Protein histidine phosphorylation has largely remained unexplored due to the challenges of analyzing relatively unstable phosphohistidine-containing proteins. We describe a procedure for determining ...the stoichiometry of histidine phosphorylation on the human histidine kinases NME1 and NME2 by intact mass spectrometry under conditions that retain this acid-labile protein modification. By characterizing these two model histidine protein kinases in the absence and presence of a suitable phosphate donor, the stoichiometry of histidine phosphorylation can be determined. The described method can be readily adapted for the analysis of other proteins containing phosphohistidine.
Aims/hypothesis
Increased inflammation and oxidative stress are associated with insulin resistance (IR) and metabolic disorders. Serum histidine levels are lower and are negatively associated with ...inflammation and oxidative stress in obese women. The objective of this study was to evaluate the efficacy of histidine supplementation on IR, inflammation, oxidative stress and metabolic disorders in obese women with the metabolic syndrome (MetS).
Methods
A total of 100 obese women aged 33–51 years with BMI ≥ 28 kg/m
2
and diagnosed with MetS were included following a health examination in the community hospital in this randomised, double-blinded, placebo-controlled trial. Participants were allocated to interventions by an investigator using sequentially numbered sealed envelopes and received 4 g/day histidine (
n
= 50) or identical placebo (
n
= 50) for 12 weeks. Participants then attended the same clinic every 2 weeks for scheduled interviews and to count tablets returned. Serum histidine, HOMA-IR, BMI, waist circumference, fat mass, serum NEFA, and variables connected to inflammation and oxidative stress were measured at baseline and 12 weeks. Participants, examining physicians and investigators assessing the outcomes were blinded to group assignment. In addition, the inflammatory mechanisms of histidine were also explored in adipocytes.
Results
At 12 weeks, a total of 92 participants completed this trail. Compared with the placebo group (
n
= 47), histidine supplementation significantly decreased HOMA-IR (−1.09 95% CI −1.49, −0.68), BMI (−0.86 kg/m
2
95% CI −1.55, −0.17), waist circumference (−2.86 cm 95% CI −3.86, −1.86), fat mass (−2.71 kg 95% CI −3.69, −1.73), serum NEFA (−173.26 μmol/l 95% CI −208.57, −137.94), serum inflammatory cytokines (TNF-α, −3.96 pg/ml 95% CI −5.29, −2.62; IL-6, −2.15 pg/ml 95% CI −2.52, −1.78), oxidative stress (superoxide dismutase, 17.84 U/ml 95% CI 15.03, 20.65; glutathione peroxidase, 13.71 nmol/ml 95% CI 9.65, 17.78) and increased serum histidine and adiponectin by 18.23 μmol/l 95% CI 11.74, 24.71 and 2.02 ng/ml 95% CI 0.60, 3.44 in histidine supplementation group (
n
= 45), respectively. There were significant correlations between changes in serum histidine and changes of IR and its risk factors. No side effects were observed during the intervention. In vitro study indicated that histidine suppresses
IL6
and
TNF
mRNA expression and nuclear factor kappa-B (NF-κB) protein production in palmitic acid-induced adipocytes in a dose-dependent manner, and these changes were diminished by an inhibitor of NF-κB.
Conclusions/interpretation
Histidine supplementation could improve IR, reduce BMI, fat mass and NEFA and suppress inflammation and oxidative stress in obese women with MetS; histidine could improve IR through suppressed pro-inflammatory cytokine expression, possibly by the NF-κB pathway, in adipocytes.
Trial registration
www.chictr.org/cn/ChiCTR-TRC-11001551
Funding
The study was supported by the National Natural Science Fund of China (No. 81202184, 81130049, 81102112), Heilongjiang Post/doctoral Fund (No. LBN-Z12193) and Key Laboratory of Nutrition and Food Hygiene (Harbin Medical University, Heilongjiang Higher Education Institutions, No. YYKFKT1202).
Two-component signal transduction systems (TCSs) are widely conserved in bacteria to respond to and adapt to the changing environment. Since TCSs are also involved in controlling the expression of ...virulence, biofilm formation, quorum sensing, and antimicrobial resistance in pathogens, they serve as candidates for novel drug targets. TCSs consist of a sensor histidine kinase (HK) and its cognate response regulator (RR). Upon perception of a signal, HKs autophosphorylate their conserved histidine residues, followed by phosphotransfer to their partner RRs. The phosphorylated RRs mostly function as transcriptional regulators and control the expression of genes necessary for stress response. HKs sense their specific signals not only in their extracytoplasmic sensor domain but also in their cytoplasmic and transmembrane domains. The signals are sensed either directly or indirectly via cofactors and accessory proteins. Accumulating evidence shows that a single HK can sense and respond to multiple signals in different domains. The underlying molecular mechanisms of how HK activity is controlled by these signals have been extensively studied both biochemically and structurally. In this article, we introduce the wide diversity of signal perception in different domains of HKs, together with their recently clarified structures and molecular mechanisms.
