Oxygen affinities of DosT and DosS sensor kinases with implications for hypoxia adaptation in Mycobacterium tuberculosis.

DosT and DosS are heme-based kinases involved in sensing and signaling O2 tension in the microenvironment of Mycobacterium tuberculosis (Mtb). Under conditions of low O2, they activate >50 dormancy-related genes and play a pivotal role in the induction of dormancy and associated drug resistance during tuberculosis infection. In this work, we reexamine the O2 binding affinities of DosT and DosS to show that their equilibrium dissociation constants are 3.3±1.0 µM and 0.46±0.08 µM respectively, which are six to eight-fold stronger than what has been widely referred to in literature. Furthermore, stopped-flow kinetic studies reveal association and dissociation rate constants of 0.84 µM-1 s-1 and 2.8 s-1, respectively for DosT, and 7.2 µM-1 s-1 and 3.3 s-1, respectively for DosS. Remarkably, these tighter O2 binding constants correlate with distinct stages of hypoxia-induced non-replicating persistence in the Wayne model of Mtb. This knowledge opens doors to deconvoluting the intricate interplay between hypoxia adaptation stages and the signal transduction capabilities of these important heme-based O2 sensors.

Mycobacterium tuberculosis DosS binds H2S through its Fe3+ heme iron to regulate the DosR dormancy regulon.

Mycobacterium tuberculosis (Mtb) senses and responds to host-derived gasotransmitters NO and CO via heme-containing sensor kinases DosS and DosT and the response regulator DosR. Hydrogen sulfide (H2S) is an important signaling molecule in mammals, but its role in Mtb physiology is unclear. We have previously shown that exogenous H2S can modulate expression of genes in the Dos dormancy regulon via an unknown mechanism(s). Here, we test the hypothesis that Mtb senses and responds to H2S via the DosS/T/R system. Using UV-Vis and EPR spectroscopy, we show that H2S binds directly to the ferric (Fe3+) heme of DosS (KDapp = 5.30 µM) but not the ferrous (Fe2+) form. No interaction with DosT(Fe2+-O2) was detected. We found that the binding of sulfide can slowly reduce the DosS heme iron to the ferrous form. Steered Molecular Dynamics simulations show that H2S, and not the charged HS- species, can enter the DosS heme pocket. We also show that H2S increases DosS autokinase activity and subsequent phosphorylation of DosR, and H2S-mediated increases in Dos regulon gene expression is lost in Mtb lacking DosS. Finally, we demonstrate that physiological levels of H2S in macrophages can induce DosR regulon genes via DosS. Overall, these data reveal a novel mechanism whereby Mtb senses and responds to a third host gasotransmitter, H2S, via DosS(Fe3+). These findings highlight the remarkable plasticity of DosS and establish a new paradigm for how bacteria can sense multiple gasotransmitters through a single heme sensor kinase.

Phosphatase-defective DevS sensor kinase mutants permit constitutive expression of DevR-regulated dormancy genes in Mycobacterium tuberculosis.

The DevR-DevS/DosR-DosS two-component system of Mycobacterium tuberculosis, that comprises of DevS sensor kinase and DevR response regulator, is essential for bacterial adaptation to hypoxia by inducing dormancy regulon expression. The dominant phosphatase activity of DevS under aerobic conditions enables tight negative control, whereas its kinase function activates DevR under hypoxia to induce the dormancy regulon. A net balance in these opposing kinase and phosphatase activities of DevS calibrates the response output of DevR. To gain mechanistic insights into the kinase-phosphatase balance of DevS, we generated alanine substitution mutants of five residues located in DHp α1 helix of DevS, namely Phe-403, Gly-406, Leu-407, Gly-411 and His-415. For the first time, we have identified kinase positive phosphatase negative (K+P-) mutants in DevS by a single-site mutation in either Gly-406 or Leu-407. M. tuberculosis Gly-406A and Leu-407A mutant strains constitutively expressed the DevR regulon under aerobic conditions despite the presence of negative signal, oxygen. These mutant proteins exhibited ∼2-fold interaction defect with DevR. We conclude that Gly-406 and Leu-407 residues are individually essential for the phosphatase function of DevS. Our study provides new insights into the negative control mechanism of DevS by demonstrating the importance of an optimal interaction between DevR and DevS, and local changes associated with individual residues, Gly-406 and Leu-407, which mimic ligand-free DevS. These K+P- mutant strains are expected to facilitate the rapid aerobic screening of DevR antagonists in M. tuberculosis, thereby eliminating the requirement for hypoxic culture conditions.

Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

Mycobacterium tuberculosis sensor kinase DosS modulates the autophagosome in a DosR-independent manner.

