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1.
J Inorg Biochem ; 257: 112576, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38761578

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Mycobacterium tuberculosis , Oxygen , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/metabolism , Oxygen/metabolism , Oxygen/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Adaptation, Physiological , Protamine Kinase/metabolism , Protamine Kinase/chemistry , Kinetics , Protein Kinases/metabolism , Protein Kinases/chemistry
2.
Redox Biol ; 52: 102316, 2022 06.
Article in English | MEDLINE | ID: mdl-35489241

ABSTRACT

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.


Subject(s)
Gasotransmitters , Mycobacterium tuberculosis , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Dioctyl Sulfosuccinic Acid/metabolism , Gasotransmitters/metabolism , Gene Expression Regulation, Bacterial , Heme/metabolism , Iron/metabolism , Mammals/genetics , Mammals/metabolism , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Protamine Kinase/chemistry , Protamine Kinase/genetics , Protamine Kinase/metabolism , Regulon
3.
Development ; 147(23)2020 12 13.
Article in English | MEDLINE | ID: mdl-33168584

ABSTRACT

DNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.


Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Endoreduplication/genetics , Membrane Proteins/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Cytoskeleton/genetics , DNA Replication/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental/genetics , Mutation/genetics , Myofibrils/genetics , Nuclear Envelope/genetics , Nuclear Matrix/genetics , Protamine Kinase/genetics
4.
Biochem J ; 477(9): 1669-1682, 2020 05 15.
Article in English | MEDLINE | ID: mdl-32309848

ABSTRACT

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.


Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Mycobacterium tuberculosis , Phosphoric Monoester Hydrolases/metabolism , Protamine Kinase/genetics , Gene Expression Regulation, Bacterial , Hypoxia , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oxygen/metabolism , Phosphorylation , Protamine Kinase/metabolism , Protein Kinases/metabolism
5.
Cell ; 180(6): 1212-1227.e14, 2020 03 19.
Article in English | MEDLINE | ID: mdl-32169215

ABSTRACT

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.


Subject(s)
Chromatin/metabolism , Protamines/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Nucleus/metabolism , Chromatin/physiology , Chromatin Assembly and Disassembly/genetics , Chromatin Assembly and Disassembly/physiology , Fertilization/genetics , Histones/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Oocytes/metabolism , Oocytes/physiology , Phosphorylation , Protamine Kinase/genetics , Protamine Kinase/metabolism , Protamines/genetics , Protein Serine-Threonine Kinases/physiology , RNA Splicing/genetics , RNA Splicing/physiology , Spermatozoa/metabolism , Transcription Factors/metabolism , Zygote/metabolism
6.
Nucleic Acids Res ; 47(19): 10086-10103, 2019 11 04.
Article in English | MEDLINE | ID: mdl-31529049

ABSTRACT

The metabolic sensor Per-Arnt-Sim (Pas) domain-containing serine/threonine kinase (PASK) is expressed predominantly in the cytoplasm of different cell types, although a small percentage is also expressed in the nucleus. Herein, we show that the nuclear PASK associates with the mammalian H3K4 MLL2 methyltransferase complex and enhances H3K4 di- and tri-methylation. We also show that PASK is a histone kinase that phosphorylates H3 at T3, T6, S10 and T11. Taken together, these results suggest that PASK regulates two different H3 tail modifications involving H3K4 methylation and H3 phosphorylation. Using muscle satellite cell differentiation and functional analysis after loss or gain of Pask expression using the CRISPR/Cas9 system, we provide evidence that some of the regulatory functions of PASK during development and differentiation may occur through the regulation of these histone modifications.


Subject(s)
DNA Methylation/genetics , DNA-Binding Proteins/genetics , Histones/genetics , Neoplasm Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Animals , Cell Differentiation/genetics , Cell Line , DNA-Binding Proteins/chemistry , HEK293 Cells , Histone Code/genetics , Histones/chemistry , Humans , Methyltransferases/genetics , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Neoplasm Proteins/chemistry , Phosphorylation/genetics , Protamine Kinase/chemistry , Protamine Kinase/genetics , Protein Serine-Threonine Kinases/chemistry , Satellite Cells, Skeletal Muscle/metabolism , Sequence Analysis, RNA
7.
Commun Biol ; 2: 349, 2019.
Article in English | MEDLINE | ID: mdl-31552302

ABSTRACT

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.


