Abstract:SummaryThe ability of CheY, the response regulator of bacterial chemotaxis, to generate clockwise rotation is regulated by two covalent modifications -phosphorylation and acetylation. While the function and signal propagation of the former are widely understood, the mechanism and role of the latter are still obscure. To obtain information on the function of this acetylation, we non-enzymatically acetylated CheY to a level similar to that found in vivo, and examined its binding to its kinase CheA, its phosphata… Show more
“…This notion was validated in the cases of the CheY and RcsB response regulators of E. coli [6] and [13], respectively). In the case of CheY, it was revealed by co-crystallization that certain lysine residues were directly involved in binding to DNA targets [27,28].…”
Section: Discussionmentioning
confidence: 93%
“…The putative phosphorylation sight is D55 [12]. Lysine residues are fairly evenly distributed within the protein but are more clustered at the C-terminus, similar to the sequence in CheY from E. coli [13]. The RprY C-terminus contains the DNA binding domain.10.1080/20002297.2018.1487743-F0003Figure 3.Amino acid sequences and conserved domains of RprY, Pat, and CobB.…”
Background: Bacteria use two-component signal transduction systems (among others) to perceive and respond to environmental changes. Within the genus Porphyromonas, we observed degeneration of these systems, as exemplified by the loss of RprX, the sensor kinase partner of the RprY.Objective: The purpose of this study was to investigate modulation of RprY function by acetylation.Design: The transcriptional activity of the rprY-pat genes were measured by RT-PCR and 5ʹ-RACE. The acetylation of RprY were detected by western blotting. Electromobility shift and in vitro ChIP assays were used to measure the DNA binding activity of RprY. The expression of RprY target genes was measured by qRT-PCR. Effects of acetylation on phosphorylation of RprY were measured by Phos-tag gels.Results: The rprY gene is cotranscribed with pat. RprY is acetylated in vivo, and autoacetylated in vitro in a reaction that is enhanced by Pat; the CobB sirtuin deacetylates RprY. Acetylation reduced the DNA binding of RprY. Induced oxidative stress decreased production of RprY in vivo, increased its acetylation and increased expression of nqrA.Conclusions: We propose that to compensate for the loss of RprX, P. gingivalis has evolved a novel mechanism to inactivate RprY through acetylation.
“…This notion was validated in the cases of the CheY and RcsB response regulators of E. coli [6] and [13], respectively). In the case of CheY, it was revealed by co-crystallization that certain lysine residues were directly involved in binding to DNA targets [27,28].…”
Section: Discussionmentioning
confidence: 93%
“…The putative phosphorylation sight is D55 [12]. Lysine residues are fairly evenly distributed within the protein but are more clustered at the C-terminus, similar to the sequence in CheY from E. coli [13]. The RprY C-terminus contains the DNA binding domain.10.1080/20002297.2018.1487743-F0003Figure 3.Amino acid sequences and conserved domains of RprY, Pat, and CobB.…”
Background: Bacteria use two-component signal transduction systems (among others) to perceive and respond to environmental changes. Within the genus Porphyromonas, we observed degeneration of these systems, as exemplified by the loss of RprX, the sensor kinase partner of the RprY.Objective: The purpose of this study was to investigate modulation of RprY function by acetylation.Design: The transcriptional activity of the rprY-pat genes were measured by RT-PCR and 5ʹ-RACE. The acetylation of RprY were detected by western blotting. Electromobility shift and in vitro ChIP assays were used to measure the DNA binding activity of RprY. The expression of RprY target genes was measured by qRT-PCR. Effects of acetylation on phosphorylation of RprY were measured by Phos-tag gels.Results: The rprY gene is cotranscribed with pat. RprY is acetylated in vivo, and autoacetylated in vitro in a reaction that is enhanced by Pat; the CobB sirtuin deacetylates RprY. Acetylation reduced the DNA binding of RprY. Induced oxidative stress decreased production of RprY in vivo, increased its acetylation and increased expression of nqrA.Conclusions: We propose that to compensate for the loss of RprX, P. gingivalis has evolved a novel mechanism to inactivate RprY through acetylation.
“…While the phosphorylation of CheY has been extensively studied, the effect of acetylation on the activity of the protein is still poorly understood (207)(208)(209). Acetylation of CheY inhibits binding to three of its interacting partners, the CheA kinase, the CheZ phosphatase, and the flagellar motor switch FliM protein (166).…”
Section: Rla Targets Whose Modifying Acetyltransferases Are Not Knownmentioning
confidence: 99%
“…Previously, acetylation of CheY was hypothesized to occur via either autoacetylation or acetyl-CoA synthetase-dependent acetylation by some unknown mechanism (206,211). To date, studies of CheY have been performed by chemical acetylation of the protein using acetic anhydride (208). However, this method does not acetylate CheY to the same extent as what is seen for the protein in vivo (166,206).…”
Section: Rla Targets Whose Modifying Acetyltransferases Are Not Knownmentioning
SUMMARY
Acylation of biomolecules (e.g., proteins and small molecules) is a process that occurs in cells of all domains of life and has emerged as a critical mechanism for the control of many aspects of cellular physiology, including chromatin maintenance, transcriptional regulation, primary metabolism, cell structure, and likely other cellular processes. Although this review focuses on the use of acetyl moieties to modify a protein or small molecule, it is clear that cells can use many weak organic acids (e.g., short-, medium-, and long-chain mono- and dicarboxylic aliphatics and aromatics) to modify a large suite of targets. Acetylation of biomolecules has been studied for decades within the context of histone-dependent regulation of gene expression and antibiotic resistance. It was not until the early 2000s that the connection between metabolism, physiology, and protein acetylation was reported. This was the first instance of a metabolic enzyme (acetyl coenzyme A [acetyl-CoA] synthetase) whose activity was controlled by acetylation via a regulatory system responsive to physiological cues. The above-mentioned system was comprised of an acyltransferase and a partner deacylase. Given the reversibility of the acylation process, this system is also referred to as
r
eversible
l
ysine
a
cylation (RLA). A wealth of information has been obtained since the discovery of RLA in prokaryotes, and we are just beginning to visualize the extent of the impact that this regulatory system has on cell function.
“…The mechanism by which acetylation inhibits Acs activity is unknown; however, reactivation occurs upon deacetylation (Starai et al, 2002), catalyzed by the sirtuin CobB (Starai et al, 2002; Starai et al, 2003). For the response regulator CheY, acetylation of several lysines influences ( i ) reversible phosphorylation of an aspartyl residue, ( ii ) rotational direction of the flagellar motor, and (iii) CheY’s ability to form complexes with 3 protein targets (Barak and Eisenbach, 2001; Barak et al, 2004; Barak et al, 1992; Barak et al, 2006; Fraiberg et al, 2014; Li et al, 2010; Liarzi et al, 2010; Yan et al, 2008). For RNase R, acetylation controls protein turnover: acetylation and thus neutralization of a lysine residue is proposed to break a salt bridge that sequesters the docking site for a chaperone that recruits proteases (Liang et al, 2011).…”
Section: The Impact Of Acetylation On Protein Function and Cellular Pmentioning
Nε-acetylation is emerging as an abundant post-translational modification of bacterial proteins. Two mechanisms have been identified: one is enzymatic, dependent on an acetyltransferase and acetyl coenzyme A; the other is non-enzymatic and depends on the reactivity of acetyl phosphate. Some, but not most, of those acetylations are reversed by deacetylases. This review will briefly describe the current status of the field and raise questions that need answering.
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