Dormancy is a key characteristic of the intracellular life-cycle of Mtb. The importance of sensor kinase DosS in mycobacteria are attributed in part to our current findings that DosS is required for both persistence and full virulence of Mtb. Here we show that DosS is also required for optimal replication in macrophages and involved in the suppression of TNF-α and autophagy pathways. Silencing of these pathways during the infection process restored full virulence in MtbΔdosS mutant. Notably, a mutant of the response regulator DosR did not exhibit the attenuation in macrophages, suggesting that DosS can function independently of DosR. We identified four DosS targets in Mtb genome; Rv0440, Rv2859c, Rv0994, and Rv0260c. These genes encode functions related to hypoxia adaptation, which are not directly controlled by DosR, e.g., protein recycling and chaperoning, biosynthesis of molybdenum cofactor and nitrogen metabolism. Our results strongly suggest a DosR-independent role for DosS in Mtb.

Oxygen triggers signal transduction in the DevS (DosS) sensor of Mycobacterium tuberculosis by modulating the quaternary structure.

A major challenge to the control and eventual eradication of Mycobacterium tuberculosis infection is this pathogen's prolonged dormancy. The heme-based oxygen sensor protein DevS (DosS) plays a key role in this phenomenon, because it is a major activator of the transcription factor DevR. When DevS is active, its histidine protein kinase region is ON and it phosphorylates and activates DevR, which can induce the transcription of the dormancy regulon genes. Here, we have investigated the mechanism by which the ligation of molecular oxygen to a heme-binding domain in DevS switches OFF its histidine protein kinase region. To shed light on the oligomerization states of this protein and possible protein-surfaces of interaction, we used analytical gel filtration, together with dynamic light scattering, fluorescence spectroscopy and chemical crosslinking. We found that DevS exists as three major species: an octamer, a tetramer and a dimer. These three states were observed for the concentration range between 0.5 and 20 µm DevS, but not below 0.1 µm. Levels of DevS in M. tuberculosis are expected to range from 5 to 26 µm. When this histidine protein kinase was OFF, the DevS was mainly tetrameric and dimeric; by contrast, when the kinase was ON, the protein was predominantly octameric. The changes in quaternary structure were rapid upon binding to the physiological signal. This finding represents a novel strategy for switching the activity of a two-component heme-based sensor. An enhanced understanding of this process might potentially lead to the design of novel regulatory agents that target the multimer interfaces for treatment of latent tuberculosis.

Target DNA stabilizes Mycobacterium tuberculosis DevR/DosR phosphorylation by the full-length oxygen sensors DevS/DosS and DosT.

Mycobacterium tuberculosis strongly relies on a latency, or nonreplicating persistence, to escape a human host's immune system. The DevR (DosR), DevS (DosS), and DosT proteins are key components of this process. Like the rhizobial FixL oxygen sensor, DevS and DosT are histidine protein kinases with a heme-binding domain. Like the FixJ partner and substrate of FixL, DevR is a classical response regulator of the two-component class. When activated by DevS or DosT during hypoxia in vivo, DevR induces a dormancy regulon of more than 40 genes. To investigate the contributions of DevS, DosT, and target DNA to the phosphorylation of DevR, we developed an in vitro assay in which the full-length, sensing, DevS and DosT proteins were used to phosphorylate DevR with ATP, in the presence of target DNAs that were introduced as oligonucleotides linked to magnetic nanoparticles. We found that the DevR phosphorylations proceeded only for the deoxy states of the sensors. The reaction was strongly inhibited by O2 , but not CO or NO. The production of phospho-DevR was enhanced sixfold by target consensus DNA or acr-DNA. The phospho-DevR bound tightly to that DNA (Kd ~ 0.8 nm toward acr-DNA), and it was only slightly displaced by a 200-fold excess of unphosphorylated DevR or of a truncated DevR with only a DNA-binding domain. To our knowledge, this represents the first in vitro study of the ligand regulation of DevR phosphorylation by full-length DevS and DosT, and demonstration of a positive effect of DNA on this reaction.

Inhibition of the DevSR Two-Component System by Overexpression of Mycobacterium tuberculosis PknB in Mycobacterium smegmatis.

The DevSR (DosSR) two-component system, which is a major regulatory system involved in oxygen sensing in mycobacteria, plays an important role in hypoxic induction of many genes in mycobacteria. We demonstrated that overexpression of the kinase domain of Mycobacterium tuberculosis (Mtb) PknB inhibited transcriptional activity of the DevR response regulator in Mycobacterium smegmatis and that this inhibitory effect was exerted through phosphorylation of DevR on Thr180 within its DNA-binding domain. Moreover, the purified kinase domain of Mtb PknB significantly phosphorylated RegX3, NarL, KdpE, TrcR, DosR, and MtrA response regulators of Mtb that contain the Thr residues corresponding to Thr180 of DevR in their DNA-binding domains, implying that transcriptional activities of these response regulators might also be inhibited when the kinase domain of PknB is overexpressed.

