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Transcriptional Regulators (transcriptional + regulator)
Kinds of Transcriptional Regulators Selected AbstractsRunx3 is involved in hair shape determinationDEVELOPMENTAL DYNAMICS, Issue 4 2005Eli Raveh Abstract Transcriptional regulators of the Runx family play critical roles in normal organ development and, when mutated, lead to genetic diseases and cancer. Runx3 functions during cell lineage decisions in thymopoiesis and neurogenesis and mediates transforming growth factor-, signaling in dendritic cells. Here, we study the function of Runx3 in the skin and its appendages, primarily the hair follicle, during mouse development. Runx3 is expressed predominantly in the dermal compartment of the hair follicles as they form and during the hair cycle, as well as in the nail and sweat gland skin appendages. Distinct expression is also detected periodically in isolated cells of the epidermis and in melanocytes, populating the hair bulb. Runx3 -deficient mice display a perturbation of the normal hair coat, which we show to be due to hair type and hair shape changes. Thus, one of the functions of Runx3 in skin may be to regulate the formation of the epithelial derived structural hair by affecting dermal to epidermal interactions. Developmental Dynamics 233:1478,1487, 2005. © 2005 Wiley-Liss, Inc. [source] The Freud-1/CC2D1A family: Transcriptional regulators implicated in mental retardationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Anastasia Rogaeva Abstract The CC2D1A gene family consists of two homologous genes, Freud-1/CC2D1A and Freud-2/CC2D1B, that share conserved domains, including several DM14 domains that are specific to this protein family, a C-terminal helix-loop-helix domain, and a C2 calcium-dependent phospholipid binding domain. Although the function of Freud-2 is unknown, Freud-1 has been shown to function as a transcriptional repressor of the serotonin-1A receptor gene that binds to a novel DNA element (FRE, 5,-repressor element). The DNA binding and repressor activities of Freud-1 are inhibited by calcium-calmodulin-dependent protein kinase. Recently, a deletion in the CC2D1A gene has been linked to nonsyndromic mental retardation. This deletion results in the truncation of the helix-loop-helix DNA binding and the C2 domains, crucial for Freud-1 repressor activity, and hence is predicted to generate an inactive or weakly dominant negative protein. The possible mechanisms by which inactivation of Freud-1 could lead to abnormal cortical development and cognitive impairment and the potential roles of Freud-1 gene targets are discussed. © 2007 Wiley-Liss, Inc. [source] Neural protein Olig2 acts upstream of the transcriptional regulator sim1 to specify diencephalic dopaminergic neuronsDEVELOPMENTAL DYNAMICS, Issue 4 2009Nataliya Borodovsky Abstract Neural factors are expressed in neural progenitors and regulate neurogenesis and gliogenesis. Recent studies suggested that these factors are also involved in determining specific neuronal fates by regulating the expression of their target genes, thereby creating transcriptional codes for neuronal subtype specification. In the present study, we show that in the zebrafish the neural gene Olig2 and the transcriptional regulator Sim1 are co-expressed in a subset of diencephalic progenitors destined towards the dopaminergic (DA) neuronal fate. While sim1 mRNA is also detected in mature DA neurons, the expression of olig2 is extinguished prior to terminal DA differentiation. Loss of function of either Olig2 or Sim1 leads to impaired DA development. Finally, Olig2 regulates the expression of Sim1 and gain of function of Sim1 rescues the deficits in DA differentiation caused by targeted knockdown of Olig2. Our findings demonstrate for the first time that commitment of basal diencephalic DA neurons is regulated by the combined action of the neural protein Olig2 and its downstream neuronal specific effector Sim1. Developmental Dynamics 238:826,834, 2009. © 2009 Wiley-Liss, Inc. [source] Drosophila female sterile (1) homeotic is a multifunctional transcriptional regulator that is modulated by Ras signalingDEVELOPMENTAL DYNAMICS, Issue 3 2008Brian L. Florence Abstract The Drosophila (fs(1)h) gene encodes small (Fs(1)hS) and large (Fs(1)hL) chromatin-binding BET protein transcription factor isoforms. Zygotic mutations cause either lethality or female sterility, whereas maternal mutations cause segmental deletions and thoracic homeotic