Sunday, November 24, 2024
11/24/2024
DESCRIPTION (provided by applicant): The ubiquitous, multiprotein, hsp90/hsp70-based chaperone machinery discovered in research on steroid receptors regulates the activity and/or turnover of more than 150 signaling proteins, including receptors, channels, transcription factors and protein kinases. The machinery regulates these hsp90 'client' proteins through the hsp70-dependent assembly of heterocomplexes with hsp90. The assembly of hsp90 heterocomplexes with signaling proteins is carried out by a multichaperone machinery present in all eukaryotic cells. We have reconstituted this nanomachinery from reticulocyte lysate and have developed a minimal system consisting of five proteins - hsp90, hsp70, Hop, hsp40, p23 - that carries out efficient signaling protein-hsp90 heterocomplex assembly. Three of these proteins self-associate to form an hsp90-Hop-hsp70 complex that carries out the ATP-dependent and K+dependent assembly of glucocorticoid receptor(GR)-hsp90 heterocomplexes while simultaneously opening the hydrophobic ligand binding cleft in the receptor to access by steroid. In this proposal, I utilize the technique of atomic force microscopy (AFM) to visualize the chaperone machinery and the different stages in assembly of the GR-hsp90 heterocomplex. In specific aim 1, AFM is used to resolve issues regarding the stoichiometry and effects of hsp90 acetylation on both the native chaperone machinery isolated from reticulocyte lysate and the machinery formed from purified proteins. In specific aim 2, the machinery is formed in stepwise fashion on the GR and imaged both in intermediate and final GR-hsp90 heterocomplexes. In specific aim 3, a flow cell will be used to determine the cofactor and chaperone requirements for hsp90 heterocomplex assembly and GR release from glucocorticoid response elements located in linear strands of DNA adsorbed to the AFM mica substrate. The effect of hsp90 acetylation state on nuclear assembly/disassembly of GR-hsp90 heterocomplexes bound to DNA will also be ascertained. Undergraduate students will play an essential role in this research and will be actively engaged in all aspects of the project, and the research infrastructure at this institution will be substantially enhanced. Students will be exposed to meaningful interdisciplinary research involving biological chemistry, pharmacology, material science, and engineering. This project seeks to advance health by promoting discovery in the development of technological capabilities in biomedical imaging and provide a structural understanding of a multi-protein machinery, its interactions, and its organization. PUBLIC HEALTH RELEVANCE: This project seeks to advance health and promote discovery by providing a structural understanding of a molecular complex, its interactions, and its organization. Events at the molecular level are often responsible for regulating the body's ability to mediate complex physiologic processes, and it is possible to identify molecular 'triggers' that promote or inhibit them. The research described in this proposal will advance our understanding of one newly identified molecular trigger (i.e. acetylation of hsp90) that presumptively affects the body's ability to facilitate a response to multiple disease states, including cancer, heart attacks, and diabetes.
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