Histone H2A T120 Phosphorylation Promotes Oncogenic Transformation via Upregulation of Cyclin D1.

How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.

DevS/DosS sensor is bifunctional and its phosphatase activity precludes aerobic DevR/DosR regulon expression in Mycobacterium tuberculosis.

Two-component systems, comprising histidine kinases and response regulators, empower bacteria to sense and adapt to diverse environmental stresses. Some histidine kinases are bifunctional; their phosphorylation (kinase) and dephosphorylation (phosphatase) activities toward their cognate response regulators permit the rapid reversal of genetic responses to an environmental stimulus. DevR-DevS/DosR-DosS is one of the best-characterized two-component systems of Mycobacterium tuberculosis. The kinase function of DevS is activated by gaseous stress signals, including hypoxia, resulting in the induction of ~ 48-genes DevR dormancy regulon. Regulon expression is tightly controlled and lack of expression in aerobic Mtb cultures is ascribed to the absence of phosphorylated DevR. Here we show that DevS is a bifunctional sensor and possesses a robust phosphatase activity toward DevR. We used site-specific mutagenesis to generate substitutions in conserved residues in the dimerization and histidine phosphotransfer domain of DevS and determined their role in kinase/phosphatase functions. In vitro and in vivo experiments, including a novel in vivo phosphatase assay, collectively establish that these conserved residues are critical for regulating kinase/phosphatase functions. Our findings establish DevS phosphatase function as an effective control mechanism to block aerobic expression of the DevR dormancy regulon. Asp-396 is essential for both kinase and phosphatase functions, whereas Gln-400 is critical for phosphatase function. The positive and negative functions perform opposing roles in DevS: the kinase function triggers regulon induction under hypoxia, whereas its phosphatase function prevents expression under aerobic conditions. A finely tuned balance in these opposing activities calibrates the dormancy regulon response output.

Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS.

Mycobacterium tuberculosis DosS is critical for the induction of M. tuberculosis dormancy genes in response to nitric oxide (NO), carbon monoxide (CO), or hypoxia. These environmental stimuli, which are sensed by the DosS heme group, result in autophosphorylation of a DosS His residue, followed by phosphotransfer to an Asp residue of the response regulator DosR. To clarify the mechanism of gaseous ligand recognition and signaling, we investigated the hydrogen-bonding interactions of the iron-bound CO and NO ligands by site-directed mutagenesis of Glu-87 and His-89. Autophosphorylation assays and molecular dynamics simulations suggest that Glu-87 has an important role in ligand recognition, whereas His-89 is essential for signal transduction to the kinase domain, a process for which Arg-204 is important. Mutation of Glu-87 to Ala or Gly rendered the protein constitutively active as a kinase, but with lower autophosphorylation activity than the wild-type in the Fe(II) and the Fe(II)-CO states, whereas the E87D mutant had little kinase activity except for the Fe(II)-NO complex. The H89R mutant exhibited attenuated autophosphorylation activity, although the H89A and R204A mutants were inactive as kinases, emphasizing the importance of these residues in communication to the kinase core. Resonance Raman spectroscopy of the wild-type and H89A mutant indicates the mutation does not alter the heme coordination number, spin state, or porphyrin deformation state, but it suggests that interdomain interactions are disrupted by the mutation. Overall, these results confirm the importance of the distal hydrogen-bonding network in ligand recognition and communication to the kinase domain and reveal the sensitivity of the system to subtle differences in the binding of gaseous ligands.

Histone modifying enzymes: novel disease biomarkers and assay development.

Histones are the chief components of chromatin. When being catalyzed by a series of histone modifying enzymes, histones may undergo various post-translational modifications such as acetylation, methylation, phosphorylation, ubiquitylation and SUMOylation. The dysregulation of histone modifying enzymes will alter the histone post-modification patterns and cause diverse diseases including cancers. Consequently, the histone modifying enzymes have emerged as the promising biomarkers for disease diagnosis and prognosis. In this review, we summarize the recent researches about the histone modifying enzymes as the disease biomarkers, and highlight the development of methods for histone modifying enzyme assays.

[SENSORS IN MYCOBACTERIA FOR THE DETECTION OF REDOX STRESS].

