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Multi-scale Modeling of Bacterial Proteasome Core Particle Assembly
Issue Date
2021-12-31Author
Itagi, Pushpa
Publisher
University of Kansas
Format
163 pages
Type
Dissertation
Degree Level
Ph.D.
Discipline
Molecular Biosciences
Rights
Copyright held by the author.
Metadata
Show full item recordAbstract
Molecular machines play a central role in cellular processes like signal transduction, motility, genome duplication, transport, protein synthesis, protein degradation, and many more. These machines cannot be synthesized directly by the cell but are assembled from individual subunits through non-covalent interactions. Most of the molecular machines studied to date have thought to have evolved from ordered and hierarchical assembly pathways. In this dissertation, we focused on the assembly of a specific molecular machine – the proteasome. The proteasome is a critical component of intracellular protein degradation and is involved in numerous processes such as cell growth, maintenance, and cell division. The proteasome consists of a proteolytically-active 20S Core Particle (CP) and a 19S Regulatory Particle (RP) that binds the CP and recognizes proteins tagged for degradation. The architecture of the CP is conserved across archaea, bacteria, and eukaryotes. The CP is formed from 14 α and 14 β subunits, arranged in a barrel-shaped complex of four stacked rings in an α7β7β7α7 arrangement. The β proteins are the catalytically active subunits, whereas the α subunits serve to bind the RP and ensure that only proteins targeted for degradation enter the CP. The proteasome CP is active only when fully assembled, which begins with the formation of Half Proteasomes (HP- α7β7), and then two HPs dimerize into a CP. All the β subunits are synthesized with an N-terminal propeptide, autocatalytically cleaved off when two HPs dimerize, and only then the CP becomes active. In this work, we elucidate different steps of CP assembly in the bacterium Rhodococcus erythroplis (Re). First, we are investigating the molecular mechanism behind the separation of time scales observed in CP assembly. Experimental work on bacterial proteasomes has shown that the HP forms completely within minutes, while HP dimerization to form CPs takes hours to complete. These studies also suggested that the β propeptide plays a role in regulating the dimerization rate. Using all-atom Molecular Dynamics (MD), we investigated the role of the β propeptide in Re CP assembly and showed that the length and polarity of a region in the propeptide impact dimerization rate. We further validated these findings experimentally. In all species, CP assembly always occurs with two HPs dimerizing, and we never observe CP with one or two missing subunits. We hypothesized that there exists some allosteric communication among the subunits to prevent dimerization of near-HP structures (α6β7, α7β6, and α6β6) with each other or an HP. Molecular simulations revealed a global conformational shift in the β subunits that causes significant conformational transitions making the near-HP structures in a non-dimerizable state. Next, we investigated the formation of HPs in three assembly CP pathways, using coarse-grained ODE mathematical models based on chemical reaction kinetics theory. Our results discuss kinetic trapping and CP assembly dynamics in different pathways. Our mathematical models reveal that there is a tradeoff between speed and robustness in these assembly pathways. Ultimately, our simulation findings have helped us address long-standing questions in the proteasome assembly field and obtain structural and molecular insights. Further, our results will lay a promising foundation for structure-based drug design for designing specific, efficient, and less toxic small-molecule proteasome assembly inhibitors to treat tuberculosis and other diseases. Lastly, our findings on self-assembling bacterial CPs will serve as a proof of concept for designing nanomachines and other nanotechnology applications.
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