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IUBMB Life Aug 2018Tuberculosis is a global health problem, with a third of the world's population infected with the bacillus, Mycobacterium tuberculosis. The problem is exacerbated by the... (Review)
Review
Tuberculosis is a global health problem, with a third of the world's population infected with the bacillus, Mycobacterium tuberculosis. The problem is exacerbated by the emergence of multidrug resistant and extensively drug resistant strains. The search for new drug targets is therefore a priority for researchers in the field. The two-component systems (TCSs) are central to the ability of the bacterium to sense and to respond appropriately to its environment. Here we summarize current knowledge on the paired TCSs of M. tuberculosis. We discuss what is currently understood regarding the signals to which each of the sensor kinases responds, and the regulons of each of the cognate response regulators. We also discuss what is known regarding attempts to inhibit the TCSs by small molecules and project their potential as pharmacological targets for the development of novel antimycobacterial agents. © 2018 IUBMB Life, 70(8):710-717, 2018.
Topics: Antitubercular Agents; Bacterial Proteins; Drug Resistance, Multiple; Humans; Molecular Targeted Therapy; Mycobacterium tuberculosis; Phosphotransferases; Signal Transduction; Tuberculosis
PubMed: 29885211
DOI: 10.1002/iub.1872 -
Life Sciences Dec 2016Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) are regulatory enzymes involved in the generation of lipid species that modulate cellular signaling pathways... (Review)
Review
Phosphatidylinositol-4,5-bisphosphate 3-kinases (PI3Ks) are regulatory enzymes involved in the generation of lipid species that modulate cellular signaling pathways through downstream effectors to influence a variety of cellular functions. Years of intensive study of PI3Ks have produced a significant body of literature in many areas, including that PI3K can mediate intracellular vesicular trafficking and through these actions contribute to a number of important physiological functions. This review focuses on the crucial roles that PI3K and AKT, a major downstream partner of PI3K, play in the regulation of vesicle trafficking during various forms of vesicular endocytosis and exocytosis.
Topics: Animals; Endocytosis; Exocytosis; Humans; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor); Proto-Oncogene Proteins c-akt; Signal Transduction
PubMed: 27760304
DOI: 10.1016/j.lfs.2016.10.018 -
ACS Synthetic Biology Nov 2022Vitamin C (l-ascorbate) deficiency is a global public health issue most prevalent in resource-limited regions, creating a need for an inexpensive detection platform....
Vitamin C (l-ascorbate) deficiency is a global public health issue most prevalent in resource-limited regions, creating a need for an inexpensive detection platform. Here, we describe efforts to engineer whole-cell and cell-free ascorbate biosensors. Both sensors used the protein UlaR, which binds to a metabolite of ascorbate and regulates transcription. The whole-cell sensor could detect lower, physiologically relevant concentrations of ascorbate, which we attributed to intact functionality of a phosphotransferase system (PTS) that transports ascorbate across the cell membrane and phosphorylates it to form UlaR's ligand. We used multiple strategies to enhance cell-free PTS functionality (which has received little previous attention), improving the cell-free sensor's performance, but the whole-cell sensor remained more sensitive. These efforts demonstrated an advantage of whole-cell sensors for detection of molecules─like ascorbate─transformed by a PTS, but also proof of principle for cell-free sensors requiring membrane-bound components like the PTS. In addition, the cell-free sensor was functional in plasma, setting the stage for future implementation of ascorbate sensors for clinically relevant biofluids in field-deployable formats.
Topics: Ascorbic Acid; Vitamins; Escherichia coli; Phosphotransferases
PubMed: 36300901
DOI: 10.1021/acssynbio.2c00335 -
MSphere Jan 2020Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However,...
Prebiotic oligosaccharides, such as fructooligosaccharides, are increasingly being used to modulate the composition and activity of the gut microbiota. However, carbohydrate utilization analyses and metagenomic studies recently revealed the ability of deleterious and uncultured human gut bacterial species to metabolize these functional foods. Moreover, because of the difficulties of functionally profiling transmembrane proteins, only a few prebiotic transporters have been biochemically characterized to date, while carbohydrate binding and transport are the first and thus crucial steps in their metabolization. Here, we describe the molecular mechanism of a phosphotransferase system, highlighted as a dietary and pathology biomarker in the human gut microbiome. This transporter is encoded by a metagenomic locus that is highly conserved in several human gut , including species. We developed a generic strategy to deeply analyze, and , the specificity and functionality of recombinant transporters in , combining carbohydrate utilization locus and host genome engineering and quantification of the binding, transport, and growth rates with analysis of phosphorylated carbohydrates by mass spectrometry. We demonstrated that the fructooligosaccharide transporter is specific for kestose, whether for binding, transport, or phosphorylation. This constitutes the biochemical proof of effective phosphorylation of glycosides with a degree of polymerization of more than 2, extending the known functional diversity of phosphotransferase systems. Based on these new findings, we revisited the classification of these carbohydrate transporters. Prebiotics are increasingly used as food supplements, especially in infant formulas, to modify the functioning and composition of the microbiota. However, little is currently known about the mechanisms of prebiotic recognition and transport by gut bacteria, while these steps are crucial in their metabolism. In this study, we established a new strategy to profile the specificity of oligosaccharide transporters, combining microbiomics, genetic locus and strain engineering, and state-of-the art metabolomics. We revisited the transporter classification database and proposed a new way to classify these membrane proteins based on their structural and mechanistic similarities. Based on these developments, we identified and characterized, at the molecular level, a fructooligosaccharide transporting phosphotransferase system, which constitutes a biomarker of diet and gut pathology. The deciphering of this prebiotic metabolization mechanism by a nonbeneficial bacterium highlights the controversial use of prebiotics, especially in the context of chronic gut diseases.
