The ubiquitin proteasome pathway regulates an astounding array of cellular events and remains essential throughout the life cycle of a cell;its dysfunction is associated with ailments as threatening as cancer and neurodegenerative diseases. The pathway functions in two stages, substrate ubiquitination, which culminates in covalent attachment of polyubiquitin to protein substrates, and proteasomal degradation, which results in the degradation of 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 ubiquitination to proteasomal degradation and the aims therefore focus on ubiquitin receptors. We use NMR to determine the structure of proteasomal and non-proteasomal receptors complexed with polyubiquitin. These studies are complemented with fluorescence spectroscopy and analytical ultracentrifugation to establish binding affinity and stoichiometry. Ultimately, the consequences of our structural data are explored by functional assays. A mechanistic understanding of how the proteasome captures and processes its substrates is in its infancy, as new ubiquitin receptors and proteasome components are still being revealed. In fact, we are part of a research team that has identified a new proteasomal ubiquitin receptor, and as part of this proposal, we determine its structure complexed with polyubiquitin as well as the functional implications of its interaction with other proteasome components. By using NMR, we can readily monitor dynamic interactions between polyubiquitin and multiple binding partners, which is a large asset, as ubiquitin receptors bind each other and such interactions are likely to provide an effective mechanism for shuttling substrates to and within the proteasome. We determine how the various receptors modulate each other's interactions with ubiquitin and affect other proteasomal events especially deubiquitination. Ultimately, our results will provide fundamental information on how the proteasome captures its ubiquitinated substrates. Public Health Relevance: As a key regulator of processes important for genome integrity, such as cell cycle regulation and gene expression, the ubiquitin proteasome pathway harbors numerous therapeutic possibilities for treating cancer and neurodegenerative diseases. Notably, inhibition of the proteasome by bortezomib/Velcade is the only treatment available to patients with multiple myeloma;however, the side effects of proteasome inhibition are severe. Our research is expected to provide fundamental information on how ubiquitinated substrates are shuttled to and within the proteasome. Such knowledge is the first step towards rationally designing inhibitors for specific protein substrates, which in the long-term could be used clinically with few side effects.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA097004-10
Application #
8244430
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Knowlton, John R
Project Start
2002-07-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
10
Fiscal Year
2012
Total Cost
$217,187
Indirect Cost
$73,355
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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