Structural Basis for DNA Recognition by the Two-Component Response Regulator RcsB.

RcsB is a highly conserved transcription regulator of the Rcs phosphorelay system, a complex two-component signal transduction system (N. Majdalani and S. Gottesman, Annu Rev Microbiol 59:379-405, 2005; A. J. Wolfe, Curr Opin Microbiol 13:204-209, 2010, https://doi.org/10.1016/j.mib.2010.01.002; D. J. Clarke, Future Microbiol 5:1173-1184, 2010, https://doi.org/10.2217/fmb.10.83). RcsB plays an important role in virulence and pathogenicity in human hosts by regulating biofilm formation. RcsB can regulate transcription alone or together with its auxiliary transcription regulators by forming heterodimers. This complexity allows RcsB to regulate transcription of more than 600 bacterial genes in response to different stresses (D. Wang et al., Mol Plant Microbe Interact 25:6-17, 2012, https://doi.org/10.1094/MPMI-08-11-0207). Despite increasing knowledge of RcsB importance, molecular mechanisms that drive the ability of RcsB to control transcription of a large number of genes remain unclear. Here, we present crystal structures of unphosphorylated RcsB in complex with the consensus DNA-binding sequence of 22-mer (DNA22) and 18-mer (DNA18) of the flhDC operon from Escherichia coli determined at 3.15- and 3.37-Å resolution, respectively. The results of our structural analysis combined with the results of in vitro binding assays provide valuable insights to the protein regulatory mechanism, demonstrate how RcsB recognizes target DNA sequences, and reveal a unique oligomeric state that allows RcsB to form homo- and heterodimers. This information will help us understand the complex mechanisms of transcriptional regulation by RcsB in bacteria.IMPORTANCE RcsB is a well-studied two-component response regulator of the Rcs phosphorelay system, conserved within the family Enterobacteriaceae, which includes many pathogens. It is a global regulator, controlling more than 5% of bacterial genes associated with capsule biosynthesis, flagellar biogenesis, cell wall biosynthesis, antibiotic resistance, biofilm formation, and virulence in pathogens. Knowledge of RcsB structure represents a unique opportunity to explore mechanisms that regulate the Rcs phosphorelay system and its role in the family Enterobacteriaceae.

Protein acetylation dynamics in response to carbon overflow in Escherichia coli.

In Escherichia coli, acetylation of proteins at lysines depends largely on a non-enzymatic acetyl phosphate-dependent mechanism. To assess the functional significance of this post-translational modification, we first grew wild-type cells in buffered tryptone broth with glucose and monitored acetylation over time by immunochemistry. Most acetylation occurred in stationary phase and paralleled glucose consumption and acetate excretion, which began upon entry into stationary phase. Transcription of rprA, a stationary phase regulator, exhibited similar behavior. To identify sites and substrates with significant acetylation changes, we used label-free, quantitative proteomics to monitor changes in protein acetylation. During growth, both the number of identified sites and the extent of acetylation increased with considerable variation among lysines from the same protein. As glucose-regulated lysine acetylation was predominant in central metabolic pathways and overlapped with acetyl phosphate-regulated acetylation sites, we deleted the major carbon regulator CRP and observed a dramatic loss of acetylation that could be restored by deleting the enzyme that degrades acetyl phosphate. We propose that acetyl phosphate-dependent acetylation is a response to carbon flux that could regulate central metabolism.

Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

Signal integration by the two-component signal transduction response regulator CpxR.

The CpxAR two-component signal transduction system in Escherichia coli and other pathogens senses diverse envelope stresses and promotes the transcription of a variety of genes that remedy these stresses. An important member of the CpxAR regulon is cpxP. The CpxA-dependent transcription of cpxP has been linked to stresses such as misfolded proteins and alkaline pH. It also has been proposed that acetyl phosphate, the intermediate of the phosphotransacetylase (Pta)-acetate kinase (AckA) pathway, can activate the transcription of cpxP in a CpxA-independent manner by donating its phosphoryl group to CpxR. We tested this hypothesis by measuring the transcription of cpxP using mutants with mutations in the CpxAR pathway, mutants with mutations in the Pta-AckA pathway, and mutants with a combination of both types of mutations. From this epistasis analysis, we learned that CpxR integrates diverse stimuli. The stimuli that originate in the envelope depend on CpxA, while those associated with growth and central metabolism depend on the Pta-AckA pathway. While CpxR could receive a phosphoryl group from acetyl phosphate, this global signal was not the primary trigger for CpxR activation associated with the Pta-AckA pathway. On the strength of these results, we contend that the interactions between central metabolism and signal transduction can be quite complex and that successful investigations of such interactions must include a complete epistatic analysis.

Olanzapine increases RGS7 protein expression via stimulation of the Janus tyrosine kinase-signal transducer and activator of transcription signaling cascade.