Topics: Bacteria; Carbohydrate Metabolism; Escherichia coli; Fermentation; Gastrointestinal Microbiome; Humans; Metabolomics; Oligosaccharides; Phosphotransferases; Prebiotics
PubMed: 31915220
DOI: 10.1128/mSphere.00771-19 -
Cancer Cell Jan 2020Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and...
Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.
Topics: Animals; CRISPR-Cas Systems; Cell Line, Tumor; Cell Proliferation; GTP Phosphohydrolases; Gene Expression Regulation, Leukemic; Humans; Leukemia, Myeloid, Acute; Membrane Proteins; Mice; Monomeric GTP-Binding Proteins; Phosphotransferases; Phosphotransferases (Alcohol Group Acceptor); Polyamines; Pyridoxal Phosphate; RNA, Small Interfering; Vitamin B 6
PubMed: 31935373
DOI: 10.1016/j.ccell.2019.12.002 -
Journal of Bacteriology Apr 2022Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to...
Streptococcus mutans is a major pathobiont involved in the development of dental caries. Its ability to utilize numerous sugars and to effectively respond to environmental stress promotes S. mutans proliferation in oral biofilms. Because of their quick action and low energetic cost, noncoding small RNAs (sRNAs) represent an ideal mode of gene regulation in stress response networks, yet their roles in oral pathogens have remained largely unexplored. We identified 15 novel sRNAs in S. mutans and show that they respond to four stress-inducing conditions commonly encountered by the pathogen in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. To better understand the role of sRNAs in S. mutans, we further explored the function of the novel sRNA SmsR4. Our data demonstrate that SmsR4 regulates the enzyme IIA (EIIA) component of the sorbitol phosphotransferase system, which transports and phosphorylates the sugar alcohol sorbitol. The fine-tuning of EIIA availability by SmsR4 likely promotes S. mutans growth while using sorbitol as the main carbon source. Our work lays a foundation for understanding the role of sRNAs in regulating gene expression in stress response networks in S. mutans and highlights the importance of the underexplored phenomenon of posttranscriptional gene regulation in oral bacteria. Small RNAs (sRNAs) are important gene regulators in bacteria, but the identities and functions of sRNAs in Streptococcus mutans, the principal bacterium involved in the formation of dental caries, are unknown. In this study, we identified 15 putative sRNAs in S. mutans and show that they respond to four common stress-inducing conditions present in human mouth: sugar-phosphate stress, hydrogen peroxide exposure, high temperature, and low pH. We further show that the novel sRNA SmsR4 likely modulates sorbitol transport into the cell by regulating SMU_313 mRNA, which encodes the EIIA subunit of the sorbitol phosphotransferase system. Gaining a better understanding of sRNA-based gene regulation may provide new opportunities to develop specific inhibitors of S. mutans growth, thereby improving oral health.
Topics: Dental Caries; Gene Expression Regulation, Bacterial; Humans; Hydrogen Peroxide; Phosphates; Phosphotransferases; RNA, Small Untranslated; Sorbitol; Streptococcus mutans; Sugars
PubMed: 35285723
DOI: 10.1128/jb.00577-21 -
Nature Communications May 2023Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two primary components of the eukaryotic membrane and play essential roles in the maintenance of membrane...
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are two primary components of the eukaryotic membrane and play essential roles in the maintenance of membrane integrity, lipid droplet biogenesis, autophagosome formation, and lipoprotein formation and secretion. Choline/ethanolamine phosphotransferase 1 (CEPT1) catalyzes the last step of the biosynthesis of PC and PE in the Kennedy pathway by transferring the substituted phosphate group from CDP-choline/ethanolamine to diacylglycerol. Here, we present the cryo-EM structures of human CEPT1 and its complex with CDP-choline at resolutions of 3.7 Å and 3.8 Å, respectively. CEPT1 is a dimer with 10 transmembrane segments (TMs) in each protomer. TMs 1-6 constitute a conserved catalytic domain with an interior hydrophobic chamber accommodating a PC-like density. Structural observations and biochemical characterizations suggest that the hydrophobic chamber coordinates the acyl tails during the catalytic process. The PC-like density disappears in the structure of the complex with CDP-choline, suggesting a potential substrate-triggered product release mechanism.
