Understanding eukaryotic proteasome assembly regulation

PROJECT SUMMARY Proteostasis of thousands of proteins is critical for cell viability, regulated in part by the cell proteasome. This is a large multi-subunit machine that degrades short-lived or abnormal proteins. Proteasome function is critical in many diseases, e.g. abnormally high in Alzheimer’s Disease or low in some cancers. In eukaryotes, the transla-

tion of subunits is initiated by transcription factor Rpn4, the subunits then assemble into the Core Particle (CP) with chaperones, and finally the CP binds to the Regulatory Particle (RP), which identifies targets for degradation. The long-term goal in this application is to help understand the mechanism that regulate proteasome assembly

in eukaryotic systems using both mathematical modeling and experimental analyses. The overall objectives in this application are to uncover the mechanisms that regulate (1) eukaryotic proteasome subunit gene expression, (2) assembly dynamics of the CP with chaperones and the RP and (3) post-translational modifications of the

proteasome. The central hypothesis is that eukaryotic cells have evolved a set of mechanisms that dynamically regulate proteasome concentration and function. The rationale for this project is that the determination of the mechanisms that regulate proteasome dynamics is likely to offer a strong framework whereby new strategies for

human disease and conditions can be developed. The central hypothesis will be tested by pursuing three specific aims: (1) determine the impact of cell signaling on proteasome pre-assembly regulation, (2) evaluate eukaryotic proteasome assembly, and (3) examine the influence of post-assembly proteasome phosphorylation. Under the

first aim, a dynamical systems model will be used to determine how Rpn4 feedback regulates proteasome sub- unit gene expression. For the second aim, an experimental approaching using yeast as a model organism will be used to investigate assembly efficiency of CP and chaperone binding. Additionally, a theoretical model of

eukaryotic proteasome assembly will also be used. In the third aim, phosphorylation rates of subunits will be experimentally tested and a generalize Monod-Wyman-Changeux mathematical model of multisite phosphory- lation will be used to analyze the conformational changes the CP attains. The research proposed in this

application is innovative, in the applicant’s opinion, because it focuses on eukaryotic proteasome assembly, which has not been sufficiently characterized to date and incorporates both mathematical modeling and experi- mental methods. The proposed research is significant because it is expected to provide strong scientific

justification for the continued development and future clinical applications of novel proteasome assembly inhibi- tors. Ultimately, such knowledge has the potential of offering new opportunities for the development of innovative therapies to treat diseases such as Alzheimer’s and cancers. Moreover, this fellowship is sponsored by Drs. Eric

J. Deeds and Jeroen Roelofs leaders in their respective fields of computational biology and biochemistry. The proposed training plan includes a strong research environment and mentoring team conducive for the applicant’s growth into an independent researcher, while promoting diversity in biosciences.