Saturday, April 20, 2024
4/20/2024
ABSTRACT M82 proteases are conserved across all kingdoms of life, and bear 4 catalytic motifs across 5 conserved C-terminal TMH (Transmembrane Helices), alongside a variable number of N-terminal TMH. PrsW from B. subtilis is the founding member of the family and performs Site-1-Proteolysis (S1P) of the sW Anti-Sigma Factor (ASF) in response to antimicrobial peptide stress. The S. aureus homolog (prsS) is upregulated in response to DNA-damaging agents, cell wall-targeting antibiotics, and during growth in serum and macrophages. prsS mutants display sensitivity to DNA-damaging agents, H2O2, and cell wall-targeting antibiotics. Importantly, prsS is also required for full virulence in S. aureus. Connected to this, a lone ECF-sigma factor, sS, exists in S. aureus. In each regard, the expression and deletion of prsS phenocopies that for sigS, which one might expect if PrsS is required for S1P of a sS ASF. However, despite our data tying the function of PrsS to sS, a candidate ASF connecting the two has not been identified. It is entirely possible that such a factor does not exist in S. aureus – there are examples of ECF s-factors that have no ASF. With that said, our PrsS data is strikingly similar to that for sS. Regardless, PrsS is clearly important to the infectious potential of S. aureus, thus determining what its proteolytic substrates are would prove highly informative. Additionally, thousands of M82 enzymes are encoded within prokaryotic genomes, yet only a handful of studies exist characterizing them. Accordingly, in this application we will interrogate PrsS and M82 peptidase function as follows: Aim #1 – Degradomic Approaches to Identify PrsS Substrates: The central way one understands how a protease contributes to cellular function is by uncovering its substrates. Therefore, in this aim we will use unbiased and cutting edge proteomic approaches to definitively explore the complete subset of cleavage events performed by PrsS in S. aureus. Aim #2 – Exploring PrsS (and M82) Protease Biology: Despite the importance of PrsS, and M82 peptidases at large, our understanding of the structural features that drive their function is sorely lacking. Accordingly, in this aim we will undertake a structure/function interrogation of PrsS, providing detailed new insight into its role, and much needed information on the M82 family in general.
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