ClpXP-dependent Proteolysis of FNR upon Loss of its O2-sensing [4Fe–4S] Cluster

Mettert, Erin L.; Kiley, Patricia J.

doi:10.1016/j.jmb.2005.09.066

Cited by 63 publications

(92 citation statements)

References 52 publications

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“…In one analyzed example, site-specific oxidation of the bacterial FNR transcriptional regulator damages its [4Fe-4S] 2+ clusters, which in turn destabilizes the FNR dimer (18); the resulting monomers are proposed to accessibly display FNR's ClpXP protease recognition tags, leading to accelerated degradation of the monomers (19). Another example is Irr, a Fe-heme-binding regulator, which is degraded under oxidizing conditions in the presence of heme (20).…”

Section: Discussionmentioning

confidence: 99%

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Ahn

Baker

2015

Proc. Natl. Acad. Sci. U.S.A.

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Peroxide operon regulator (PerR) is a broadly conserved hydrogen peroxide sensor in bacteria, and oxidation of PerR at its regulatory metal-binding site is considered irreversible. Here, we tested whether this oxidation specifically targets PerR for proteolysis. We find that oxidizing conditions stimulate PerR degradation in vivo, and LonA is the principal AAA+ (ATPases associated with diverse cellular activities) protease that degrades PerR. Degradation of PerR by LonA is recapitulated in vitro, and biochemical dissection of this degradation reveals that the presence of regulatory metal and PerR-binding DNA dramatically extends the half-life of the protein. We identified a LonA-recognition site critical for oxidation-controlled PerR turnover. Key residues for LonA-interaction are exposed to solvent in PerR lacking metal, but are buried in the metal-bound form. Furthermore, one residue critical for Lon recognition is also essential for specific DNAbinding by PerR, thus explaining how both the metal and DNA ligands prevent PerR degradation. This ligand-controlled allosteric mechanism for protease recognition provides a compelling explanation for how the oxidation-induced conformational change in PerR triggers degradation. Interestingly, the critical residues recognized by LonA and exposed by oxidation do not function as a degron, because they are not sufficient to convert a nonsubstrate protein into a LonA substrate. Rather, these residues are a conformation-discriminator sequence, which must work together with other residues in PerR to evoke efficient degradation. This mechanism provides a useful example of how other proteins with only mild or localized oxidative damage can be targeted for degradation without the need for extensive oxidation-dependent protein denaturation.oxidative damage | proteolysis | degradation signals | Lon

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Section: Discussionmentioning

confidence: 99%

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Ahn

Baker

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…A shift from anaerobic to aerobic conditions oxidizes the Fe-S cluster to cause its disassembly in the Fnr protein, whereby Fnr loses the activity of a transcription factor (10). Moreover, the Fnr regulator that lacks the Fe-S cluster becomes vulnerable to ClpXP protease-mediated proteolysis (28). Although aconitase B (AcnB) is a tricarboxylic acid cycle enzyme carrying a Fe-S cluster, the apo form of AcnB plays a regulatory role by binding to the acnB mRNA (29).…”

Section: Discussionmentioning

confidence: 99%

The FeoC Protein Leads to High Cellular Levels of the Fe(II) Transporter FeoB by Preventing FtsH Protease Regulation of FeoB in Salmonella enterica

2013

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In the gammaproteobacteria, the FeoA, FeoB, and FeoC proteins constitute the Feo system, which mediates ferrous iron [Fe(II)] import. Of these Feo proteins, FeoB is an inner membrane Fe(II) transporter that is aided by the small protein FeoA. However, the role of another small protein, FeoC, has remained unknown. Here we report that the FeoC protein is necessary for FeoB protein-mediated Fe(II) uptake in Salmonella experiencing low levels of oxygen and iron. The FeoC protein was found to directly bind to the FeoB transporter, leading to high cellular levels of FeoB. Depletion of the FtsH protease enabled high levels of FeoB in the absence of FeoC, suggesting that the FeoC protein protects the FeoB transporter from FtsH-mediated proteolysis. Our present study provides a singular example of bacteria that can control expression of iron uptake systems posttranslationally by employing a small iron transporter-binding protein.

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“…1A). Residues 5 to 11 were previously identified as a ClpXP protease binding site, which functions in the regulated turnover of the apoform of FNR under aerobic conditions (16). Protease analysis demonstrated that the N-terminal region is susceptible to limited trypsin digestion, suggesting that this region is either unstructured or surface exposed (17).…”

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confidence: 99%

“…2). It seems unlikely that these truncation mutants have accelerated the ClpXP-dependent proteolysis of FNR, since neither FNR⌬2-12 nor FNR⌬2-13 contains the N-terminal ClpXP binding site (residues 5 to 11), and the second ClpXP binding site (residues 249 and 250) is not sufficient to target FNR for ClpXP-dependent proteolysis (16). It also seems unlikely that decreased transcription can explain the reduction in protein levels, since transcription of these mutant genes is driven by plasmid sequences and should be unaffected by the truncation mutations.…”

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confidence: 99%

“…The values shown for ␤-galactosidase activity (gray bars) are means of results from three independent experiments, expressed as percentages relative to the activity for full-length FNR (the average activity of wild-type FNR [expressed from pET11a] was 250 Miller units, and the average error from triplicate samples was less than 10%). Cells were also subjected to Western blot analysis (16) to measure the amount of full-length FNR and various FNR truncation mutants after growth under the same conditions as those for the ß-galactosidase assays. Proteins were detected with UV, and the amounts of FNR were quantified using Molecular Dynamics ImageQuant software and expressed as percentages relative to the level for full-length FNR (black bars (13).…”

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confidence: 99%

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Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR

Yan

Kiley

2008

J Bacteriol

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ClpXP-dependent Proteolysis of FNR upon Loss of its O2-sensing [4Fe–4S] Cluster

Cited by 63 publications

References 52 publications

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

Oxidization without substrate unfolding triggers proteolysis of the peroxide-sensor, PerR

The FeoC Protein Leads to High Cellular Levels of the Fe(II) Transporter FeoB by Preventing FtsH Protease Regulation of FeoB in Salmonella enterica

Dissecting the Role of the N-Terminal Region of the Escherichia coli Global Transcription Factor FNR

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