Investigating the mechanism of ClpX-mediated degradation in mycobacteria - PROJECT SUMMARY AND ABSTRACT Mycobacterium tuberculosis (Mtb), which causes the disease tuberculosis (TB), is one of the deadliest human pathogens. This is due, in part, to its inherent resistance to several classes of antibiotics and its ability to evolve resistance through mutations. Current treatment for TB is also lengthy and causes significant morbidity, underscoring the need for novel antibiotics. Bacterial proteolysis targeting chimeras (bacPROTACs), which work by targeting essential proteins for degradation by proteolytic machinery, are beginning to be explored as a new tool for combatting TB. ClpX is an essential mycobacterial protein that is known to unfold and target numerous proteins for degradation in other bacteria, and given its essentiality, ClpX is a promising candidate for antibiotic PROTAC development in Mtb. Still, relatively little is known about which substrates it can target, what processes it regulates, or why it is essential in mycobacteria. By understanding the underlying biology of ClpX, we can develop ClpX-based bacPROTACs that target its most suitable substrates, synergize with existing antibiotics, and exploit essential interactions to prevent the evolution of drug resistance. In Aim 1 of this proposal, I will characterize the substrates of ClpX in vivo using quantitative proteomics and validate that ClpX directly mediates the degradation of these proteins using in vitro degradation assays. Of the proteins modulated by ClpX in vivo and degraded by ClpX in vitro, I will evaluate which ones can be targeted for degradation by ClpX using induced proximity. Finally, I will use machine learning to identify protein features that predict proximity-mediated degradation to help identify additional targets for ClpX-based bacPROTACs. In Aim 2, I will test the hypothesis that ClpX is essential in mycobacteria because it regulates DNA replication. I will first validate the interactions between ClpX and replication proteins using reciprocal co-immunoprecipitation. I will then test whether ClpX mediates the degradation of its binding partners in vitro and in vivo. Finally, I will test whether these DNA replication proteins can be targeted for degradation through induced proximity to ClpX and whether this affects bacterial viability. Together, these experiments will help determine the role of ClpX in an essential biological process and globally define its substrates in mycobacteria. This work will serve as an important foundation for the future development of novel PROTAC antibiotics to treat TB.
