Kante, A. | bioRxiv Science

bioRxiv2023

In vitro reconstitution of the M.tb proteasome core particle reveals conserved aspects of bacterial proteasome assembly

According to the WHO, one in three people in the world has a latent tuberculosis infection. Tuberculosis is caused by the bacterium Mycobacterium tuberculosis (M.tb). The development of multi-drug resistant (MDR) tuberculosis indicates a need for novel treatments. Hence, it is important to find a second line of treatment for patients infected with MDR tuberculosis. The proteasome is known to be necessary for survival under stress and pathogenicity in M.tb. However, our ability to use the proteasome as drug target has been limited by our abilities to screen for inhibitor compounds in vitro. The proteasome is a protease complex that degrades proteins and is crucial for the maintenance of protein homeostasis within cells. Like many protein complexes, the proteasome must assemble into a specific quaternary structure in order to be active. Specifically, the proteolytically-active proteasome Core Particle (CP) consists of 28 subunits (14 and 14 {beta}) that must assemble into a barrel-like structure in order to become catalytically active. Hence, understanding the assembly process in not only important from a basic cell biological perspective, but may also serve as the basis for the discovery of novel assembly inhibitors. In this study, we have established for the first time a protocol to express and purify the M.tb and {beta} subunits separately in vitro. The subunits are soluble monomers on purification and only assemble into active CPs upon reconstitution. Our assembly experiments revealed that M.tb CP assembly pathway is almost certainly identical to that seen in previous experiments on the CP from the bacterium Rhodococcus erythropolis (R.e), but assembly in M.tb is much slower. Interestingly, we found that subunits from M.tb and R.e spontaneously self-assembled into active hybrid proteasomes on reconstitution with each other, despite having only 65% sequence similarity. Our work thus strongly suggests that the CP assembly pathway is conserved across bacteria, and the ability to perform in vitro assembly experiments on the M.tb proteasome opens up the possibility of performing critical experiments, including screening for potential molecules that could inhibit assembly, directly in this clinically-relevant organism.

biochemistry↗

bioRxiv2022

Understanding the Separation of Timescales in Rhodococcus erythropolis Proteasome Core Particle Assembly

The 20S proteasome Core Particle (CP) is a molecular machine that is a key component of cellular protein degradation pathways. Like other molecular machines, it is not synthesized in an active form, but rather as a set of subunits that assemble into a functional complex. The CP is conserved across all domains of life and is composed of 28 subunits, 14 and 14 {beta}, arranged in four stacked 7-member rings (7{beta}7{beta}77). While details of CP assembly vary across species, the final step in the assembly process is universally conserved: two half proteasomes (HP: 7{beta}7) dimerize to form the CP. In the bacterium Rhodococcus erythropolis, experiments have shown that the formation of the HP is completed within minutes, while the dimerization process takes hours. The N-terminal propeptide of the {beta} subunit, which is autocatalytically cleaved off after CP formation, plays a key role in regulating this separation of time scales. However, the detailed molecular mechanism of how the propeptide achieves this regulation is unclear. In this work, we used Molecular Dynamics (MD) simulations to characterize HP conformations and found that the HP exists in two states: one where the propeptide interacts with key residues in the HP dimerization interface and likely blocks dimerization, and one where this interface is free. We found that a propeptide mutant that dimerizes extremely slowly is essentially always in the non-dimerizable state, while the WT rapidly transitions between the two. Based on these simulations, we designed a propeptide mutant that favored the dimerizable state in MD simulations. In vitro assembly experiments confirmed that this mutant dimerizes significantly faster than WT. Our work thus provides unprecedented insight into how this critical step in CP assembly is regulated, with implications both for efforts to inhibit proteasome assembly and for the evolution of hierarchical assembly pathways.

biophysics↗