Mycobacterium species are exposed to oxidative and nitrosylative stress from environments within and outside the host cells. After the host is infected with the bacilli, macrophages produce superoxide molecules via NADPH oxidase activity and nitric oxide (NO) via inducible NO synthase activity to kill the bacilli. The pathogenic bacilli can successfully survive in host cells via anti-oxidative and anti-nitrosylative mechanisms. In particular, Mycobacterium tuberculosis persisters pose a great problem for chemotherapy because most anti-mycobacterial drugs are ineffective against mycobacteria that are in the persistent state. In accordance with the changes in redox balance, the bacilli change their metabolic pathways from aerobic to anaerobic ones, thereby leading to a change from an actively growing state to a dormant state. Therefore, M. tuberculosis is expected to be equipped with sensors that detect redox stress in the environment such that it can switch to the dormant state and change its metabolic pathways accordingly. In this review, roles of the mycobacterial O2, NO, and CO gas sensors, DosS and DosT, consisting of the DosR regulon, and mycobacterial DNA binding proteins WhiBs, which contain iron-sulfur clusters, in latent infection are discussed.

DosS Is required for the complete virulence of mycobacterium tuberculosis in mice with classical granulomatous lesions.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within granulomas to persist. DosR, in concert with sensor kinases DosS and DosT, regulates the response to hypoxia. Yet Mtb lacking functional DosR colonize the lungs of C57Bl/6 mice, presumably owing to the lack of organized lesions with sufficient hypoxia in that model. We compared the phenotype of the Δ-dosR, Δ-dosS, and Δ-dosT mutants to Mtb using C3HeB/FeJ mice, an alternate mouse model where lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol. The progression of infection was analyzed by tissue bacterial burden and histopathology. A measure of the comparative global immune responses was also analyzed. Although Δ-dosR and Δ-dosT grew comparably to wild-type Mtb, Δ-dosS exhibited a significant defect in bacterial burden and pathology in vivo, accompanied by ablated proinflammatory response. Δ-dosS retained the ability to induce DosR. The Δ-dosS mutant was also attenuated in murine macrophages ex vivo, with evidence of reduced expression of the proinflammatory signature. Our results show that DosS, but not DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice. The attenuation of Δ-dosS is not due to its inability to induce the DosR regulon, nor is it a result of the accumulation of hypoxia. That the in vivo growth restriction of Δ-dosS could be mimicked ex vivo suggested sensitivity to macrophage oxidative burst. Anoxic caseous centers within tuberculosis lesions eventually progress to cavities. Our results provide greater insight into the molecular mechanisms of Mtb persistence within host lungs.

Smoking cessation reverses DNA double-strand breaks in human mononuclear cells.

OBJECTIVE: Cigarette smoking is a major risk factor for atherosclerotic cardiovascular disease, which is responsible for a significant proportion of smoking-related deaths. However, the precise mechanism whereby smoking induces this pathology has not been fully delineated. Based on observation of DNA double-strand breaks (DSBs), the most harmful type of DNA damage, in atherosclerotic lesions, we hypothesized that there is a direct association between smoking and DSBs. The goal of this study was to investigate whether smoking induces DSBs and smoking cessation reverses DSBs in vivo through examination of peripheral mononuclear cells (MNCs). APPROACH AND RESULTS: Immunoreactivity of oxidative modification of DNA and DSBs were increased in human atherosclerotic lesions but not in the adjacent normal area. DSBs in human MNCs isolated from the blood of volunteers can be detected as cytologically visible "foci" using an antibody against the phosphorylated form of the histone H2AX (γ-H2AX). Young healthy active smokers (n = 15) showed increased γ-H2AX foci number when compared with non-smokers (n = 12) (foci number/cell: median, 0.37/cell; interquartile range [IQR], 0.31-0.58 vs. 4.36/cell; IQR, 3.09-7.39, p<0.0001). Smoking cessation for 1 month reduced the γ-H2AX foci number (median, 4.44/cell; IQR, 4.36-5.24 to 0.28/cell; IQR, 0.12-0.53, p<0.05). A positive correlation was noted between γ-H2AX foci number and exhaled carbon monoxide levels (r = 0.75, p<0.01). CONCLUSIONS: Smoking induces DSBs in human MNCs in vivo, and importantly, smoking cessation for 1 month resulted in a decrease in DSBs to a level comparable to that seen in non-smokers. These data reinforce the notion that the cigarette smoking induces DSBs and highlight the importance of smoking cessation.

Regulation of MDA-MB-231 cell proliferation by GSK-3ß involves epigenetic modifications under high glucose conditions.

