CssRS is a two-component system that plays a pivotal role in mediating the secretion stress response in Bacillus subtilis. This system upregulates the synthesis of membrane-bound HtrA family proteases that cope with misfolded proteins that accumulate within the cell envelope as a result of overexpression or heat shock. Recent studies have shown the induction of CssRS-regulated genes in response to cell envelope stress. We investigated the induction of the CssRS-regulated htrA promoter in the presence of different cell wall- and membrane-active substances and observed induction of the CssRS-controlled genes by glycopeptides (vancomycin and teicoplanin), polymyxins B and E, certain β-lactams, and detergents. Teicoplanin was shown to elicit remarkably stronger induction than vancomycin and polymyxin B. Teicoplanin and polymyxin B induced the spxO gene expression in a CssRS-dependent fashion, resulting in increased activity of Spx, a master regulator of disulfide stress in Bacillus subtilis. The CssRS signaling pathway and Spx activity were demonstrated to be involved in Bacillus subtilis resistance to teicoplanin and polymyxin B.
- MeSH
- Anti-Bacterial Agents * pharmacology MeSH
- Bacillus subtilis * genetics drug effects metabolism MeSH
- Bacterial Proteins * genetics metabolism MeSH
- Polymyxin B * pharmacology MeSH
- Promoter Regions, Genetic MeSH
- Gene Expression Regulation, Bacterial * drug effects MeSH
- Signal Transduction MeSH
- Teicoplanin * pharmacology MeSH
- Publication type
- Journal Article MeSH
The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.
- MeSH
- Bacterial Proteins chemistry metabolism MeSH
- Phosphorylation MeSH
- Heme chemistry MeSH
- Histidine Kinase chemistry metabolism MeSH
- Mass Spectrometry MeSH
- Crystallography, X-Ray MeSH
- Protein Structure, Quaternary MeSH
- Oxygen metabolism MeSH
- Models, Molecular MeSH
- Myxococcales metabolism MeSH
- Oxidation-Reduction MeSH
- Protein Domains MeSH
- Signal Transduction MeSH
- Deuterium Exchange Measurement MeSH
- Ferric Compounds chemistry MeSH
- Ferrous Compounds chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.
- MeSH
- Aeromonas salmonicida genetics metabolism MeSH
- Bacterial Proteins chemistry genetics metabolism MeSH
- Escherichia coli genetics metabolism MeSH
- Phosphorylation MeSH
- Heme chemistry metabolism MeSH
- Histidine chemistry metabolism MeSH
- Histidine Kinase chemistry genetics metabolism MeSH
- Cloning, Molecular MeSH
- Aspartic Acid chemistry metabolism MeSH
- Oxygen chemistry metabolism MeSH
- Myxococcales chemistry enzymology MeSH
- Protein Domains MeSH
- Recombinant Proteins chemistry genetics metabolism MeSH
- Protein Structure, Secondary MeSH
- Signal Transduction * MeSH
- Structural Homology, Protein MeSH
- Deuterium Exchange Measurement MeSH
- Iron chemistry metabolism MeSH
- Publication type
- Journal Article MeSH
Bordetella pertussis is the causative agent of human whooping cough, a highly contagious respiratory disease which despite vaccination programs remains the major cause of infant morbidity and mortality. The requirement of the RNA chaperone Hfq for virulence of B. pertussis suggested that Hfq-dependent small regulatory RNAs are involved in the modulation of gene expression. High-throughput RNA sequencing revealed hundreds of putative noncoding RNAs including the RgtA sRNA. Abundance of RgtA is strongly decreased in the absence of the Hfq protein and its expression is modulated by the activities of the two-component regulatory system BvgAS and another response regulator RisA. Whereas RgtA levels were elevated under modulatory conditions or in the absence of bvg genes, deletion of the risA gene completely abolished RgtA expression. Profiling of the ΔrgtA mutant in the ΔbvgA genetic background identified the BP3831 gene encoding a periplasmic amino acid-binding protein of an ABC transporter as a possible target gene. The results of site-directed mutagenesis and in silico analysis indicate that RgtA base-pairs with the region upstream of the start codon of the BP3831 mRNA and thereby weakens the BP3831 protein production. Furthermore, our data suggest that the function of the BP3831 protein is related to transport of glutamate, an important metabolite in the B. pertussis physiology. We propose that the BvgAS/RisA interplay regulates the expression of RgtA which upon infection, when glutamate might be scarce, attenuates translation of the glutamate transporter and thereby assists in adaptation of the pathogen to other sources of energy.