Subject(s)
Autophagosomes/metabolism , Autophagosomes/microbiology , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/physiology , Protamine Kinase/metabolism , Protein Kinases/metabolism , Tuberculosis/metabolism , Tuberculosis/microbiology , Autophagosomes/immunology , Autophagy , Bacterial Proteins/genetics , DNA-Binding Proteins , Gene Expression Profiling , Gene Silencing , Host-Pathogen Interactions/immunology , Humans , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Mutation , Mycobacterium tuberculosis/enzymology , Phagocytes/immunology , Phagocytes/metabolism , Phagocytes/microbiology , Phosphorylation , Protamine Kinase/genetics , Protein Kinases/genetics , Tuberculosis/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism , Virulence
8.
FEBS J ; 286(21): 4278-4293, 2019 11.
Article in English | MEDLINE | ID: mdl-31254441

ABSTRACT

Tuberculosis is one of the oldest known infectious diseases, responsible for millions of deaths annually around the world. The ability of Mycobacterium tuberculosis (Mtb) to enter into a dormant state has been considered integral to the success of this bacterium as a human pathogen. One of the key systems involved in regulating the entrance into dormancy is the differentially expressed in virulent strain sensor protein (DevS) [(dormancy survival sensor protein (DosS)]. However, the physiological signal for DevS has remained unclear since it was first shown to be a heme-based sensor with conflicting reports on whether it is a redox or an oxygen sensor. To address this question and provide a better understanding of the electronic properties of this protein, we present here, for the first time, a series of spectroelectrochemistry measurements of the full-length holo DevS in anaerobic conditions as well as bound to CO, NO, imidazole (Imz), cyanide, and O2 . An interesting feature of this protein is its ability to bind Imz even in the ferrous state, implying small-molecule analogues could be designed as potential regulators. Nonetheless, a midpoint potential (Em ) value of +10 mV [vs normal hydrogen electrode (NHE)] for DevS as measured under anaerobic conditions is much higher than the expected cytosolic potential for Mtb or even within stimulated macrophages (~ -270 mV vs NHE), indicating this sensor works in a reduced ferrous state. These data, along with the high oxygen affinity and very slow auto-oxidation rate of DevS, provides evidence that it is not a redox sensor. Overall, this study validates the biological function of DevS as an oxygen sensor directly involved in the dormancy/latency of Mtb.


Subject(s)
Bacterial Proteins/genetics , Biosensing Techniques , Mycobacterium tuberculosis/metabolism , Protamine Kinase/genetics , Tuberculosis/metabolism , Bacterial Proteins/chemistry , Carbon Monoxide/chemistry , Cyanides/chemistry , Heme , Humans , Imidazoles/chemistry , Mycobacterium tuberculosis/pathogenicity , Nitric Oxide/chemistry , Oxidation-Reduction , Oxygen/chemistry , Protamine Kinase/chemistry , Tuberculosis/microbiology , Tuberculosis/pathology
9.
FEBS J ; 286(3): 479-494, 2019 02.
Article in English | MEDLINE | ID: mdl-30570222

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Heme/chemistry , Mycobacterium tuberculosis/drug effects , Oxygen/pharmacology , Protamine Kinase/chemistry , Protein Kinases/genetics , Bacterial Proteins/metabolism , Chromatography, Gel , Cloning, Molecular , DNA-Binding Proteins , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Heme/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Latent Tuberculosis/microbiology , Latent Tuberculosis/pathology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Mycobacterium tuberculosis/metabolism , Oxygen/chemistry , Oxygen/metabolism , Phosphorylation , Protamine Kinase/genetics , Protamine Kinase/metabolism , Protein Interaction Domains and Motifs , Protein Kinases/metabolism , Protein Multimerization , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Regulon , Signal Transduction , Spectrometry, Fluorescence , Transcription, Genetic/drug effects
10.
FEBS J ; 284(22): 3954-3967, 2017 11.
Article in English | MEDLINE | ID: mdl-28977726

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , DNA/metabolism , Mycobacterium tuberculosis/metabolism , Oxygen/metabolism , Protamine Kinase/metabolism , Protein Kinases/metabolism , Bacterial Proteins/chemistry , DNA/chemistry , DNA-Binding Proteins , Gene Expression Regulation, Bacterial , Humans , Magnetite Nanoparticles/chemistry , Mycobacterium tuberculosis/growth & development , Phosphorylation , Protamine Kinase/chemistry , Protein Kinases/chemistry , Regulon
11.
Mol Cells ; 40(9): 632-642, 2017 Sep 30.
Article in English | MEDLINE | ID: mdl-28843272

ABSTRACT

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.


