Identification of ClpS mediated N-end rule degradation in Mycolicibacterium smegmatis
Date
2023
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Publisher
University of Delaware
Abstract
Tuberculosis (TB) has been and remains one of the most significant human infectious diseases, and currently causes more global death than any other single infectious agent aside from SARS-Cov-2. Advancements in public health have had a marked impact on prognosis and on the overall number of TB cases, however TB remains a persistent challenge in areas of the world lacking strong public health infrastructure or where public health programs have been disrupted by conflict. Adding to this burden, multidrug resistant strains (MDR-TB) of the causative bacterium, Mycobacterium tuberculosis (Mtb), have emerged, demanding the development of novel drugs and the exploration of novel drug targets. ☐ One promising target is the strictly essential caseinolytic peptidase complex, ClpC1P1P2 protease, which uses chemical energy from ATP to degrade proteins in the mycobacterial cytosol. The ClpC1 unfoldase component of this protease has emerged as a particularly attractive molecular target, in part because no direct orthologs exists in humans. However, few ClpC1-specific substrates, proteolytic adaptors or proteolytic programs have been identified to date, hampering efforts to develop targeted screens for novel ClpC1 inhibitors. The discovery of additional ClpC1- specific degradation pathways would improve our ability to discover and characterize compounds that selectively inhibit ClpC1P1P2. ☐ In the model bacterium Escherichia coli (E. coli), the closest homolog to ClpC1P1P2 is the protease ClpAP, which cooperates with a unique proteolytic adaptor: ClpS. E. coli ClpS facilitates a degradation pathway called the N-end rule in which polypeptides that display the amino acids leucine, phenylalanine, tyrosine, or tryptophan at their N-terminus are recognized by ClpS and delivered to the unfoldase ClpA. ClpA then unfolds the polypeptide and passes it into the associated ClpP peptidase for destruction. Mtb and the surrogate model mycobacterium Mycolicibacterium smegmatis (Msm) both possess an ortholog of ClpS, but it has not yet been reported whether an analogous N-end rule pathways exists in these organisms. ☐ In Chapter 2, we tested the hypothesis that ClpS cooperates with ClpC1P1P2 to carry out N-end rule proteolysis in mycobacteria. To address this, we looked at three features of the Clp protease system; accessing the conservation of mycobacterial ClpS to the well-studied E. coli ClpS, the interaction between mycobacterial ClpS and the unfoldase ClpC1, and finally the profile of N-end amino acids prioritized for degradation by ClpC1P1P2. Sequence and structural analysis demonstrate that EcoClpS and MsmClpS share pronounced similarities, including key features associated with N-end rule substrate binding. Biophysical studies with purified MsmClpS demonstrate its ability to bind model peptides bearing canonical N-end rule amino acids, and these interactions are confirmed by co-crystal structures with model N-end rule peptides. Finally, a cell based proteolytic reporter assay implemented in Msm establishes that model substrates displaying the four canonical N-end rule amino acids are expressed at lower steady state levels than substrates displaying any of the remaining 16 amino acids. Together, the data indicates that ClpS binds substrates presenting canonical N-end rule residues in Msm, and mediates an N-end rule degradation pathway in a similar pattern to E. coli. By identifying a novel substrate degradation pathway, we open a new avenue for drug screening that targets against this essential protease. Furthermore, the methodology and techniques used in this project can serve as a baseline for future substrate screens of the mycobacterial Clp protease. Additional work will be required to understand the interaction of ClpS to ClpC1 as well as the role this pathway plays in mycobacterial protein homeostasis. ☐ In Chapter 3, we report proof-of-principle studies that demonstrate the feasibility of using APEX2-facilitated proximity labeling to identify novel substrates and interaction partners of Clp proteases within mycobacteria. Mycobacterial Clp proteases, including ClpC1P1P2, are known to be essential for cell viability, but the specific protease function responsible are not well understood. Identifying novel substrates, adaptors, and regulators would expand our understanding of protease function and regulation in these cells. APEX2 proximity labeling is a method of covalently tagging nearby interaction partners of a protein of interest with biotin or other chemical handles. Here, we engineer fusion constructs of the APEX2 peroxidase with several proteins of interest. Furthermore, we establish that APEX2- mediated labeling can be carried out in mycobacterial cytosol and intracellular transport is not fundamentally impeded by the cell wall. Finding novel interaction partners of Clp protease will ultimately help determine how this essential protease is regulated and help to inform drug discovery through exploitation of these interaction surfaces.
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Keywords
N-end rule degradation, Proteins, Proteolysis, Escherichia coli, Mycobacterium tuberculosis, Tuberculosis