Hyperglycemia is a critical risk factor for development and progression of breast cancer. We have recently reported that high glucose induces phosphorylation of histone H3 at Ser 10 as well as de-phosphorylation of GSK-3ß at Ser 9 in MDA-MB-231 cells. Here, we elucidate the mechanism underlying hyperglycemia-induced proliferation in MDA-MB-231 breast cancer cells. We provide evidence that hyperglycemia led to increased DNA methylation and DNMT1 expression in MDA-MB-231 cells. High glucose condition led to significant increase in the expression of PCNA, cyclin D1 and decrease in the expression of PTPN 12, p21 and PTEN. It also induced hypermethylation of DNA at the promoter region of PTPN 12, whereas hypomethylation at Vimentin and Snail. Silencing of GSK-3ß by siRNA prevented histone H3 phosphorylation and reduced DNMT1 expression. We show that chromatin obtained after immunoprecipitation with phospho-histone H3 was hypermethylated under high glucose condition, which indicates a cross-talk between DNA methylation and histone H3 phosphorylation. ChIP-qPCR analysis revealed up-regulation of DNMT1 and metastatic genes viz. Vimentin, Snail and MMP-7 by phospho-histone H3, which were down-regulated upon GSK-3ß silencing. To the best of our knowledge, this is the first report which shows that interplay between GSK-3ß activation, histone H3 phosphorylation and DNA methylation directs proliferation of breast cancer cells.

Antibody inhibition of protein activity in starfish oocytes.

Antibodies are widely utilized in cell and molecule biology for immunoblots, immunostaining, immunoprecipitation, immunoaffinity purification, and immunoassay. Some antibodies can be used for in vivo inhibition experiments. These antibodies bind to their target molecules and neutralize their functions, providing functional information in the study of their biological role. Here, we describe our methods for obtaining inhibitory antibodies against desired proteins. We then describe in the starfish oocyte system how to inhibit a target protein, even in the nucleus, by injection of antibody into the cytoplasm, and how to evaluate antibody inhibition of cell cycle regulators in small numbers of oocytes.

Distribution pattern of histone H3 phosphorylation at serine 10 during mitosis and meiosis in Brachiaria species.

Histones are the major eukaryotic DNA-binding proteins. Posttranslational modifications on N-terminal tails of histones that form nucleosomes are often associated with distinct biological functions. Some theories suggest that one of these modifications, the phosphorylation of histone H3 at serine 10 (H3S10ph) plays a role in both chromosome condensation and sister chromatid cohesion. Although histones and some of their modifications are highly conserved, studies have shown that role and distribution of H3S10ph may differ between species. We evaluated the pattern of H3 phosphorylation using immunodetection during mitosis and meiosis in both diploid and tetraploid genotypes of Brachiaria species. Results revealed differences in chromosome distribution of H3S10ph when mitosis and meiosis were compared. Whole chromosomes were phosphorylated during meiosis I, whereas phosphorylation was restricted to the pericentromeric region in both meiosis II and mitosis. There was no variation in phosphorylation patterns between Brachiaria species and diploid and tetraploid genotypes. Regarding spatiotemporal coordination in the Brachiaria species evaluated, H3S10ph is related to maintenance of sister chromatid cohesion during cell divisions.

Activation of ATP binding for the autophosphorylation of DosS, a Mycobacterium tuberculosis histidine kinase lacking an ATP lid motif.

The sensor histidine kinases of Mycobacterium tuberculosis, DosS and DosT, are responsible for sensing hypoxic conditions and consist of sensor and kinase cores responsible for accepting signals and phosphorylation activity, respectively. The kinase core contains a dimerization and histidine phosphate-accepting (DHp) domain and an ATP binding domain (ABD). The 13 histidine kinase genes of M. tuberculosis can be grouped based on the presence or absence of the ATP lid motif and F box (elements known to play roles in ATP binding) in their ABDs; DosS and DosT have ABDs lacking both these elements, and the crystal structures of their ABDs indicated that they were unsuitable for ATP binding, as a short loop covers the putative ATP binding site. Although the ABD alone cannot bind ATP, the kinase core is functional in autophosphorylation. Appropriate spatial arrangement of the ABD and DHp domain within the kinase core is required for both autophosphorylation and ATP binding. An ionic interaction between Arg(440) in the DHp domain and Glu(537) in the short loop of the ABD is available and may open the ATP binding site, by repositioning the short loop away from the site. Mutations at Arg(440) and Glu(537) reduce autophosphorylation activity. Unlike other histidine kinases containing an ATP lid, which protects bound ATP, DosS is unable to accept ATP until the ABD is properly positioned relative to the histidine; this may prevent unexpected ATP reactions. ATP binding can, therefore, function as a control mechanism for histidine kinase activity.