Subject(s)
Cell Hypoxia/genetics , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Protein Serine-Threonine Kinases/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA-Binding Proteins , Gene Expression Regulation, Bacterial/genetics , Humans , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Mycobacterium tuberculosis/pathogenicity , Oxygen/chemistry , Oxygen/metabolism , Phosphorylation , Protamine Kinase/genetics , Protamine Kinase/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Tuberculosis/genetics , Tuberculosis/microbiology
12.
Arch Biochem Biophys ; 612: 1-8, 2016 Dec 15.
Article in English | MEDLINE | ID: mdl-27729224

ABSTRACT

DosS is a sensor in Mycobacterium tuberculosis that differentially responds to O2, NO, and CO, as well as to changes in the redox state of the prosthetic heme iron atom. The ferrous protein and its Fe(II)NO and Fe(II)CO complexes undergo autophosphorylation and subsequently transfer the phosphate group to DosR, a nuclear factor, to activate it. In contrast, autophosphorylation is negligible with the ferric protein and the Fe(II)O2 complex. To clarify the basis for this differential response to gases, we have determined the crystal structures of the NO and COcomplexes of the DosS GAF-A domain, which contains the heme to which the gases bind. Comparison of these crystal structures with those reported for the phosphorylation-inactive ferric GAF-A domain suggest that the GAF-A domain is in a dynamic equilibrium between active and inactive states, and that the position of Glu87 in the heme cavity, which depends on the which gas is bound, acts as a modulator of the equilibrium, and therefore of catalytic activity.


Subject(s)
Bacterial Proteins/chemistry , Carbon Monoxide/chemistry , Iron/chemistry , Mycobacterium tuberculosis/chemistry , Nitric Oxide/chemistry , Oxygen/chemistry , Protamine Kinase/chemistry , Amino Acids/chemistry , Catalysis , Crystallography, X-Ray , Heme/chemistry , Hydrogen Bonding , Oxidation-Reduction , Phosphorylation , Protein Domains , Protein Multimerization , Protein Structure, Secondary , Signal Transduction
13.
Mol Cell ; 64(1): 176-188, 2016 10 06.
Article in English | MEDLINE | ID: mdl-27716482

ABSTRACT

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.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin D1/genetics , Gene Expression Regulation, Neoplastic , Histones/genetics , Intracellular Signaling Peptides and Proteins/genetics , Protein Processing, Post-Translational , Protein Serine-Threonine Kinases/genetics , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Chromatin/chemistry , Chromatin/metabolism , Cyclin D1/metabolism , Drosophila Proteins , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , HeLa Cells , Histones/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Methylation , Mice , Oligopeptides/genetics , Oligopeptides/metabolism , Phosphorylation , Protamine Kinase/genetics , Protamine Kinase/metabolism , Protein Serine-Threonine Kinases/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Threonine/metabolism , Ubiquitination
14.
Mol Cell ; 63(4): 544-546, 2016 08 18.
Article in English | MEDLINE | ID: mdl-27540854

ABSTRACT

Regulatory roles of protein and DNA modifications in gene expression during host defense have long been appreciated. In a recent article published in Nature Immunology, Li et al. (2016) provide a unique glimpse of yet another aspect of coordinated DNA methylation and protein acetylation in host response to pathogenic stimuli. They elegantly demonstrate that DNA methylation and transcriptional activation at the HDAC9 promoter by DNMT3a, along with lysine deacetylation of TBK1 by HDAC9, are essential events during host defense.


Subject(s)
Histone Deacetylases , Protamine Kinase , Acetylation , DNA , DNA Methylation , Histones , Humans , Methyltransferases , Polynucleotide 5'-Hydroxyl-Kinase , Promoter Regions, Genetic
15.
FEBS J ; 283(15): 2949-62, 2016 08.
Article in English | MEDLINE | ID: mdl-27327040

