Identifying new sequence markers for proteolysis by Escherichia coli Clp proteases
Date
2020
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
ATP-dependent proteases are large enzymatic complexes that catalyze the degradation of folded proteins in the cytosol. These proteases are ubiquitous in nature, being present in all domains of life. Whereas eukaryotic organisms generally possess a single centralized proteolytic complex, bacteria possess multiple separate ATP-dependent proteases, each with distinct specificities for protein substrates (Sauer & Baker, 2011). Bacterial ATP-dependent proteases participate in a range of cellular functions, including general protein quality control and homeostasis (Susan Gottesman et al., 1997); adaptation to changing environmental conditions and nutrient availability (Goldberg & St John, 1976); and regulation of specific cellular pathways (Little et al., 1980). Importantly, these enzymes regulate virulence phenotypes in a variety of bacterial pathogens (Frees et al., 2003; Kwon et al., 2004), and specific proteases are strictly essential for the viability of the major global pathogen, Mycobacterium tuberculosis (Raju et al., 2014). ☐ Escherichia coli, the model system where these enzymes have been most extensively studied, has five ATP-dependent proteases: ClpAP, ClpXP, FtsH, HslUV and Lon. Although their details differ, each protease incorporates two functional components: a ring-shaped ATPase that recognizes and unfolds protein substrates, and a self-compartmentalized peptidase that hydrolyzes unfolded polypeptides into short peptide products (Sauer & Baker, 2011). The unfoldases play a critical role in regulating proteolysis overall through their ability to selectively recognize substrate proteins and ignore other proteins in the cellular proteome. Multiple mechanisms may be used to identify substrates, but it is thought that many are identified through direct recognition of short peptide degradation signals, called degrons, present at either terminus or within the polypeptide chain (Varshavsky, 2019). A relatively small number of degrons have been identified and characterized in E. coli and other bacteria, although major questions surrounding degron recognition remain unanswered. How many unique classes of degrons exist in a given cell? Do all proteases possess unique degrons? Do proteases have overlapping recognition of some degrons sequences? How does the stringency and affinity for degron sequences vary among proteases? The work presented here is part of an effort to address these questions. ☐ Few experiments have attempted to globally identify proteolytic substrates and degrons within bacteria. Most have employed “trapping” approaches, in which mutagenically inactivated proteases are used to capture substrates (Erbse et al., 2006; Graham et al., 2013; Liao & Van Wijk, 2019). While these types of experiments have revealed putative novel degrons, trap experiments have limitations. First, they are unlikely to reveal substrates that bind weakly or occur in low abundance. Second, expression of an inactive protease may compromise cell viability in cases where the protease is essential. Third, inactivating mutations can interfere with autocatalytic processing (Fischer et al., 2015) and/or self-assembly of peptidase monomers into oligomeric structures (Kunjappu & Hochstrasser, 2014). Thus, there is a need for complementary approaches that overcome the above mentioned constraints, and better allow us to characterize the entire degron landscape. ☐ The Schmitz lab has devised a novel cell-based degron discovery approach that links cell survival to degron recognition. In this approach, E. coli cells express a protein toxin in frame with a short randomized terminal peptide sequences. In cases where the tag is not a degron, toxin accumulates and leads to cell death. By contrast, if the tag is a valid degron, endogenous protease machinery destroys the toxin, allowing cell survival. My project aims to validate the toxin-based degron discovery approach by i) testing the hypothesis that growth rate in the context of the cell-based screen correlates with proteolysis rate in vitro, and ii) testing the hypothesis that candidate degrons are recognized by individual cellular proteases. ☐ Initial tests with the toxin-based system revealed that expression of toxin bearing a known E. coli degron, the ssrA tag (Farrell et al., 2005), permitted near wild-type cell survival. I identified substitutions in the last position of the ssrA tag that permitted either slower growth or no survival at all. To test whether the degree of survival correlated with the degree of recognition and proteolysis of tagged toxin, I assayed proteolysis in vitro of mCherry model substrates bearing these three degrons. Michaelis-Menten analysis revealed that the modified ssrA tags were degraded with weaker KM and/or slower Vmax, providing fundamental validation for the toxin-based degron screening approach. ☐ Toxin-based screening against a library of randomized terminal sequences revealed several candidate degrons that confer cell survival. I hypothesized that individual degrons would be recognized by individual proteases. To test this, I used bacterial recombineering to generate a panel of isogenic E. coli strains incorporating scarless deletions of ATP-dependent protease genes clpP, clpS, or clpA, alone or in combination. I expressed toxin bearing the above mentioned candidate degrons in these different genetic backgrounds, and examined the pattern of survival and cell death. I observed that three of the seven degrons tested required wild-type clpP for survival, implicating either ClpXP or ClpAP in degradation. None of these three degrons conferred survival in the context of a clpS-clpA deletion, strongly suggesting that ClpXP recognizes the degron in each case. These observations support the hypothesis that most degrons are specific to individual protease, and additionally suggest that ClpXP plays a dominant role in degron-directed proteolysis in E. coli, ☐ This project provides new insight into the details of ATP-dependent proteolysis in bacteria, and provides essential validation for a novel degron discovery approach. The workflows optimized here for constructing deletions strains, for testing the protease specificities of candidate degrons, and for evaluating proteolysis in vivo lay the groundwork for future efforts to explore bacterial proteolysis on a larger scale.
Description
Keywords
ATP-dependent proteases, Bacteria, E. coli, In-vitro proteolysis assay, Knock-out genes using recombineering