemother Pharmacol

267

254

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Purpose The anti-apoptotic function of the 70 kDa family of heat shock proteins and their role in cancer is well documented. Dual targeting of Hsc70 and Hsp70 with siRNA induces proteasome-dependent degradation of Hsp90 client proteins and extensive tumor specific apoptosis as well as the potentiation of tumor cell apoptosis following pharmacological Hsp90 inhibition. Methods We have previously described the discovery and synthesis of novel adenosine-derived inhibitors of the 70 kDa family of heat shock proteins; the first inhibitors described to target the ATPase binding domain. The in vitro activity of VER-155008 was evaluated in HCT116, HT29, BT474 and MDA-MB-468 carcinoma cell lines. Cell proliferation, cell apoptosis and caspase 3/7 activity was determined for VER-155008 in the absence or presence of small molecule Hsp90 inhibitors. Results VER-155008 inhibited the proliferation of human breast and colon cancer cell lines with GI 50 s in the range 5.3-14.4 lM, and induced Hsp90 client protein degradation in both HCT116 and BT474 cells. As a single agent, VER-155008 induced caspase-3/7 dependent apoptosis in BT474 cells and non-caspase dependent cell death in HCT116 cells. VER-155008 potentiated the apoptotic potential of a small molecule Hsp90 inhibitor in HCT116 but not HT29 or MDA-MB-468 cells. In vivo, VER-155008 demonstrated rapid metabolism and clearance, along with tumor levels below the predicted pharmacologically active level. Conclusion These data suggest that small molecule inhibitors of Hsc70/Hsp70 phenotypically mimic the cellular mode of action of a small molecule Hsp90 inhibitor and can potentiate the apoptotic potential of a small molecule Hsp90 inhibitor in certain cell lines. The factors determining whether or not cells apoptose in response to Hsp90 inhibition or the combination of Hsp90 plus Hsc70/ Hsp70 inhibition remain to be determined.

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“…The structures of the ATPase domains from several Hsp70s have been solved (Flaherty et al, 1990;Harrison et al, 1997;Sriram et al, 1997). These structures resemble that of actin and hexokinase, consisting of two lobes with a deep cleft between them in which nucleotide binds.…”

Section: Introductionmentioning

confidence: 99%

Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

Pfund¹,

Huang²,

Lopez-Hoyo

et al. 2001

MBoC

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Ssbs of Saccharomyces cerevisiae are ribosome-associated molecular chaperones, which can be cross-linked to nascent polypeptide chains. Because Ssbs are members of a divergent subclass of Hsp70s found thus far only in fungi, we asked if the structural requirements for in vivo function were similar to those of “classic” Hsp70s. An intact peptide-binding domain is essential and an alteration of a conserved residue in the peptide-binding cleft (V442) affects function. However, Ssb tolerates a number of alterations in the peptide-binding cleft, revealing a high degree of flexibility in its functional requirements. Because binding of Ssb to peptide substrates in vitro was undetectable, we assessed the importance of substrate binding using the chimera BAB, in which the peptide binding domain of Ssb is exchanged for the analogous domain of the more “classical” Hsp70, Ssa. BAB, which binds peptide substrates in vitro, can substitute for Ssb in vivo. Alteration of a residue in the peptide-binding cleft of BAB creates a protein with a reduced affinity for peptide and altered ribosome binding that is unable to substitute for Ssb in vivo. These results indicate that Ssb's ability to bind unfolded polypeptides is likely critical for its function. This binding accounts, in part, for its stable interaction with translating ribosomes, even although it has a low affinity for peptides that detectably bind to other Hsp70s in vitro. These unusual properties may allow Ssb to function efficiently as a chaperone for ribosome-bound nascent chains.

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Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain

Cited by 137 publications

References 47 publications

Primase Activity of Human DNA Polymerase α-Primase

Primase Activity of Human DNA Polymerase α-Primase

A novel, small molecule inhibitor of Hsc70/Hsp70 potentiates Hsp90 inhibitor induced apoptosis in HCT116 colon carcinoma cells

Divergent Functional Properties of the Ribosome-Associated Molecular Chaperone Ssb Compared with Other Hsp70s

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