ABSTRACT

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.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/enzymology , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/metabolism , Protamine Kinase/chemistry , Protamine Kinase/metabolism , Protein Kinases/metabolism , Aerobiosis , Amino Acid Motifs , Amino Acid Sequence , Bacterial Proteins/genetics , Conserved Sequence , DNA/metabolism , DNA-Binding Proteins , Gene Expression Regulation, Bacterial , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Phosphoprotein Phosphatases/genetics , Protamine Kinase/genetics , Protein Binding , Protein Domains , Regulon
16.
J Biol Chem ; 291(31): 16100-11, 2016 07 29.
Article in English | MEDLINE | ID: mdl-27235395

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Carbon Monoxide , Mycobacterium tuberculosis , Nitric Oxide , Protamine Kinase , Signal Transduction/physiology , Amino Acid Substitution , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbon Monoxide/chemistry , Carbon Monoxide/metabolism , Hydrogen Bonding , Mutation, Missense , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Nitric Oxide/chemistry , Nitric Oxide/metabolism , Phosphorylation , Protamine Kinase/chemistry , Protamine Kinase/genetics , Protamine Kinase/metabolism
17.
Clin. transl. oncol. (Print) ; 18(2): 113-124, feb. 2016. ilus
Article in English | IBECS | ID: ibc-148215

ABSTRACT

Twist proteins are members of basic helix-loop-helix family and are major regulators of embryogenesis. In adult humans, Twist proteins are mainly expressed in precursor cells, including myogenic, osteoblastic, chondroblastic and myelomonocytic lineages, maintaining their undifferentiated state. In addition, they play important roles in lymphocyte function and maturation. Recently, several studies have reported regulatory roles for Twist in the function and development of hematopoietic cells as well as in survival and development of numerous hematological malignancies. It is activated by numerous signal transduction pathways, including Akt, nuclear factor κB, Wnt, signal transducer and activator of transcription 3, mitogen-activated protein kinase and Ras signaling. Activated Twist has an anti-apoptotic role and protects cancer cells from apoptotic cell death. In addition, overexpression of Twist promotes the process of epithelial-mesenchymal transition, which has an essential role in cancer metastasis. Hereby, we review the aberrant expression of Twist in hematopoietic malignancies such as leukemias, lymphomas and myelodysplastic syndrome, which is related with poor prognosis and drug resistance in these disorders. Inactivation of Twist by small RNAs technology or chemotherapeutic inhibitors targeting Twist and upstream or downstream molecules of Twist signaling pathways may be helpful in management of disease to improve treatment strategies in malignancies (AU)


No disponible


Subject(s)
Humans , Male , Female , Biomarkers/metabolism , Bone Marrow Neoplasms/metabolism , Bone Marrow Neoplasms/pathology , Hematologic Neoplasms/blood , Hematologic Neoplasms/genetics , Protamine Kinase/administration & dosage , Epithelium/abnormalities , Leukemia/blood , Stem Cell Niche/genetics , Biomarkers/analysis , Bone Marrow Neoplasms/complications , Hematologic Neoplasms/metabolism , Hematologic Neoplasms/pathology , Protamine Kinase , Protamine Kinase/metabolism , Apoptosis/physiology , Epithelium/pathology , Leukemia/drug therapy , Stem Cell Niche/physiology
18.
Expert Rev Mol Diagn ; 16(3): 297-306, 2016.
Article in English | MEDLINE | ID: mdl-26750583

ABSTRACT

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.


Subject(s)
Biomarkers, Tumor/metabolism , Histone Acetyltransferases/metabolism , Histone Deacetylases/metabolism , Histone Demethylases/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Neoplasms/diagnosis , Protamine Kinase/metabolism , Animals , Enzyme Assays/methods , Histone Methyltransferases , Humans , Molecular Diagnostic Techniques/methods
20.
Biochim Biophys Acta ; 1859(3): 476-85, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26657617

ABSTRACT

Many metazoan cell types differentially express multiple non-allelic amino acid sequence variants of histone H1. Although early work revealed that H1 variants, collectively, are phosphorylated during interphase and mitosis, differences between individual H1 variants in the sites they possess for mitotic and interphase phosphorylation have been elucidated only relatively recently. Here, we review current knowledge on the regulation and function of interphase H1 phosphorylation, with a particular emphasis on how differences in interphase phosphorylation among the H1 variants of mammalian cells may enable them to have differential effects on transcription and other chromatin processes.


Subject(s)
Chromatin/physiology , Histones/physiology , Interphase , Animals , Histones/chemistry , Humans , Phosphorylation , Protamine Kinase/physiology , Protein Processing, Post-Translational , Protein Structure, Tertiary , Transcription, Genetic
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