- MeSH
- Bacterial Proteins genetics metabolism MeSH
- Bordetella pertussis genetics metabolism MeSH
- Glutamates metabolism MeSH
- Humans MeSH
- RNA, Small Untranslated genetics MeSH
- Gene Expression Regulation, Bacterial MeSH
- Signal Transduction * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Our understanding of the origin of animals has been transformed by characterizing their most closely related, unicellular sisters: the choanoflagellates, filastereans, and ichthyosporeans. Together with animals, these lineages make up the Holozoa [1, 2]. Many traits previously considered "animal specific" were subsequently found in other holozoans [3, 4], showing that they evolved before animals, although exactly when is currently uncertain because several key relationships remain unresolved [2, 5]. Here we report the morphology and transcriptome sequencing from three novel unicellular holozoans: Pigoraptor vietnamica and Pigoraptor chileana, which are related to filastereans, and Syssomonas multiformis, which forms a new lineage with Corallochytrium in phylogenomic analyses. All three species are predatory flagellates that feed on large eukaryotic prey, and all three also appear to exhibit complex life histories with several distinct stages, including multicellular clusters. Examination of genes associated with multicellularity in animals showed that the new filastereans contain a cell-adhesion gene repertoire similar to those of other species in this group. Syssomonas multiformis possessed a smaller complement overall but does encode genes absent from the earlier-branching ichthyosporeans. Analysis of the T-box transcription factor domain showed expansion of T-box transcription factors based on combination with a non-T-box domain (a receiver domain), which has not been described outside of vertebrates. This domain and other domains we identified in all unicellular holozoans are part of the two-component signaling system that has been lost in animals, suggesting the continued use of this system in the closest relatives of animals and emphasizing the importance of studying loss of function as well as gain in major evolutionary transitions.
- MeSH
- Biological Evolution * MeSH
- Eukaryota classification genetics physiology MeSH
- Fetal Proteins genetics metabolism MeSH
- Evolution, Molecular MeSH
- Predatory Behavior * MeSH
- T-Box Domain Proteins genetics metabolism MeSH
- RNA, Ribosomal, 18S genetics MeSH
- Signal Transduction * MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.
- MeSH
- Amyloid beta-Protein Precursor genetics metabolism MeSH
- Cell Membrane metabolism MeSH
- Endocytosis MeSH
- Endosomes chemistry metabolism MeSH
- HEK293 Cells MeSH
- Cadherins genetics metabolism MeSH
- Humans MeSH
- Membrane Proteins genetics metabolism MeSH
- Brain Chemistry MeSH
- Brain metabolism MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neurons cytology metabolism MeSH
- Presenilins genetics metabolism MeSH
- ADAM10 Protein genetics metabolism MeSH
- Nerve Tissue Proteins genetics metabolism MeSH
- Receptors, Notch genetics metabolism MeSH
- Gene Expression Regulation MeSH
- Amyloid Precursor Protein Secretases genetics metabolism MeSH
- Signal Transduction MeSH
- Two-Hybrid System Techniques MeSH
- Tetraspanins genetics metabolism MeSH
- Protein Transport MeSH
- Protein Binding MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Cytokinins (CKs) and ethylene (ET) are among the most ancient organic chemicals on Earth. A wide range of organisms including plants, algae, fungi, amoebae, and bacteria use these substances as signaling molecules to regulate cellular processes. Because of their ancestral origin and ubiquitous occurrence, CKs and ET are also considered to be ideal molecules for inter-kingdom communication. Their signal transduction pathways were first historically deciphered in plants and are related to the two-component systems, using histidine kinases as primary sensors. Paradoxically, although CKs and ET serve as signaling molecules in different kingdoms, it has been supposed for a long time that the canonical CK and ET signaling pathways are restricted to terrestrial plants. These considerations have now been called into question following the identification over recent years of genes encoding CK and ET receptor homologs in many other lineages within the tree of life. These advances shed new light on the dissemination and evolution of these hormones as both intra- and inter-specific communication molecules in prokaryotic and eukaryotic organisms.