transformations. Here, we describe novel fs(1)h embryonic phenotypes: homeosis of the head in zygotic mutants and deletion of head and tail regions in maternal mutants, similar to those caused by dominant torso (torD) alleles. tor activates transcription of tailless (tll) and hückebein (hkb) by means of a canonical Ras pathway, through inactivation of Groucho (Gro), Capicua (Cic) and, possibly, Grainy-head (Grh) repressors. Expression of both tailless and hückebein are de-repressed in fs(1)h maternal mutants, as in torD, gro, grh, and cic mutant animals, indicating fs(1)h is also necessary for tll and hkb repression. These data link Ras signaling with modulation of a chromatin-binding transcription factor, Fs(1)h, suggesting a novel mechanism by which Ras can modulate gene expression. Developmental Dynamics 237:554,564, 2008. © 2008 Wiley-Liss, Inc. [source] Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Daniel W. Seufert Abstract Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx -injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development. Developmental Dynamics 232:313,324, 2005. © 2004 Wiley-Liss, Inc. [source] Expression of a non-DNA-binding Ikaros isoform exclusively in B cells leads to autoimmunity but not leukemogenesisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2007Heather Wojcik Abstract Ikaros is a transcriptional regulator whose function is essential for B cell development. It is expressed in the hematopoietic stem cell (HSC) through the mature B cell stage. Using genetically engineered mice in which the endogenous Ikaros gene is disrupted, it has been shown that a lack of Ikaros leads to a block in B cell development and that its severe diminution results in a hyperresponsive B cell compartment. Ikaros expression within the HSC has led to speculation as to whether the role of Ikaros in B cell biology is largely accomplished prior to B cell specification. In addition, widespread expression of Ikaros in hematopoietic cells leads to the possibility that some or all of the observed defects are not B cell autonomous. In this report, we demonstrate that over-expression of a dominant interfering Ikaros isoform exclusively in B cells has profound effects on mature B cell function. We provide evidence that continued high-level expression of Ikaros is essential for homeostasis of peripheral lymphocytes and maintenance of B cell tolerance. We also show that deregulation of Ikaros activity does not rapidly result in B cell leukemogenesis as it does with 100% penetrance within the T cell lineage. [source] Activity-dependent subcellular localization of NAC1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2005Laxman Korutla Abstract The expression of the transcriptional regulator NAC1 is increased in the nucleus accumbens of rats withdrawn from cocaine self-administration, and in vivo studies indicate that the up-regulation is a compensatory mechanism opposing the acute effects of cocaine. Both mammalian two-hybrid assay and punctate localization largely in the nucleus suggest NAC1 is a transcriptional regulator. However, in this report it is shown that in differentiated PC12 and Neuro2A cells, as well as in primary cortical neurons, NAC1 is diffusely expressed not only in the cell nucleus but also in cytoplasm. Blockade of spontaneous electrical activity by tetrodotoxin prevented the diffuse expression of NAC1, and depolarization with high potassium concentrations induced diffuse cellular localization in non-differentiating cells. The use of protein kinase C (PKC) inhibitors and activator, as well as the systematic mutation of potential PKC phosphorylation sites in NAC1, demonstrated that phosphorylation of residue S245 by PKC is a necessary event inducing diffuse NAC1 expression outside of the nucleus. These observations indicate a potential non-transcriptional role for NAC1 in the brain. [source] Identification and characterization of a novel transcriptional regulator, MatR, for malonate metabolism in Rhizobium leguminosarum bv. trifoliiFEBS JOURNAL, Issue 24 2000Hwan Young Lee A novel gene, matR, located upstream of matABC, transcribed in the opposite direction, and encoding a putative regulatory protein by sequence analysis was discovered from Rhizobium leguminosarum bv. trifolii. The matA, matB, and matC genes encode malonyl-CoA decarboxylase, malonyl-CoA synthetase, and a presumed malonate transporter, respectively. Together, these enzymes catalyze the uptake and conversion of malonate to acetyl-CoA. The