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
• antibakteriální látky * farmakologie   MeSH
• Bacillus subtilis * genetika   účinky léků   metabolismus   MeSH
• bakteriální proteiny * genetika   metabolismus   MeSH
• polymyxin B * farmakologie   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií * účinky léků   MeSH
• signální transdukce MeSH
• teikoplanin * farmakologie   MeSH
• Publikační typ
• časopisecké články 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
• bakteriální proteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• hem chemie   MeSH
• histidinkinasa chemie   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• kyslík metabolismus   MeSH
• molekulární modely MeSH
• Myxococcales metabolismus   MeSH
• oxidace-redukce MeSH
• proteinové domény MeSH
• signální transdukce MeSH
• vodík-deuteriová výměna MeSH
• železité sloučeniny chemie   MeSH
• železnaté sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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 genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fosforylace MeSH
• hem chemie   metabolismus   MeSH
• histidin chemie   metabolismus   MeSH
• histidinkinasa chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• kyselina asparagová chemie   metabolismus   MeSH
• kyslík chemie   metabolismus   MeSH
• Myxococcales chemie   enzymologie   MeSH
• proteinové domény MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• signální transdukce * MeSH
• strukturní homologie proteinů MeSH
• vodík-deuteriová výměna MeSH
• železo chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články 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
• bakteriální proteiny genetika   metabolismus   MeSH
• Bordetella pertussis genetika   metabolismus   MeSH
• glutamáty metabolismus   MeSH
• lidé MeSH
• malá nekódující RNA genetika   MeSH
• regulace genové exprese u bakterií MeSH
• signální transdukce * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• biologická evoluce * MeSH
• Eukaryota klasifikace   genetika   fyziologie   MeSH
• fetální proteiny genetika   metabolismus   MeSH
• molekulární evoluce MeSH
• predátorské chování * MeSH
• proteiny T-boxu genetika   metabolismus   MeSH
• RNA ribozomální 18S genetika   MeSH
• signální transdukce * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články 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
• cytokininy metabolismus   MeSH
• ethyleny metabolismus   MeSH
• Eukaryota metabolismus   MeSH
• lidé MeSH
• prokaryotické buňky metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy 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
• biologické modely MeSH
• buněčný cyklus genetika   MeSH
• lidé MeSH
• regulace genové exprese MeSH
• savci MeSH
• signální transdukce MeSH
• stochastické procesy MeSH
• systémová biologie * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• Arabidopsis metabolismus   MeSH
• fosforylace MeSH
• fyziologie buňky MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Hypoxia-inducible factors (HIFs) are transcription factors controlling energy, iron metabolism, erythropoiesis, and development. Dysregulation of these proteins contributes to tumorigenesis and cancer progression. Recent findings revealed the important role of HIFs in the pathogenesis of neuroendocrine tumors, especially pheochromocytoma (PHEO) and paraganglioma (PGL). PHEOs and PGLs are catecholamine-producing tumors arising from sympathetic- or parasympathetic-derived chromaffin tissue. To date, eighteen PHEO/PGL susceptibility genes have been identified. Based on the main signaling pathways, PHEOs/PGLs have been divided into two clusters, pseudohypoxic cluster 1 and cluster 2, rich in kinase receptor signaling and protein translation pathways. Recent data suggest that both clusters are interconnected via the HIF signaling and its role in tumorigenesis is supported by newly described somatic and germline mutations in HIF2A gene in patients with PHEOs/PGLs associated with polycythemia, and in some of them also with somatostatinoma. Moreover, HIFalpha signaling has also been shown to be upregulated in neuroendocrine tumors other than PHEO/PGL. Some of these tumors are components of hereditary tumor syndromes which can be associated with PHEO/PGL, but also in ileal carcinoids or melanoma. HIF signaling appears to be one of the crucial players in tumorigenesis, which could suggest new therapeutic approaches for treatment of neuroendocrine tumors.

• MeSH
• faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus   MeSH
• feochromocytom genetika   metabolismus   patologie   MeSH
• lidé MeSH
• nádory nadledvin genetika   metabolismus   patologie   MeSH
• neuroendokrinní nádory genetika   metabolismus   patologie   MeSH
• prognóza MeSH
• signální transdukce * MeSH
• transkripční faktory bHLH genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Multistep phosphorelay (MSP) signaling mediates responses to a variety of important stimuli in plants. In Arabidopsis MSP, the signal is transferred from sensor histidine kinase (HK) via histidine phosphotransfer proteins (AHP1-AHP5) to nuclear response regulators. In contrast to ancestral two-component signaling in bacteria, protein interactions in plant MSP are supposed to be rather nonspecific. Here, we show that the C-terminal receiver domain of HK CKI1 (CKI1(RD) ) is responsible for the recognition of CKI1 downstream signaling partners, and specifically interacts with AHP2, AHP3 and AHP5 with different affinities. We studied the effects of Mg²⁺, the co-factor necessary for signal transduction via MSP, and phosphorylation-mimicking BeF₃⁻ on CKI1(RD) in solution, and determined the crystal structure of free CKI1(RD) and CKI1(RD) in a complex with Mg²⁺. We found that the structure of CKI1(RD) shares similarities with the only known structure of plant HK, ETR1(RD) , with the main differences being in loop L3. Magnesium binding induces the rearrangement of some residues around the active site of CKI1(RD) , as was determined by both X-ray crystallography and NMR spectroscopy. Collectively, these results provide initial insights into the nature of molecular mechanisms determining the specificity of MSP signaling and MSP catalysis in plants.

• MeSH
• Arabidopsis enzymologie   genetika   fyziologie   MeSH
• fosforylace MeSH
• fosfotransferasy genetika   metabolismus   MeSH
• histidin metabolismus   MeSH
• krystalografie rentgenová MeSH
• mapování interakce mezi proteiny MeSH
• molekulární modely MeSH
• mutace MeSH
• proteinkinasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• proteiny huseníčku chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní fúzní proteiny MeSH
• senzitivita a specificita MeSH
• signální transdukce fyziologie   MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH