Systems Biology Approaches to Identify HSP90 Functions and Substrates

Bennington College, Bennington VT

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Abstract

DESCRIPTION (provided by applicant): The molecular chaperone Hsp90 is an essential protein, ubiquitous throughout eukaryotes, that comprises 1-2% of total cytosolic protein in the yeast Saccharomyces cerevisiae. Hsp90 is required for viability, even under non-stress conditions. This, coupled with its abundance, implies a role for Hsp90 in numerous cellular processes. To date, however, only a handful of folding "clients" for the yeast Hsp90 chaperone machinery have been identified. In mammalian cells, Hsp90 folds a variety of cell signaling molecules such as kinases and steroid hormone receptors. In order to uncover the scope of requisite cellular functions and substrates of Hsp90, we propose to utilize novel technologies to perform whole-genome screens in yeast, extensive bioinformatic analyses of the resulting data, and biological validation of the findings. This new and exciting approach circumvents existing limitations to the identification of Hsp90 substrates through traditional means, allowing for the possibility of high-impact results that will define the essential cellular functions of Hsp90. This multi-pronged approach circumvents existing limitations to identifying Hsp90 substrates, allowing for results that uncover the scope of requisite cellular functions and substrates of Hsp90. The homology between yeast and mammalian Hsp90 permits the translation of these new data to humans, allowing for the identification of and intervention in human diseases whose underlying cause is Hsp90- dependent. Specific Aims: i. Identify cellular targets of Hsp90 using genome-wide chemical-genetic screens. ii. Develop bioinformatic approaches to analyze Hsp90 targets and interaction networks derived from chemical-genetic screens. iii. Validate experimentally putative Hsp90 targets identified in Aims (i.) and (ii.) The use of existing homozygous and heterozygous yeast deletion strain collections, and the technology to identify each deletion strain upon the hybridization of its genomic DNA to high- density oligonucleotide arrays, allows the rapid identification of Hsp90-dependent cellular substrates and processes. Specific Aims ii. and iii. subject deletion strains sensitive to Hsp90- inhibition to bioinformatic and biological analyses, facilitating the construction of a complete picture of Hsp90 function in yeast. PUBLIC HEALTH RELEVANCE: The increasing relevance of the molecular chaperone Hsp90 to numerous human disease states, most notably cancers and neurodegenerative disorders, makes it an attractive target for intervention. Yet, the activity of Hsp90 is critical for cell viability. It is therefore crucial to generate a complete picture of the cellular range of essential Hsp90 functions and substrates to facilitate design of the most informed and intelligent therapeutic pursuits targeting Hsp90.