deduced amino-acid sequence of matR showed sequence similarity with GntR from Bacillus subtilis in the N-terminal region encoding a helix-turn-helix domain. Electrophoretic mobility shift assay indicated that MatR bound to a fragment of DNA corresponding to the mat promoter region. The addition of malonate or methylmalonate increased the association of MatR and DNA fragment. DNase I footprinting assays identified a MatR binding site encompassing 66 nucleotides near the mat promoter. The mat operator region included an inverted repeat (TCTTGTA/TACACGA) centered ,46.5 relative to the transcription start site. Transcriptional assays, using the luciferase gene, revealed that MatR represses transcription from the mat promoter and malonate alleviates MatR-mediated repression effect on the expression of Pmat -luc+ reporter fusion. [source] DNA bending and looping in the transcriptional control of bacteriophage ,29FEMS MICROBIOLOGY REVIEWS, Issue 5 2010Ana Camacho Abstract Recent studies on the regulation of phage ,29 gene expression reveal new ways to accomplish the processes required for the orderly gene expression in prokaryotic systems. These studies revealed a novel DNA-binding domain in the phage main transcriptional regulator and the nature and dynamics of the multimeric DNA,protein complex responsible for the switch from early to late gene expression. This review describes the features of the regulatory mechanism that leads to the simultaneous activation and repression of transcription, and discusses it in the context of the role of the topological modification of the DNA carried out by two phage-encoded proteins working synergistically with the DNA. [source] A new physiological role for Pdr12p in Saccharomyces cerevisiae: export of aromatic and branched-chain organic acids produced in amino acid catabolismFEMS YEAST RESEARCH, Issue 6 2006Lucie A. Hazelwood Abstract Saccharomyces cerevisiae can use a broad range of compounds as sole nitrogen source. Many amino acids, such as leucine, tyrosine, phenylalanine and methionine, are utilized through the Ehrlich pathway. The fusel acids and alcohols produced from this pathway, along with their derived esters, are important contributors to beer and wine flavor. It is unknown how these compounds are exported from the cell. Analysis of nitrogen-source-dependent transcript profiles via microarray analysis of glucose-limited, aerobic chemostat cultures revealed a common upregulation of PDR12 in cultures grown with leucine, methionine or phenylalanine as sole nitrogen source. PDR12 encodes an ABC transporter involved in weak-organic-acid resistance, which has hitherto been studied in the context of resistance to exogenous organic acids. The hypothesis that PDR12 is involved in export of natural products of amino acid catabolism was evaluated by analyzing the phenotype of null mutants in PDR12 or in WAR1, its positive transcriptional regulator. The hypersensitivity of the pdr12, and war1, strains for some of these compounds indicates that Pdr12p is involved in export of the fusel acids, but not the fusel alcohols derived from leucine, isoleucine, valine, phenylalanine and tryptophan. [source] Inhibition of Proinflammatory Cytokine Expression by NF-,B (p65) Antisense Oligonucleotide in Helicobacter pylori -Infected MiceHELICOBACTER, Issue 6 2005Sang Gyun Kim ABSTRACT Background.,Helicobacter pylori induces the expression of proinflammatory cytokines in vitro by activating nuclear factor-,B, a transcriptional regulator. However, it has not been clarified whether H. pylori -induced proinflammatory cytokines are also mediated through nuclear factor-,B in vivo. The aim of this study was to evaluate the role of nuclear factor-,B on the expressions of proinflammatory cytokines in H. pylori -infected mice. Materials and Methods., We evaluated nuclear factor-,B (p65) activation in the H. pylori -infected gastric mucosa of mice by immunofluorescent staining using antip65 polyclonal antibody, and the expressions of proinflammatory cytokines with inhibition of nuclear factor-,B pathway by using phosphorothioate antisense and sense oligonucleotide against the nuclear factor-,B (p65). Results., In the H. pylori -infected gastric mucosa of mice, immunofluorescent staining using antip65 polyclonal antibody showed nuclear factor-,B (p65) activation, which was particularly localized to epithelial cells. Tumor necrosis factor-, and interleukin-1, concentrations in gastric mucosa by enzyme-linked immunosorbent