Andrew Truman - US grants

Project Summary BiP/Kar2 is a universally conserved molecular chaperone based in the endoplasmic reticulum that performs a variety of functions in the cell including protein folding of both newly synthesized and denatured protein ?clients? and targeted degradation of terminally misfolded proteins. In yeast, Kar2 function is regulated by co-chaperones such as Sec63, Jem1 and Scj1. While Scj1 and Jem1 appear to have some redundant functions, previous studies have demonstrated phenotypic differences between cells lacking Scj1 or Jem1. The specific roles of Jem1 and Scj1 in activating Kar2 and their particular client portfolio remains undetermined. All organisms require correct and accurate replication of DNA to grow and proliferate. Misregulation of DNA replication can result in either cell death or cancer. Our recent studies have uncovered a role for Scj1 and Kar2 in regulating genome integrity. While ER chaperone function and genome integrity are fundamental cellular processes, no connection between them has previously been established. Our recent studies suggest that this ER chaperone-genome integrity connection may be conserved in mammalian cells as loss of ERdj1 (co-chaperone of mammalian Kar2, BiP) sensitizes cells to DNA replication inhibitors such as triapine and hydroxyurea. Any strategy that lowers the rate of DNA replication in cells may form the basis of novel anticancer therapies. In this proposal, we expect to gain further mechanistic insight into how ER chaperones and co- chaperones control DNA replication. We propose to use both molecular biology and state-of-the-art mass spectrometric techniques in and yeast and cancer cells to achieve the aims of the objectives in our proposal. The scope of this work has broad implications for a variety of diseases associated with DNA replication and ER molecular chaperone function, including many types of cancer, viral infection and malaria. !

Project Summary All organisms require maintenance of DNA integrity to grow and proliferate. Replication and repair of DNA damage requires the increased synthesis of DNA nucleotides, a process that is dependent on the activity of the kinases such as ATM and ATR. Misregulation of DNA replication can result in either cell death or cancer. Studies by our lab and others have shown that many DNA damage response (DDR) proteins (such as ATM, ATR, XRCC1 and RNR) are stabilized by the molecular chaperones Hsp70 and Hsp90. These proteins perform a variety of functions in the cell including protein folding of both newly synthesized and denatured proteins, protein transport across membranes and disaggregation of oligomerized proteins. Research has primarily focused on how chaperone function specificity arises through regulation of expression, isoform differences and the variety of co- chaperone proteins that bind to the Hsp70 and Hsp90 molecules. Despite the identification of several phosphorylation sites on both yeast and mammalian Hsp70 through global proteomic screens (known as the chaperone code), the biological function of these remains unclear. Our studies published in Cell determined that CDK-mediated phosphorylation of a single site on Hsp70 can regulate chaperone function by altering both co- chaperone and client protein interactions. In this proposal, we aim to understand how the activation of DDR can promote changes in the pattern of Hsp70 chaperone code. We predict that in line with several Hsp90-kinase interactions, Hsp70 phosphorylation during DNA damage creates a feedback system whereby chaperone phosphorylation increases stability of DDR proteins, amplifying the signal of the DNA damage response. We propose to use both molecular biology and state-of-the-art mass spectrometric techniques on both Saccharomyces cerevisiae and mammalian cell culture cells to achieve the aims of the objectives in our proposal. Identification and study of functional phosphorylation sites on Hsp70 in both yeast and mammalian cells will provide us with a completely novel way to target chaperone activity. Hsp70 activity may be suppressed using specific phosphatase/kinase inhibitors. It may be possible to target specific ?client? proteins though alteration of Hsp70 phosphorylation status and specific Hsp70 phospho-species may have a higher susceptibility to inhibitors. The scope of this work has broad implications for a variety of diseases associated with both the DNA damage response and molecular chaperone function, including many types of cancer and neurodegenerative illnesses caused by protein aggregation (Huntington?s disease, Alzheimer?s disease and Creutzfeld-Jakob disease).