- MeSH
- Cytokinins metabolism MeSH
- Ethylenes metabolism MeSH
- Eukaryota metabolism MeSH
- Humans MeSH
- Prokaryotic Cells metabolism MeSH
- Signal Transduction physiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
We propose a new framework for rigorous robustness analysis of stochastic biochemical systems that is based on probabilistic model checking techniques. We adapt the general definition of robustness introduced by Kitano to the class of stochastic systems modelled as continuous time Markov Chains in order to extensively analyse and compare robustness of biological models with uncertain parameters. The framework utilises novel computational methods that enable to effectively evaluate the robustness of models with respect to quantitative temporal properties and parameters such as reaction rate constants and initial conditions. We have applied the framework to gene regulation as an example of a central biological mechanism where intrinsic and extrinsic stochasticity plays crucial role due to low numbers of DNA and RNA molecules. Using our methods we have obtained a comprehensive and precise analysis of stochastic dynamics under parameter uncertainty. Furthermore, we apply our framework to compare several variants of two-component signalling networks from the perspective of robustness with respect to intrinsic noise caused by low populations of signalling components. We have successfully extended previous studies performed on deterministic models (ODE) and showed that stochasticity may significantly affect obtained predictions. Our case studies demonstrate that the framework can provide deeper insight into the role of key parameters in maintaining the system functionality and thus it significantly contributes to formal methods in computational systems biology.
- MeSH
- Models, Biological MeSH
- Cell Cycle genetics MeSH
- Humans MeSH
- Gene Expression Regulation MeSH
- Mammals MeSH
- Signal Transduction MeSH
- Stochastic Processes MeSH
- Systems Biology * MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Multistep phosphorelay (MSP) pathways mediate a wide spectrum of adaptive responses in plants, including hormonal and abiotic stress regulations. Recent genetic evidence suggests both partial redundancy and possible functional cross-talk on the one hand and a certain level of specificity on the other. Here, we discuss recent achievements improving our understanding of possible molecular mechanisms of specificity in MSP. We consider a certain evolutionary conservation of ancestral two-component signalling systems from bacteria in a process of molecular recognition that, as we have recently shown, could be applied also to a certain extent in the case of plant MSP. Furthermore, we discuss possible roles of kinase and phosphatase activities, kinetics of both these enzymatic reactions, and phosphorylation lifetime. We include also recent findings on the expression specificity of individual members of MSP pathways and, finally, based on our recent findings, we speculate about a possible role of magnesium in regulation of MSP pathways in plants. All these mechanisms could significantly influence specificity and signalling output of the MSP pathways.
In pleiotropic diseases, multiple organ systems are affected causing a variety of clinical manifestations. Here, we report a pleiotropic disorder with a unique constellation of neurological, endocrine, exocrine, and haematological findings that is caused by biallelic MADD variants. MADD, the mitogen-activated protein kinase (MAPK) activating death domain protein, regulates various cellular functions, such as vesicle trafficking, activity of the Rab3 and Rab27 small GTPases, tumour necrosis factor-α (TNF-α)-induced signalling and prevention of cell death. Through national collaboration and GeneMatcher, we collected 23 patients with 21 different pathogenic MADD variants identified by next-generation sequencing. We clinically evaluated the series of patients and categorized the phenotypes in two groups. Group 1 consists of 14 patients with severe developmental delay, endo- and exocrine dysfunction, impairment of the sensory and autonomic nervous system, and haematological anomalies. The clinical course during the first years of life can be potentially fatal. The nine patients in Group 2 have a predominant neurological phenotype comprising mild-to-severe developmental delay, hypotonia, speech impairment, and seizures. Analysis of mRNA revealed multiple aberrant MADD transcripts in two patient-derived fibroblast cell lines. Relative quantification of MADD mRNA and protein in fibroblasts of five affected individuals showed a drastic reduction or loss of MADD. We conducted functional tests to determine the impact of the variants on different pathways. Treatment of patient-derived fibroblasts with TNF-α resulted in reduced phosphorylation of the extracellular signal-regulated kinases 1 and 2, enhanced activation of the pro-apoptotic enzymes caspase-3 and -7 and increased apoptosis compared to control cells. We analysed internalization of epidermal growth factor in patient cells and identified a defect in endocytosis of epidermal growth factor. We conclude that MADD deficiency underlies multiple cellular defects that can be attributed to alterations of TNF-α-dependent signalling pathways and defects in vesicular trafficking. Our data highlight the multifaceted role of MADD as a signalling molecule in different organs and reveal its physiological role in regulating the function of the sensory and autonomic nervous system and endo- and exocrine glands.
- MeSH
- Phenotype MeSH
- Humans MeSH
- Mutation MeSH
- Nervous System Diseases genetics MeSH
- Death Domain Receptor Signaling Adaptor Proteins genetics MeSH
- Signal Transduction genetics MeSH
- Protein Transport genetics MeSH
- Guanine Nucleotide Exchange Factors genetics MeSH
- Developmental Disabilities genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