assay (ELISA) were elevated in the infected group versus the uninfected group. Pretreatment with nuclear factor-,B (p65) antisense oligonucleotide inhibited the activation of nuclear factor-,B and the expressions of tumor necrosis factor-, and interleukin-1, in H. pylori -infected gastric mucosa. Sense oligonucleotide did not influence on the expression of proinflammatory cytokines. Conclusions.,H. pylori infection was found to activate the expressions of proinflammatory cytokines via nuclear factor-,B in vivo, and this may play an important role in the initiation of H. pylori- induced gastric inflammation. [source] Integrative nuclear FGFR1 signaling (INFS) as a part of a universal "feed-forward-and-gate" signaling module that controls cell growth and differentiationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2003Michal K. Stachowiak Abstract A novel signaling mechanism is described through which extracellular signals and intracellular signaling pathways regulate proliferation, growth, differentiation, and other functions of cells in the nervous system. Upon cell stimulation, fibroblast growth factor receptor-1 (FGFR1), a typically plasma membrane-associated protein, is released from ER membranes into the cytosol and translocates to the cell nucleus by an importin-,-mediated transport pathway along with its ligand, FGF-2. The nuclear accumulation of FGFR1 is activated by changes in cell contacts and by stimulation of cells with growth factors, neurotransmitters and hormones as well as by a variety of different second messengers and thus was named integrative nuclear FGFR1 signaling (INFS). In the nucleus, FGFR1 localizes specifically within nuclear matrix-attached speckle-domains, which are known to be sites for RNA Pol II-mediated transcription and co-transcriptional pre-mRNA processing. In these domains, nuclear FGFR1 colocalizes with RNA transcription sites, splicing factors, modified histones, phosphorylated RNA Pol II, and signaling kinases. Within the nucleus, FGFR1 serves as a general transcriptional regulator, as indicated by its association with the majority of active nuclear centers of RNA synthesis and processing, by the ability of nuclear FGFR1 to activate structurally distinct genes located on different chromosomes and by its stimulation of multi-gene programs for cell growth and differentiation. We propose that FGFR1 is part of a universal "feed-forward-and-gate" signaling module in which classical signaling cascades initiated by specific membrane receptors transmit signals to sequence specific transcription factors (ssTFs), while INFS elicited by the same stimuli feeds the signal forward to the common coactivator, CREB-binding protein (CBP). Activation of CBP by INFS, along with the activation of ssTFs by classical signaling cascades brings about coordinated responses from structurally different genes located at different genomic loci. © 2003 Wiley-Liss, Inc. [source] Regulation of protein phosphatase 1, activity in hypoxia through increased interaction with NIPP1: Implications for cellular metabolismJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kathrina M. Comerford Eukaryotic cells sense decreased oxygen levels and respond by altering their metabolic strategy to sustain non-respiratory ATP production through glycolysis, and thus promote cell survival in a hypoxic environment. Protein phosphatase 1 (PP1) has been recently implicated in the governance of the rational use of energy when metabolic substrates are abundant and contributes to cellular recovery following metabolic stress. Under conditions of hypoxia, the expression of the gamma isoform of PP1 (PP1,), is diminished, an event we have hypothesized to be involved in the adaptive cellular response to hypoxia. Decreased PP1, activity in hypoxia has a profound impact on the activity of the cAMP response element binding protein (CREB), a major transcriptional regulator of metabolic genes and processes. Here, we demonstrate a further mechanism leading to inhibition of PP1 activity in hypoxia which occurs at least in part through increased association with the nuclear inhibitor of PP1 (NIPP1), an event dependent upon decreased basal cAMP/PKA-dependent signaling. Using a dominant negative NIPP1 construct, we provide evidence that NIPP1 plays a major role in the regulation of both CREB protein expression and CREB-dependent transcription in hypoxia. Furthermore, we demonstrate functional sequellae of such events including altered gene expression and recovery of cellular ATP levels. In summary, we demonstrate that interaction with NIPP1 mediates decreased PP1, activity in hypoxia, an event which may constitute an inherent part of the cellular oxygen-sensing machinery and may play a role in physiologic adaptation to hypoxia. J. Cell. Physiol. 