The Role Of p97-Chaperone Complexes In Protein Quality Control

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DESCRIPTION (provided by applicant): The long-term vision for my research is to determine how the ubiquitin proteasome system (UPS) can be targeted for therapy in diseases such as cancer; the short-term goals of this proposal are to understand the fundamentals of how the UPS regulates protein quality control. My past research has focused on how cellular stress; both intrinsic and extrinsic activate adaptive responses via activation of phosphorylation networks and ubiquitin-mediated signaling pathways. My graduate work with Melanie Cobb at UT Southwestern focused on elucidating signaling pathways involving a poorly understood family of kinases known as TAOs. My work led to the important finding that these kinases were important for the activation of DNA damage responsive signaling pathways. I continued in this vein when I joined Wade Harper's lab at Harvard and initially studied how DNA damage led to the ubiquitin-mediated degradation of a key cell cycle protein known CDT1. Importantly, I developed a screening platform that allowed me to globally screen for genes involved in CDT1 degradation using siRNA mediated knockdown of all the genes in the human genome and high-content imaging. I am currently interested in applying quantitative mass- spectrometry to understand how cellular stress globally activates pathways leading to regulated protein degradation. In this regard the environment at Harvard is key, I have already been exposed to state of the art screening technologies at the Institute of Chemistry and Cell Biology (ICCB) at Harvard. The Harper lab's long- standing collaboration with Steve Gygi's lab at Harvard has enabled members of the Harper lab to learn and apply various methods in mass spectrometry to biological problems. The department of Cell Biology at Harvard has numerous resources for post-doctoral fellows to interact with each other and with faculty. The department has a unique program that helps senior post-docs on the job market with acquiring the skills to successfully navigate the job application process. I believe that such an environment allows me not only to ask important questions, but also provides the facilities and research expertise to help me answer them. The goal of this proposal is to identify the mechanisms that maintain protein quality control in cells. As proteins are translated off the ribosome, chaperone systems are in place to bind the polypeptide and assist in their folding and targeting. This is particularly important for proteins with hydrophobic regions that are normally buried in the folded state, but are exposed during protein synthesis. If these regions are not appropriately shielded from the cytosol, they will aggregate and lead to toxicity. A growing body of research indicates that aneuploid tumor cells express the proteins encoded in their extra chromosomes. Expression of these proteins may be potentially harmful to cells because it imbalances the normal repertoire of cellular proteins and overburdens chaperone systems. Cancer cells are thought to overcome this adversity by up-regulating chaperones for assisted folding. This has been termed a form of 'non-oncogene' addiction. In general, during cases of proteomic stress, the excess of newly synthesized proteins that fail to fold is ubiquitinated and destroyed by the proteasome. Recently a complex of chaperones has been identified that associate with the translating ribosome and bind to hydrophobic regions of proteins when they are released into the cytosol. This system, nucleated on the BAG6 chaperone aids in the insertion of these proteins into endoplasmic reticulum (ER) where they will be processed for their final destination. It has been shown that when hydrophobic domain containing proteins fail to translocate into the ER, the BAG6 chaperone complex aids in their ubiquitination and degradation. However, little is known about the proteins that ubiquitinate and facilitate degradation of BAG6 client proteins. Importantly, most of the studies have been performed with a handful of reporter substrates, so it is unclear what the full cohort of BAG6 substrates in cells are. Through a proteomic screen, we have identified two new components of this pathway that associate with BAG6. The studies proposed here will attempt to identify the role of these complexes in the degradation of newly synthesized proteins, reconstitute their activity in vitro and identify their relevant targets in cancer cells. Overall tis proposal will elaborate on the mechanisms that maintain proteostais and identify new avenues for intervention in cancer and other aggregation prone diseases.