Topics: Humans; Ethanolamines; Choline; Phosphatidylcholines; Cytidine Diphosphate Choline; Phosphotransferases; Catalysis
PubMed: 37137909
DOI: 10.1038/s41467-023-38290-2 -
Advances in Biological Regulation Jan 2021Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol...
Inositol phosphate encompasses a large multifaceted family of signalling molecules that originate from the combinatorial attachment of phosphate groups to the inositol ring. To date, four distinct inositol kinases have been identified, namely, IPK, ITPK, IPPK (IP5-2K), and PPIP5K. Although, ITPKs have recently been identified in archaea, eukaryotes have taken advantage of these enzymes to create a sophisticated signalling network based on inositol phosphates. However, it remains largely elusive what fundamental biochemical principles control the signalling cascade. Here, we present an evolutionary approach to understand the development of the 'inositol phosphate code' in eukaryotes. Distribution analyses of these four inositol kinase groups throughout the eukaryotic landscape reveal the loss of either ITPK, or of PPIP5K proteins in several species. Surprisingly, the loss of IPPK, an enzyme thought to catalyse the rate limiting step of IP (phytic acid) synthesis, was also recorded. Furthermore, this study highlights a noteworthy difference between animal (metazoan) and plant (archaeplastida) lineages. While metazoan appears to have a substantial amplification of IPK enzymes, archaeplastida genomes show a considerable increase in ITPK members. Differential evolution of IPK and ITPK between plant and animal lineage is likely reflective of converging functional adaptation of these two types of inositol kinases. Since, the IPK family comprises three sub-types IPMK, IP6K, and IP3-3K each with dedicated enzymatic specificity in metazoan, we propose that the amplified ITPK group in plant could be classified in sub-types with distinct enzymology.
Topics: Animals; Eukaryotic Cells; Humans; Inositol Phosphates; Multigene Family; Phosphotransferases; Phylogeny; Plants; Signal Transduction
PubMed: 33422459
DOI: 10.1016/j.jbior.2020.100782 -
PloS One 2020Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the...
Cells respond to changes in environmental conditions by activating signal transduction pathways and gene expression programs. Here we present a dataset to explore the relationship between environmental stresses, kinases, and global gene expression in yeast. We subjected 28 drug-sensitive kinase mutants to 10 environmental conditions in the presence of inhibitor and performed mRNA deep sequencing. With these data, we reconstructed canonical stress pathways and identified examples of crosstalk among pathways. The data also implicated numerous kinases in novel environment-specific roles. However, rather than regulating dedicated sets of target genes, individual kinases tuned the magnitude of induction of the environmental stress response (ESR)-a gene expression signature shared across the set of perturbations-in environment-specific ways. This suggests that the ESR integrates inputs from multiple sensory kinases to modulate gene expression and growth control. As an example, we provide experimental evidence that the high osmolarity glycerol pathway is an upstream negative regulator of protein kinase A, a known inhibitor of the ESR. These results elaborate the central axis of cellular stress response signaling.
Topics: Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Fungal; Mitogen-Activated Protein Kinases; Phosphotransferases; RNA, Messenger; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Signal Transduction; Stress, Physiological; Transcription Factors
PubMed: 32160258
DOI: 10.1371/journal.pone.0230246 -
Chembiochem : a European Journal of... Jan 2021Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its...
Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of exon 1 of the Huntingtin protein (Httex1) play important roles in modulating its cellular properties and functions in health and disease. In particular, phosphorylation of threonine and serine residues (T3, S13, and/or S16) has been shown to inhibit Htt aggregation in vitro and inclusion formation in cellular and animal models of Huntington's disease (HD). In this paper, we describe a new and simple methodology for producing milligram quantities of highly pure wild-type or mutant Httex1 proteins that are site-specifically phosphorylated at T3 or at both S13 and S16. This advance was enabled by 1) the discovery and validation of novel kinases that efficiently phosphorylate Httex1 at S13 and S16 (TBK1), at T3 (GCK) or T3 and S13 (TNIK and HGK), and 2) the development of an efficient methodology for producing recombinant native Httex1 proteins by using a SUMO-fusion expression and purification strategy. As a proof of concept, we demonstrate how this method can be applied to produce Httex1 proteins that are both site-specifically phosphorylated and fluorescently or isotopically labeled. Together, these advances should increase access to these valuable tools and expand the range of methods and experimental approaches that can be used to elucidate the mechanisms by which phosphorylation influences Httex1 or HTT structure, aggregation, interactome, and function(s) in health and disease.
Topics: Exons; Humans; Huntingtin Protein; Mutation; Phosphorylation; Phosphotransferases; Protein Aggregates; Protein Conformation; Recombinant Proteins
PubMed: 32805086
DOI: 10.1002/cbic.202000508