209: 211,218, 2006. © 2006 Wiley-Liss, Inc. [source] Hypoxia suppresses runx2 independent of modeled microgravityJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Christopher Ontiveros Bone loss is a consequence of skeletal unloading as seen in bed rest and space flight. Unloading decreases oxygenation and osteoblast differentiation/function in bone. Previously we demonstrated that simulation of unloading in vitro, by culturing differentiating mouse osteoblasts in a horizontal rotating wall vessel (RWV), results in suppressed expression of runx2, a master transcriptional regulator of osteoblast differentiation. However, the RWV is able to reproduce in a controlled fashion at least two aspects of disuse that are directly linked, model microgravity and hypoxia. Hypoxia in the RWV is indicated by reduced medium oxygen tension and increased expression of GAPDH and VEGF. To uncouple the role of model microgravity from hypoxia in suppressed runx2 expression, we cultured osteoblasts under modeled microgravity (oxygenated, horizontal RWV rotation), hypoxia (vertical RWV rotation), or both conditions (horizontal RWV rotation). The expression, DNA binding activity and promoter activity of runx2, was suppressed under hypoxic but not normoxic modeled microgravity RWV conditions. Consistent with a role for hypoxia in suppression of runx2, direct exposure to hypoxia alone is sufficient to suppress runx2 expression in osteoblasts grown in standard tissue culture plates. Taken together, our findings indicate that hypoxia associated with skeletal unloading could be major suppressor of runx2 expression leading to suppressed osteoblast differentiation and bone formation. © 2004 Wiley-Liss, Inc. [source] Autophagy and amino acid homeostasis are required for chronological longevity in Saccharomyces cerevisiaeAGING CELL, Issue 4 2009Ashley L. Alvers Summary Following cessation of growth, yeast cells remain viable in a nondividing state for a period of time known as the chronological lifespan (CLS). Autophagy is a degradative process responsible for amino acid recycling in response to nitrogen starvation and amino acid limitation. We have investigated the role of autophagy during chronological aging of yeast grown in glucose minimal media containing different supplemental essential and nonessential amino acids. Deletion of ATG1 or ATG7, both of which are required for autophagy, reduced CLS, whereas deletion of ATG11, which is required for selective targeting of cellular components to the vacuole for degradation, did not reduce CLS. The nonessential amino acids isoleucine and valine, and the essential amino acid leucine, extended CLS in autophagy-deficient as well as autophagy-competent yeast. This extension was suppressed by constitutive expression of GCN4, which encodes a transcriptional regulator of general amino acid control (GAAC). Consistent with this, GCN4 expression was reduced by isoleucine and valine. Furthermore, elimination of the leucine requirement extended CLS and prevented the effects of constitutive expression of GCN4. Interestingly, deletion of LEU3, a GAAC target gene encoding a transcriptional regulator of branched side chain amino acid synthesis, dramatically increased CLS in the absence of amino acid supplements. In general, this indicates that activation of GAAC reduces CLS whereas suppression of GAAC extends CLS in minimal medium. These findings demonstrate important roles for autophagy and amino acid homeostasis in determining CLS in yeast. [source] Chemokine IL-8 induction by particulate wear debris in osteoblasts is mediated by NF-,BJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2005Elizabeth A. Fritz Abstract Chemokines, or chemotactic cytokines, are major regulators of the inflammatory response and have been identified as pathogenic factors in the periprosthetic soft tissue. Particulate wear debris induced NF-kB activation, the major transcriptional regulator of IL-8 and MCP-1 pro-inflammatory genes and, indeed, both IL-8 and MCP-1 chemokine gene expressions were upregulated in titanium particulate-stimulated human osteoblasts. Here, we demonstrate that phagocytosed particles activate the IL-8 gene promoter via a NF-kB-mediated mechanism. Transfection of a dominant negative mutant IkB, protein that cannot be serine phosphorylated led to suppression of IL-8 promoter activity. The p65/RelA NF-kB subunit activity was affected in both a time- and titanium particle concentration-dependent fashion. Titanium particles led to increased ERK, JNK, and p38 activation in MG-63 osteoblast cells, and IL-8 protein release was suppressed by specific inhibitors of the ERK and p38 MAPK pathways. Together, our results suggest that wear debris particles induce chemokine expression in osteoblasts via NF-kB-mediated transcriptional activation, which is controlled by the MAPK signal transduction pathway. