Understanding Mycobacterium tuberculosis 20S proteasome assembly

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PROJECT SUMMARY Tuberculosis affects over 8,000 individuals in the United States every year, with Mycobacterium tuberculosis (Mtb) able to resist the immune system in part through proteasome function. The proteasome is the macromolecular structure responsible for the degradation of misfolded or short-lived proteins in cells, and is composed of four stacked heptameric rings in a barrel-like structure. The long-term goal of this work is to help understand the mechanisms that regulate proteasome assembly in the Mtb system using both mathematical modeling and experimental analyses. The overall objectives in this application are to (1) elucidate the mechanism(s) by which assembly dynamics regulate proteasome formation and (2) determine their role in Mtb proteasome assembly. The central hypothesis is that Mtb proteasome has evolved a set of mechanisms that maximize yield and thus bacterial immune resistance. The rationale for this project is that determination of the mechanisms that regulate Mtb proteasome yield is likely to offer a strong scientific framework whereby new strategies for tuberculosis therapies in patients can be developed. The central hypothesis will be tested by pursuing three specific aims: (1) Evaluate the assembly kinetics of the Mtb proteasome using mathematical and experimental analyses, (2) Develop a biophysical framework to understand interactions between intermediate rings in proteasome assembly and (3) Analyze structures of intermediate rings in Mtb proteasome assembly. In the first aim, a mathematical model will be used to determine the role kinetic parameters play in ring formation and ultimately proteasome assembly. Additionally, Mtb monomers will be used to experimentally measure the kinetics of assembly. For the second aim, a biophysical framework will be developed to study the interactions between monomers and intermediate rings based on their size and structure. Furthermore, a mass-spectrometry approach will be used to identify the size and composition of intermediate rings formed during Mtb proteasome assembly. In the third aim, a cryo-Electron Microscopy approach will be used to analyze the structures of assembly intermediates with atomic resolution. The research proposed in this application is innovative because it focuses on the Mtb proteasome, which has not been sufficiently characterized to date, and because it incorporates both mathematical modeling and experimental methods. The proposed research is significant because it is expected to provide a foundation for the development and future clinical applications of novel Mtb proteasome assembly inhibitors. Ultimately, such knowledge has the potential of offering new opportunities for the development of innovative therapies to treat tuberculosis and other bacterial infections. Moreover, this fellowship is sponsored by Drs. Eric J. Deeds and Joseph A. Loo, who are leaders in their respective fields of computational biology and mass spectrometry. The proposed training plan includes a strong research environment and mentoring team conducive to the applicant’s growth into a highly successful independent researcher.