Allosteric regulation of the proteasomal ATPases

West Virginia University, Morgantown WV

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Abstract

? DESCRIPTION (provided by applicant): The proteasome is solely responsible for targeted degradation of specific proteins in eukaryotes, and thus the process of substrate selection and initiation of degradation is the most critical step in regulation of this critical process. A complx of ATPases in the 26S proteasome regulate this critical step, however, it is not known how substrate binding to the proteasome regulates proteasome activity to modulate substrate processing, nor is it known if structural changes in the ATPase's N-domain allow for these regulatory mechanisms. The current working hypothesis is that substrate binding to the extreme N-domain of the ATPase allosterically communicates to the ATPase domains to trigger substrate processing activities. To determine the conformational changes that occur in the N-domain of the proteasomal ATPases, point-specific crosslinks will be used to determine how substrate and nucleotide binding alter the range of N-domain conformational states that exist. Furthermore, to determine the role of the N-domain's conformational changes in proteasome function, conformational changes will be restricted using these established crosslinks and biochemical and biophysical techniques will be employed to assess the functions of these conformational changes. This work is significant because it will define a dynamic mechanism that allows substrates to activate proteasome function, allowing one to understand how the proteasomal ATPases communicate distant messages through the N-domains at a molecular level. This will provide a mechanistic framework to understand how substrate degradation by the proteasome could be perturbed in human diseases. This research is innovative because the tractable in vitro model system has allowed the generation of highly novel preliminary data that offer critical insights into understanding how the N-domains regulate proteasome function.