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Characterization of a Novel RING Finger Gene OsRFP1, which is Induced by Ethylene, Salicylic Acid and Blast Fungus Infection in RiceJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2008Shanyue Zhou Abstract OsRFP1, a C3H2C3 -type zinc finger gene, was isolated through screening a blast-induced rice cDNA library. The full-length cDNA of the OsRFP1 gene is 1393 bp with an open reading frame (ORF) encoding 302 amino acid residues. The deduced amino acid sequence of OsRFP1 contains an N-terminal Pfam:zf-CHY domain and a C-terminal C3H2C3 -type RING signature. OsRFP1 was found localizing in the nucleus based on the fluorescence emitted by OsRFP1-GFP fusion protein expressed in onion epidermal cells. GAL4 DNA-binding vector pBD-containing OsRFP1 could activate expression of the reporter genes of His/Ade/LacZ in yeast strain AH109 indicating that OsRFP1 has the transcriptional activation activity. RNA blot analysis showed that expression of the OsRFP1 gene was significantly induced by ethylene (ET), salicylic acid (SA) and blast fungus infection. Together, these results indicate that the OsRFP1 may function as a transcriptional regulator in ET-dependent signal pathway in plant defense. [source] Burkholderia pseudomallei RpoS regulates OxyR and the katG-dpsA operon under conditions of oxidative stressMICROBIOLOGY AND IMMUNOLOGY, Issue 7 2010Witawat Jangiam ABSTRACT Burkholderia pseudomallei, the causative agent of the potentially fatal tropical disease melioidosis, is known to be highly resistant to oxidative stress although the mechanism of this resistance remains to be fully elucidated. Previous studies have shown that an OxyR is involved in the regulation of oxidative stress via the katG and dpsA genes encoding KatG and DpsA and that the alternative sigma factor, RpoS, plays a critical role in resistance to oxidative stress by regulating katG and katE genes. Here it is shown that RpoS is essential for expression of the oxidative stress regulator OxyR, since a mutant strain lacking RpoS failed to induce oxyR expression both during normal growth and under conditions of oxidative stress. It is further demonstrated that the RpoS acts as a positive transcriptional regulator of oxyR and dpsA expression, while OxyR acts as a negative transcriptional regulator of the katG-dpsA operon via OxyR repressor under normal growth conditions, and as a positive transcriptional regulator via OxyR under conditions of oxidative stress. Therefore both RpoS and OxyR are required to promote expression of both the katG-dpsA operon and dpsA gene. [source] A novel bacterial signalling system with a combination of a Ser/Thr kinase cascade and a His/Asp two-component systemMOLECULAR MICROBIOLOGY, Issue 2 2005Renate Lux Summary Prokaryotes and eukaryotes have long been thought to use very different types of kinases (the His kinases of the ,bacterial' two-component systems versus the ,eukaryotic' Ser/Thr/Tyr kinases) to carry out signal transduction. This paradigm no longer holds true, because both systems are now found together in an increasing number of prokaryotic organisms and ,two-component' His kinase are present in eukaryotes. Pioneering work on bacterial protein serine threonine kinases (PSTKs) has been performed in Myxococcus xanthus, a soil bacterium with a complex life cycle that possesses orthologues of signalling-related kinases ,typical' of both the prokaryotic and the eukaryotic kingdoms. In the work reported in this volume of Molecular Microbiology, Nariya and Inouye describe a PSTK cascade that modulates the biochemical activity of MrpC, a CRP-like transcriptional regulator for essential developmental signalling pathways in M. xanthus whose transcription is under the control of a two-component system. This is the first report of both