To investigate the inhibitory impact of chlorogenic acid (CGA) on the growth of Morganella psychrotolerans and its ability to form histamine.
The antimicrobial effect of CGA on M. psychrotolerans was ...evaluated using the minimum inhibitory concentration (MIC) method, revealing an MIC value of 10 mg ml-1. The alkaline phosphatase (AKP) activity, cell membrane potential, and scanning electron microscopy images revealed that CGA treatment disrupted cell structure and cell membrane. Moreover, CGA treatment led to a dose-dependent decrease in crude histidine decarboxylase (HDC) activity and gene expression of histidine decarboxylase (hdc). Molecular docking analysis demonstrated that CGA interacted with HDC through hydrogen bonds. Furthermore, in situ investigation confirmed the efficacy of CGA in controlling the growth of M. psychrotolerans and significantly reducing histamine formation in raw tuna.
CGA had good activity in controlling the growth of M. psychrotolerans and histamine formation.
Autophosphorylation of histidine kinases (HK) is the first step for signal transduction in bacterial two-component signalling systems. As HKs dimerize, the His residue is phosphorylated in cis or ...trans depending on whether the ATP molecule used in the reaction is bound to the same or the neighboring subunit, respectively. The cis or trans autophosphorylation results from an alternative directionality in the connection between helices α1 and α2 in the HK DHp domain, in such a way that α2 could be oriented almost 90° counterclockwise or clockwise with respect to α1. Sequence and length variability of this connection appears to lie behind the different directionality and is implicated in partner recognition with the response regulator (RR), highlighting its importance in signal transduction. Despite this mechanistic difference, HK autophosphorylation appears to be universal, involving conserved residues neighboring the phosphoacceptor His residue. Herein, we describe a simple protocol to determine both autophosphorylation directionality of HKs and the roles of the catalytic residues in these protein kinases.
Abstract
Azotobacter vinelandii is a soil bacterium that produces alginates, a family of polymers of biotechnological interest. In A. vinelandii, alginate production is controlled by the ...two-component system GacS/GacA. GacS/GacA, in turn, regulates the Rsm post-transcriptional regulatory system establishing a cascade that regulates alginate biosynthesis by controlling the expression of the algD biosynthetic gene. In Pseudomonas aeruginosa, GacS/GacA is influenced by other histidine-kinases constituting a multicomponent signal transduction system. In this study, we explore the presence of GacS-related histidine-kinases in A. vinelandii and discover a novel histidine-kinase (Avin_34990, renamed HrgS). This histidin-kinase acts as a negative regulator of alginate synthesis by controlling the transcription of the sRNAs belonging to the Rsm post-transcriptional regulatory system, for which a functional GacS is required.
Regulation of alginate synthesis by the novel histidine-kinase HrgS.
Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted ...by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2) receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.
Pseudomonas aeruginosa
is an opportunistic gram-negative pathogenic microorganism that poses a significant challenge in clinical treatment. Antibiotics exhibit limited efficacy against mature ...biofilm, culminating in an increase in the number of antibiotic-resistant strains. Therefore, novel strategies are essential to enhance the effectiveness of antibiotics against
Pseudomonas aeruginosa
biofilms. D-histidine has been previously identified as a prospective anti-biofilm agent. However, limited attention has been directed towards its impact on
Pseudomonas aeruginosa
. Therefore, this study was undertaken to explore the effect of D-histidine on
Pseudomonas aeruginosa
in vitro. Our results demonstrated that D-histidine downregulated the mRNA expression of virulence and quorum sensing (QS)-associated genes in
Pseudomonas aeruginosa
PAO1 without affecting bacterial growth. Swarming and swimming motility tests revealed that D-histidine significantly reduced the motility and pathogenicity of PAO1. Moreover, crystal violet staining and confocal laser scanning microscopy demonstrated that D-histidine inhibited biofilm formation and triggered the disassembly of mature biofilms. Notably, D-histidine increased the susceptibility of PAO1 to amikacin compared to that in the amikacin-alone group. These findings underscore the efficacy of D-histidine in combating
Pseudomonas aeruginosa
by reducing biofilm formation and increasing biofilm disassembly. Moreover, the combination of amikacin and D-histidine induced a synergistic effect against
Pseudomonas aeruginosa
biofilms, suggesting the potential utility of D-histidine as a preventive strategy against biofilm-associated infections caused by
Pseudomonas aeruginosa
.
Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine ...kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme’s residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.
Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic ...reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.