Members of the oncogenic Polyomavirus family, such as Simian Virus 40 (SV40) and the human BK and JC viruses, require only one virally-encoded protein to transform mammalian cells: T antigen. T antigen is also required for viral replication, and the opportunistic growth of ICV and BKV in humans leads to disease. In addition, SV40 has been identified in several human cancers, induces brain tumors in rodents, and causes chromosomal re-arrangements in human fibroblasts. Three domains within T antigen are essential for these viruses to replicate and transform cells, two of which mediate binding to members of the p53 and Rb tumor suppressor families. The function of the third domain was mysterious until the discovery that this domain functioned in vitro as a DnaJ/Hsp40 molecular chaperone; members of the DnaJ/Hsp40 family activate Hsp70 chaperones and with Hsp70 can function as molecular """"""""machines"""""""" that catalyze protein transport, folding, degradation, and activation in vivo and in vitro. The existing in vitro assay for T antigen chaperone activity measures the protein's ability to enhance the chaperone properties of a yeast Hsp70 chaperone. Notably, mutations that compromised T antigen chaperone activity also inhibited viral replication and tumorigenesis, a result that provided the first direct link between chaperone function and viral-mediated tumor formation. ? ? Because of this link, it was surmised that small molecule inhibitors of the interaction between DnaJ/Hsp40 and Hsp70 might block T antigen function. As a first step toward this goal, novel chaperone modulators have been identified through computational methods and the in vitro chaperone assay describe above. To identify specific, high affinity chaperone modulators that can ultimately be developed for therapeutic intervention, the Specific Aims of this grant application are to:1. Better define the structural motifs in a group of related organic compounds that compromise Hsp70 function in order to obtain more potent chaperone modulators.2. Assay existing and newly obtained Hsp70-modulators for their ability to inhibit T antigen activation of Hsp70 activity in vitro, to prevent the chaperone-dependent release of a polypeptide substrate from Hsp70, and to inhibit the chaperone-mediated transport of a precursor protein into yeast ER-derived microsomal vesicles.
| Fewell, Sheara W; Smith, Christine M; Lyon, Michael A et al. (2004) Small molecule modulators of endogenous and co-chaperone-stimulated Hsp70 ATPase activity. J Biol Chem 279:51131-40 |
