Actinobacterial Clp protease evolutionary diversity and substrate recognition
Date
2022
Authors
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Publisher
University of Delaware
Abstract
The Actinomycetota (synonym Actinobacteria) are one of the largest bacterial phyla and are widely distributed in nature, playing roles in nutrient cycling, as microbiome members, and as human pathogens. The actinobacterial species Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), which is one of the leading causes of infectious mortality worldwide. Although public health and medical advances have reduced TB levels in past centuries, TB is still a major global health challenge due to its ability its highly communicable nature the high increase of drug resistance. Although most TB infections can successfully be treated with a limited arsenal of antibiotics, about 5% of new TB infections are at least partially drug resistant. Most antibiotics currently used against TB target pathways associated with growth and synthesis of macromolecules such as DNA and proteins. However, in many TB infections, Mtb can exist in a metabolically dormant state for years. Thus, there is interest in identifying drugs that kill Mtb without targeting growth-associated processes. Importantly, Mtb possess several ATP-dependent proteases that play important roles in protein homeostasis and survival against the host cell immune system. Among these proteases, the Clp proteases are notably essential for M. tuberculosis growth, and have emerged as promising drug targets. To target these enzymes effectively, is imperative to develop further fundamental understanding of the biochemical characteristics and physiological functions of the Clp proteases. In this thesis, we aim to provide new insights on how Clp proteases carry out precise regulation of specific cellular pathways, explore the interactions between ATP-dependent Clp proteases and their substrates, and develop robust assay methods for identifying antibiotics that target these enzymes. ☐ In Chapter 2, seek to understand sequence conservation of ClpC enzymes across Actinobacteria by stringently sorting bona fide ClpC orthologs from the closely related unfoldase ClpB. To accomplish this, we developed a robust bioinformatic method to classify ClpC paralogs and characterize their specialized function based on their sequence characteristics. Unexpectedly, we identified a novel and conserved group of actinobacterial paralogs, distinct from ClpC and ClpB, which we term ClpI. ClpI sequences are similar to ClpC, but possess a more variable N-terminal domain (NTD), and ClpI subtypes exist with and without the LGF loops required to interact with a peptidase partner. This work expands our understanding of the protein quality control landscape in Actinobacteria, and provides a novel methodology for paralog discrimination that may be applied to other enzymes and clades. ☐ In Chapter 3, we demonstrate the existence of an N-end rule proteolytic pathway in mycobacteria. Some distantly related bacteria possess a well-defined proteolytic pathway in which the ClpS adaptor recognizes substrates based on the identity of their N-terminal residue, and delivers them to a Clp protease for destruction. Prior biochemical experiments demonstrated that mycobacterial ClpS can collaborate with ClpC1•ClpP1P2 protease to degrade some canonical N-end rule substrates. We sought to establish whether N-end rule proteolysis occurs in mycobacteria, and determine which N-terminal amino acids are recognized by mycobacterial ClpS. We used a panel of fluorescent model substrates to test for N-end rule proteolysis in mycobacterial cells. Moreover, structural and biophysical binding studies were used to examine the interaction between the N-terminal residue of a substrate and the ClpS adaptor. Our studies demonstrate that the four canonical N-end rule amino acids – Leu, Phe, Tyr, and Trp – are recognized by a mycobacterial N-end rule pathway. ☐ In Chapter 4, we described the development of novel fluorogenic folded protein substrates with optimal characteristics for high-throughput in vitro screening for protease-targeting antibiotics. Our strategy involves covalently attaching short AMC-bearing peptides to a native protein substrate, creating a branched fluorogenic substrate. We aimed to monitor the AMC fluorescence by using ClpX/ClpC1•ClpP1P2 for proteolysis of AMC-labeled proteins. This novel strategy has advantages over existing methods in efficiently identifying compounds that block or dysregulate proteolysis of folded proteins substrates.
Description
Keywords
Actinobacteria, Communicable diseases, Protein interaction, Target inhibition, Proteolytic pathway, Bacterial proteases, Antibiotics, Tuberculosis