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

Abstract

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 α and 14 β) 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 α and β 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.