LINKING THE PROTEASOME ACTIVITY TO XPC BINDING PROTEIN HHR23A

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ubiquitin proteasome pathway regulates an astounding array of cellular events and remains essential throughout the life cycle of a cell. It is therefore not surprising that its dysfunction is associated with ailments as threatening as cancer and neurodegenerative diseases. This pathway functions in two stages, substrate ubiquitylation, which culminates in the covalent attachment of polyubiquitin to a protein substrate, and proteasomal degradation, which results in the degradation of the protein substrate into immunocompetent peptides. The connection between these two events requires ubiquitin receptors. The goal of this project is to determine the mechanistic pathways connecting substrate ubiquitylation to proteasomal degradation. We use NMR spectroscopy to determine the structure of proteasomal and non-proteasomal ubiquitin receptors alone and complexed with polyubiquitin. These structures will provide fundamental information on how receptors recognize their ubiquitylated substrates. NMR is the ideal methodology for these studies, as we can readily monitor dynamic interactions between polyubiquitin and multiple binding partners. This attribute is a large asset, as ubiquitin receptors bind each other and such interactions likely provide an effective mechanism for shuttling ubiquitylated substrates to and within the proteasome. By using NMR, we will monitor polyubiquitin as it passed from one receptor to another, and thereby determine which interactions are mutually exclusive or prioritized. Such studies will lead to models of how ubiquitylated substrates are captured by their receptors and delivered to the proteasome. The outcome of this research could be used to reveal points of specificity in the ubiquitin-proteasome pathway. Notably, the clinical importance of this pathway has been demonstrated by the success of a specific proteasome inhibitor (bortezomib/Velcade) in treating patients with multiple myeloma. A mechanistic understanding of the ubiquitin proteasome pathway is in its infancy, as new ubiquitin receptors and proteasome components are still being revealed. We are part of a research team that has identified a new proteasomal ubiquitin receptor. As part of this proposal, we determine this protein¿s structure and define how it interacts with ubiquitin and other receptors.