YjbH-Enhanced Proteolysis of Spx by ClpXP in Bacillus subtilis Is Inhibited by the Small Protein YirB (YuzO)

Kommineni, Sushma; Garg, Saurabh; Chan, Chio Mui; Zuber, Peter

doi:10.1128/jb.01350-10

Cited by 23 publications

(32 citation statements)

References 52 publications

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“…YjbH bears a His-Cys-rich region on the N terminus which is important for Zn metal binding and for enhancement of proteolysis of Spx by ClpXP (Garg et al, 2009 (Kommineni et al, 2011). However, no Spx-related phenotype has been uncovered in the yirB deletion mutant.…”

Section: Discussionmentioning

confidence: 96%

“…Previous studies (Garg et al, 2009;Kommineni et al, 2011;Larsson et al, 2007) showed that YjbH acts as a substrate recognition factor to enhance Spx proteolysis by ClpXP.…”

Section: Discussionmentioning

confidence: 99%

“…Protein samples (20 mg) were resolved by 15 % SDS-PAGE. The protein was transferred onto a nitrocellulose membrane and immunoblotting was performed to detect Spx, BsYjbH and GtYjbH with anti-Spx (Nakano et al, 2001), anti-BsYjbH (Kommineni et al, 2011) and anti-His antibodies, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Alternative explanations for regulation of YjbH-mediated proteolysis of Spx may have to be considered. For example, a small protein, YirB, has been shown to inhibit the Spxproteolysis-enhancing activity of YjbH activity in vivo and in vitro through direct YjbH interaction (Kommineni et al, 2011), although YirB's physiological role is uncertain at this time.…”

Section: Introductionmentioning

confidence: 99%

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Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

et al. 2012

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Proteolytic control can govern the levels of specific regulatory factors, such as Spx, a transcriptional regulator of the oxidative stress response in Gram-positive bacteria. Under oxidative stress, Spx concentration is elevated and upregulates transcription of genes that function in the stress response. When stress is alleviated, proteolysis of Spx catalysed by ClpXP reduces Spx concentration. Proteolysis is enhanced by the substrate recognition factor YjbH, which possesses a His-Cys-rich region at its N terminus. However, mutations that generate H12A, C13A, H14A, H16A and C31/34A residue substitutions in the N terminus of Bacillus subtilis YjbH (BsYjbH) do not affect functionality in Spx proteolytic control in vivo and in vitro. Because of difficulties in obtaining soluble BsYjbH, the Geobacillus thermodenitrificans yjbH gene was cloned, which yielded soluble GtYjbH protein. Despite its lack of a His-Cys-rich region, GtYjbH complements a B. subtilis yjbH null mutant, and shows high activity in vitro when combined with ClpXP and Spx in an approximately 30 : 1 (ClpXP/Spx : GtYjbH) molar ratio. In vitro interaction experiments showed that Spx and the protease-resistant Spx DD (in which the last two residues of Spx are replaced with two Asp residues) bind to GtYjbH, but deletion of 12 residues from the Spx C terminus (SpxDC) significantly diminished interaction and proteolytic degradation, indicating that the C terminus of Spx is important for YjbH recognition. These experiments also showed that Spx, but not GtYjbH, interacts with ClpX. Kinetic measurements for Spx proteolysis by ClpXP in the presence and absence of GtYjbH suggest that YjbH overcomes non-productive Spx-ClpX interaction, resulting in rapid degradation.

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Section: Discussionmentioning

confidence: 96%

“…Previous studies (Garg et al, 2009;Kommineni et al, 2011;Larsson et al, 2007) showed that YjbH acts as a substrate recognition factor to enhance Spx proteolysis by ClpXP.…”

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

et al. 2012

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“…It is not fully understood how the adaptor activity of YjbH is regulated. However, recently is has been reported that in B. subtilis the 54-amino-acid protein YirB may function as an antiadaptor protein (18). It is suggested that YirB functions by interacting with YjbH, causing a subsequent release of Spx from YjbH (18).…”

mentioning

confidence: 99%

The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

Engman¹,

Rogstam²,

Frees³

et al. 2011

Journal of Bacteriology

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Spx is a global regulator that is widespread among the low-G؉C-content Gram-positive bacteria. Spx has been extensively studied in Bacillus subtilis, where it acts as an activator and a repressor of transcription in response to disulfide stress. Under nonstress conditions, Spx is rapidly degraded by the ClpXP protease. This degradation is enhanced by the YjbH adaptor protein. Upon disulfide stress, the amount of Spx rapidly increases due to a decrease in degradation. In the opportunistic pathogen Staphylococcus aureus, Spx is a global regulator influencing growth, biofilm formation, and general stress protection, and cells lacking the spx gene exhibit poor growth also under nonstress conditions. To investigate the mechanism by which the activity of Spx is regulated, we identified a homolog in S. aureus of the B. subtilis yjbH gene. The gene encodes a protein that shows approximately 30% sequence identity to YjbH of B. subtilis. Heterologous expression of S. aureus yjbH in a B. subtilis yjbH mutant restored Spx to wild-type levels both under nonstress conditions and under conditions of disulfide stress. From these studies, we conclude that the two YjbH homologues have a conserved physiological function. Accordingly, inactivation of yjbH in S. aureus increased the level of Spx protein and transcription of the Spx-regulated gene trxB. Notably, the yjbH mutant exhibited reduced growth and increased pigmentation, and both phenotypes were reversed by complementation of the yjbH gene.

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Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry

et al. 2016

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Adaptor proteins assist proteases in degrading specific proteins under appropriate conditions. The adaptor protein YjbH promotes the degradation of an important global transcriptional regulator Spx, which controls the expression of hundreds of genes and operons in response to thiol-specific oxidative stress in Bacillus subtilis. Under normal growth conditions, the transcription factor is bound to the adaptor protein and therefore degraded by the AAA+ protease ClpXP. If this binding is alleviated during stress, the transcription factor accumulates and turns on genes encoding stress-alleviating proteins. The adaptor protein YjbH is thus a key player involved in these interactions but its structure is unknown. To gain insight into its structure and interactions we have used chemical crosslinking mass spectrometry. Distance constraints obtained from the crosslinked monomer were used to select and validate a structure model of YjbH and then to probe its interactions with other proteins. The core structure of YjbH is reminiscent of DsbA family proteins. One lysine residue in YjbH (K177), located in one of the α-helices outside the thioredoxin fold, crosslinked to both Spx K99 and Spx K117, thereby suggesting one side of the YjbH for the interaction with Spx. Another lysine residue that crosslinked to Spx was YjbH K5, located in the long and presumably very flexible N-terminal arm of YjbH. Our crosslinking data lend support to a model proposed based on site-directed mutagenesis where the YjbH interaction with Spx can stabilize and present the C-terminal region of Spx for protease recognition and proteolysis. Proteins 2016; 84:1234-1245. © 2016 Wiley Periodicals, Inc.

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YjbH-Enhanced Proteolysis of Spx by ClpXP in Bacillus subtilis Is Inhibited by the Small Protein YirB (YuzO)

Cited by 23 publications

References 52 publications

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP

The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

Exploring structure and interactions of the bacterial adaptor protein YjbH by crosslinking mass spectrometry

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