The mechanism of action of a NudC domain small heat shock protein

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DESCRIPTION (provided by applicant): A wide range of important human diseases are characterized by protein misfolding. In the cell, protein chaperones ensure correct folding of newly synthesized proteins as well as the refolding of stress-denatured proteins. It follows that a full understanding of protein misfolding diseases depends on understanding chaperone mechanisms. The goal of this project is to understand the biological functions of BOBBER1 (BOB1), a recently identified Arabidopsis protein chaperone. BOB1 is the first chaperone of its kind with roles in both development and in temperature responses suggesting that it has important and unique functions. BOB1 contains an evolutionarily conserved NudC protein domain. Because NudC domain chaperones like BOB1 are found in many organisms including humans, understanding how BOB1 functions will contribute to a fundamental understanding of cellular mechanisms of protein quality control. This proposal involves investigating 1) how changes in BOB1 protein structure affect its function, and 2) identifying other proteins and genes which interact with BOB1. The approach for understanding BOB1 function involves using a series of mutants to systematically determine the effects of each mutation on organismal thermotolerance, on chaperone activity, and on BOB1s sub-cellular localization. At high temperatures BOB1 is incorporated into heat shock granules (HSGs), stress-induced sub-cellular structures whose function and behavior are poorly characterized. Observation of BOB1 protein in living cells will be used to learn more about the function and composition of HSGs. In order to identify proteins and genes which interact with BOB1 biochemical and genetic approaches will be used. BOB1 will be affinity purified and interacting proteins will be identified using mass spectrometry. Interacting proteins are predicted to include chaperone substrates and HSG proteins, and these interactors will be characterized in order to understand the cellular context in which BOB1 functions. To identify genes and genetic pathways that interact with BOB1 a sensitized screen in a bob1-3 mutant background will be performed. The proteins and genes identified using these approaches will contribute to a mechanistic understanding of BOB1 function. This will enhance understanding of NudC chaperone functions, and more broadly will provide novel insights into cellular mechanisms of protein folding. PUBLIC HEALTH RELEVANCE: This project involves determining how the BOBBER1 protein enables plants to survive at high temperatures as well as ensuring that plants grow and develop normally. BOBBER1 is a molecular chaperone which helps other proteins fold and function correctly and BOBBER1-like proteins are found in most organisms including humans. Protein misfolding is a characteristic feature of many human diseases, so understanding how BOBBER1 functions will provide valuable insights into this important biological process.