Atypical antipsychotics such as olanzapine have high affinity for multiple monoamine neurotransmitter receptors and are the mainstay of pharmacological therapy for treatment of schizophrenia. In addition to blocking monoamine receptors, these drugs also affect intracellular signaling cascades. We now report that 24-h treatment with 300 nM olanzapine causes desensitization of serotonin (5-HT)(2A) receptors in A1A1v cells, a rat cortical cell line, as indicated by a reduction in inositol phosphate accumulation following stimulation with a 5-HT(2A/2C) receptor agonist (-)-1-(2,5-dimethoxy-4-lodophenyl)-2-aminopropane HCl. Olanzapine treatment for 24 h increased the levels of 5-HT(2A) receptors in both cytosol (234 +/- 34% of control level) and membrane fractions (206 +/- 14% of control levels) and RGS7 proteins in both cytosol (193 +/- 32% of control levels) and membrane fractions (160 +/- 18% of control levels) as measured on Western blots. Increased phosphorylation of Janus tyrosine kinase (JAK) 2 and increased phosphorylation and nuclear translocation of signal transducer and activator of transcription (STAT) 3 with 24-h olanzapine treatment demonstrate activation of the JAK-STAT signaling cascade. Pretreatment with a JAK inhibitor, AG490 [alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide], prevented the olanzapine-induced increase in membrane RGS7 protein levels; AG490 alone had no effect on RGS7 protein levels. We verified that treatment with AG490 reduced phosphorylation of JAK2 and inhibited the nuclear localization of phospho-STAT3. Interestingly, treatment with the JAK inhibitor had no effect on 5-HT(2A) receptor protein levels. These data suggest that olanzapine-induced activation of the JAK-STAT signaling cascade causes increased expression of RGS7 protein, which in turn could mediate desensitization of 5-HT(2A) receptor signaling caused by olanzapine because RGS7 binds to Galpha(q) protein and accelerates GTP hydrolysis.

Phosphorylation of Galpha11 protein contributes to agonist-induced desensitization of 5-HT2A receptor signaling.

Agonist treatment causes desensitization of many G protein-coupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of Galpha q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT2A) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT(2A/2C) receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of Galpha q/11 proteins, as demonstrated by immunoprecipitation of Galpha q/11 and both incorporation of 32P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of Galpha q/11 proteins, suggesting that different mechanisms are involved in desensitization after short- and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in Galpha11 significantly reduced DOI-stimulated phosphorylation of Galpha11 and DOI-induced desensitization of 5-HT2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of Galpha q/11 proteins and DOI-induced desensitization of 5-HT2A receptors. Expression of Galpha11 S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of Galpha11 protein by PKC and CaMK contributes to agonist-induced homologous desensitization of 5-HT2A receptor signaling as well as heterologous desensitization. The phosphorylation of Galpha protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.

Alterations in 5-HT2A receptor signaling in male and female transgenic rats over-expressing either Gq or RGS-insensitive Gq protein.

Serotonin 2A (5-HT2A) receptors are coupled to Galphaq and Galpha11 proteins to activate phospholipase C (PLC). Regulators of G-protein signaling proteins (RGS) modulate G-protein signaling by accelerating the intrinsic GTPase activity of Galphaq and Galpha11. This study investigated the effects of over-expression of wild-type Galphaq proteins (Gq-Tg) and over-expression of RGS-insensitive Galphaq proteins (G188S, RGSi-Tg) on 5-HT2A receptor mediated signaling in transgenic rats. Over-expression of wild-type Galphaq and RGS insensitive mutant Galphaq did not produce significant alterations in the levels of Galpha11, RGS2, RGS4, RGS7, RGS16 or 5-HT2A proteins. RGSi-Tg rats had higher oxytocin and corticosterone responses to (-)DOI, a 5-HT2A/2C receptor agonist, compared to Gq-Tg rats. RGSi-Tg and Gq-Tg rats had higher ACTH responses to (-)DOI compared to control rats. Similarly, 5-HT-stimulated PLC activity in the frontal cortex was higher in RGSi-Tg and Gq-Tg rats compared to control rats. In contrast, GTPgammaS-stimulated PLC activity was higher in Gq-Tg rats but not in RGSi-Tg rats compared to control rats. There was a small but statistically significant increase in the affinity of [125I]-DOI labeled 5-HT2A receptors in RGSi-Tg rats and Gq-Tg rats compared to controls. There were no significant differences in Bmax and Kd of [3H] ketanserin labeled 5-HT2A receptors among the three groups. These data suggest that the effect of RGS proteins on 5-HT2A receptor signaling is cell type specific. In transgenic rats over-expressing Galphaq, endogenous RGS proteins have a negative effect on 5-HT2A receptor-mediated oxytocin release. In contrast, endogenous RGS protein had no impact on 5-HT2A receptor-mediated ACTH release in transgenic rats.