The N-degradome of Escherichia coli

Humbard, Matthew A.; Surkov, Serhiy; Donatis, Gian Marco De; Jenkins, Lisa M. Miller; Maurizi, Michael R.

doi:10.1074/jbc.m113.492108

Cited by 45 publications

(28 citation statements)

References 38 publications

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“…For instance, the N-terminal residues of PhoP matched part of the ClpX recognition motif present in the ClpX substrate DadA (Flynn et al, 2003) (Figure S1A, blue bar). Likewise, the N-terminal residues of PhoP include those normally recognized by the adaptor ClpS (Dougan et al, 2010; Humbard et al, 2013) (Figure S1A, red bar). In addition, PhoP has a motif that resembles the ClpA substrate Dps (Figure S1A, green bar) (Ninnis et al, 2009).…”

Section: Resultsmentioning

confidence: 99%

“…Degradation of certain ClpS-dependent substrates requires the amino acyl transferase (Aat) protein, which attaches a leucine or phenylalanine residue to the N-terminal basic amino acids lysine or arginine, thereby allowing recognition by ClpS (Humbard et al, 2013). However, Aat is not necessary for ClpS-dependent degradation of PhoP because PhoP levels were not altered upon deletion of the aat gene (Figure S1B).…”

Section: Resultsmentioning

confidence: 99%

“…In all organisms, regulated degradation of specific proteins by the recognition of a degron governs a broad range of physiological functions (Varshavsky, 2011). Given that the N-end rule pathway is widely conserved in eukaryotes (Choi et al, 2010; Varshavsky, 2011) and prokaryotes (Erbse et al, 2006; Ninnis et al, 2009), the ClpS adaptor is essential for degradation of several N-end rule substrates in bacteria (Humbard et al, 2013), and the widespread phylogenetic distribution of ClpS-mediated proteolysis (Kirstein et al, 2009), we anticipate that analogous mechanisms may provide differential stability to other substrates dependent on ClpS and potentially other adaptors.…”

Section: Discussionmentioning

confidence: 99%

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Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate

2017

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Summary According to the N-end rule, the N-terminal residue of a protein determines its stability. In bacteria, the adaptor ClpS mediates proteolysis by delivering substrates bearing specific N-terminal residues to the protease ClpAP. We now report that the Salmonella adaptor ClpS binds to the N-terminus of the regulatory protein PhoP, resulting in PhoP degradation by ClpAP. We establish that the PhoP-activated protein MgtC protects PhoP from degradation by outcompeting ClpS for binding to PhoP. MgtC appears to act exclusively on PhoP as it did not alter the stability of a different ClpS-dependent ClpAP substrate. Removal of five N-terminal residues rendered PhoP stability independent of both the clpS and mgtC genes. By preserving PhoP protein levels, MgtC enables normal temporal transcription of PhoP-activated genes. The identified mechanism provides a simple means to spare specific substrates from an adaptor-dependent protease.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate

2017

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“…While the DBCO reaction was carried out in cells, the assay was carried out in lysates, as is common for AaT, which lacks a clear activity-dependent phenotype (even the TS351G AaT-deletion cell line does not exhibit an obvious phenotype). [13, 17b, 21] A discussion of potential improvements to this method is given below.…”

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confidence: 99%

Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

2014

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Unnatural amino acids with bioorthogonal reactive groups have the potential to provide a rapid and specific mechanism for covalently inhibiting a protein of interest. Here, we use unnatural mutagenesis to insert an azide (Z) into the target protein at positions such that a “click” reaction with an alkyne modulator (X) will alter protein function. This bioorthogonal reactive pair can engender specificity of X for the Z-containing protein even if the target is otherwise identical to another protein, allowing for rapid target validation in living cells. We demonstrate our method using inhibition of the E. coli enzyme aminoacyl transferase by both active site occlusion and allosteric mechanisms. This “clickable magic bullet” strategy should be generally applicable to protein inhibition, within the limits of unnatural amino acid mutagenesis.

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“…However, it is also possible that protein unfolding or damage could expose polypeptide regions that that would be subject to nonspecific cleavage in the cell. These new N-termini may themselves be destabilizing or may be further modified by amino acid transferases to generate N-end rule degrons [65,66]. Regardless of how these destabilizing N-termini are generated, the presence of these N-end rule degrons signal immediate degradation through ClpS/ClpAP.…”

Section: Adaptor Dependent Proteolysis In Regulation and Quality Controlmentioning

confidence: 99%

Selective adaptor dependent protein degradation in bacteria

Kuhlmann

Chien

2017

Current Opinion in Microbiology

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Energy dependent proteolysis is essential for all life, but uncontrolled degradation leads to devastating consequences. In bacteria, oligomeric AAA+ proteases are responsible for controlling protein destruction and are regulated in part by adaptor proteins. Adaptors are regulatory factors that shape protease substrate choice by either restricting or enhancing substrate recognition in several ways. In some cases, protease activity or assembly itself requires adaptor binding. Adaptors can also alter specificity by acting as scaffolds to tether particular substrates to already active proteases. Finally, hierarchical assembly of adaptors can use combinations of several activities to enhance the protease’s selectivity. Because the lifetime of the constituent proteins directly affects the duration of a particular signaling pathway, regulated proteolysis impacts almost all cellular responses. In this review, we describe recent progress in regulated protein degradation, focusing on fundamental principles of adaptors and how they perform critical biological functions, such as promoting cell cycle progression and quality control.

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The N-degradome of Escherichia coli

Cited by 45 publications

References 38 publications

Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate

Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate

Specific Modulation of Protein Activity by Using a Bioorthogonal Reaction

Selective adaptor dependent protein degradation in bacteria

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