Hsp70 molecular chaperones play critical roles in the pathogenesis of many human diseases, including cancer and viral replication. Hsp70s bind polypeptides and couple ATP hydrolysis to alter substrate conformation and function. However, ATP hydrolysis by Hsp70 is weak, but can be stimulated by J domain-protein chaperones. To identify new targets of chaperone action, I performed a multi-copy suppressor screen for genes that improved the slow growth defect of yeast lacking YDJ1 but expressing a defective YDJ1 chimera. Among the genes identified were MID2, which regulates cell wall integrity, and PKC1, which encodes protein kinase C, which is also linked to cell wall biogenesis. Consistent with these data, I found that ydj1Δ yeast and yeast with temperature sensitive mutations in Hsp90 exhibit phenotypes consistent with cell wall defects but these phenotypes were improved by Mid2p or Pkc1p over-expression. Mid2p over-expression thickened the ydj1Δ cell wall, which is likely the basis for suppression of the ydj1Δ growth defect. These data provide the first link between cytoplasmic chaperones and cell wall integrity, and suggest that chaperones orchestrate the biogenesis of this structure.
Another J domain-protein is the Large Tumor Antigen (TAg) in the polyomavirus Simian Virus 40 (SV40). TAg is required for viral replication and cellular transformation, and binds Hsp70. Because of their roles in cancer and SV40 function, small molecule modulators that inhibit Hsp70 or J-protein activity might represent novel anti-cancer and/or anti-viral agents. To identify such agents, I screened a bank of small molecules and identified a compound, MAL3-101, that had no effect on endogenous Hsp70 ATPase activity, but inhibited TAg stimulation of Hsp70 ATPase activity and reduced breast cancer cell proliferation. Forty-two derivatives of MAL3-101 were then synthesized and twelve compounds inhibited breast cancer cell proliferation at lower concentrations than MAL3-101. Reduction of cell proliferation correlated with reduced TAg stimulation of Hsp70 in vitro. Intriguingly, one compound, MAL2-11B, also inhibited the ATPase activity of TAg. This compound inhibited viral replication almost five-fold and SV40 DNA replication in vitro. These data show that J-protein inhibitors may be viable treatments for breast cancer and polyomavirus infection.
|Advisor:||Brodsky, Jeffrey L.|
|School:||University of Pittsburgh|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 68/10, Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics, Cellular biology|
|Keywords:||Cell wall, Chaperone modulators, Chaperones, Hsp70, J-domain, Simian virus 40, Viral replication|
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