Tumor suppressors are regulatory proteins that receive and integrate diverse signals and function to exert control over key cellular processes such as cell proliferation, differentiation, and apoptosis. Because loss or perturbation of their activity often results in cancer or other diseases, and because of their central role in governing organismal development and tissue homeostasis, these proteins are of great interest. The retinoblastoma protein (pRb) is a well characterized tumor suppressor that, in concert with two related proteins, p130and p107,control cell cycle entry and exit, in part, by regulating the activity of the E2F family of transcription factors. Many viruses, including Simian virus 40 (SV40) encode oncoproteins that bind Rb-family members and interfere with their ability to regulate E2Fs. The large tumor antigen (T antigen) encoded by SV40 binds to pRb, p107, and p130via an LXCXE motif and blocks the ability of these proteins to inhibit E2F-dependent transcription and to induce growth arrest. The retinoblastoma family has been studied intensively, yet little is known about the molecular basis by which viruses, such as SV40, block their action. In fact, interaction with T antigen has different consequences for each Rb protein. For example, p130 is degraded following SV40 infection or transformation, while the levels of pRb remain unchanged. Thus, T antigen appears to be able to distinguish different Rb-E2F complexes, but the basis for this discrimination is unknown. Like many regulatory proteins pRb and E2F transcription factors do not exist in isolation. Rather they function as part of large multiprotein assemblages that include chromatin modifiers, the basal transcription apparatus, as well as other factors, and the dynamic assembly and disassembly of these complexes is critical to their regulation. T antigen has a J domain and has been shown to function as a DnaJ molecular chaperone. The J domain is required for a vital DNA replication, transcriptional control, and virion assembly. Importantly, the J domain is required for T antigen to block the function of Rb proteins and thus to activate E2F-dependent transcription. This application seeks to understand the mechanistic and structural basis for the action of T antigen's recognition and disruption of Rb-E2F complexes.'First, biochemical studies will explore the ability of the T antigen chaperone machine to distinguish and act upon p130-E2F4-DP1, pRb- E2F4-DP1, and pRb-E2F1-DP1 complexes. Second, the role of J domain orientation and flexibility will be examined using a combination of NMR and X-ray crystallography. Finally, a combined genetic and biochemical approach will be used to identify additional protein participants in the chaperone reaction. These studies will enhance our understanding of how these tumor suppressors govern cell proliferation and survival, and how subversion of these mechanisms by viruses or genetic mutation, contribute to cancer.
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