a functional PSTK cascade in bacteria and the use of both PSTK and two-component systems to control a single complex bacterial signalling event. [source] Roles of CmpR, a LysR family transcriptional regulator, in acclimation of the cyanobacterium Synechococcus sp. strain PCC 7942 to low-CO2 and high-light conditionsMOLECULAR MICROBIOLOGY, Issue 3 2004Yukari Takahashi Summary The cmp operon of Synechococcus sp. strain PCC 7942, encoding a high-affinity bicarbonate transporter, is induced under low CO2 conditions by a LysR family protein CmpR. CmpR was found to be required also for induction of the operon by transfer of the cells from low-light to high-light conditions, indicating involvement of a common mechanism in the high-light- and low-CO2 -responsive regulation. Expression of the high-light inducible genes psbAII and psbAIII, on the other hand, was found to be induced also by low-CO2 conditions. A single promoter was responsible for the high-light and low-CO2 induction of each of psbAII and psbAIII, suggesting involvement of a common regulatory mechanism in the light and CO2 responses of the psbA genes. CmpR was, however, not required for the induction of psbAII and psbAIII, indicating the presence of multiple mechanisms for induction of genes under high-light and low-CO2 conditions. The CmpR-deficient mutant nevertheless showed lower levels of the psbAII and psbAIII transcripts than the wild-type strain under all the light and CO2 conditions examined. Gel shift assays showed that CmpR binds to the enhancer elements of psbAII and psbAIII, through specific interaction with a sequence signature conforming to the binding motif of similar LysR family proteins. These findings showed that CmpR acts as a trans -acting factor that enhances transcription of the photosystem II genes involved in acclimation to high light, revealing a complex network of gene regulation in the cyanobacterium. [source] Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulenceMOLECULAR MICROBIOLOGY, Issue 2 2002Yaowei Kang Summary As reported previously for Ralstonia solanacearum strain GMI1000, wild-type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching-positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43-amino-acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10-fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10-fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities. [source] Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl-lipid desaturaseMOLECULAR MICROBIOLOGY, Issue 5 2002Larisa E. Cybulski Summary The Des pathway of Bacillus subtilis regulates the expression of the acyl-lipid desaturase, Des, thereby controlling the synthesis of unsaturated fatty acids (UFAs) from saturated phospholipid precursors. Previously, we showed that the master switch for the Des pathway is a two-component regulatory system composed of a membrane-associated kinase, DesK, and a soluble transcriptional regulator, DesR, which stringently controls transcription of the des gene. Activation of this pathway takes place when cells are shifted to low growth temperature. Here, we report on the mechanism by which isoleucine regulates the Des pathway. We found that exogenous isoleucine sources, as well as its ,-keto acid derivative, which is a branched-chain fatty acid precursor, negatively regulate the expression of the des gene at 37°C. The DesK,DesR two-component system mediates this response, as both partners are required to sense and transduce the isoleucine signal at 37°C. Fatty acid profiles strongly indicate that isoleucine affects the signalling state of the DesK sensor protein by dramatically increasing the incorporation of the lower-melting-point anteiso-branched-chain fatty acids into membrane phospholipids. We propose that both a decrease in membrane fluidity at constant temperature and a temperature downshift induce des by the same mechanism. Thus, the Des pathway would provide a novel mechanism to optimize membrane lipid fluidity at a constant temperature. [source] Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factorsMOLECULAR MICROBIOLOGY, Issue 5 2002David L. Hava Summary Streptococcus pneumoniae (the pneumococcus) is carried in the nasopharynx of healthy individuals, but can spread to other host sites and lead to pneumonia, bacteraemia, otitis media and meningitis. Although it is logical to think a priori that differential gene expression would contribute to the ability of this patho-gen to colonize different sites, in fact very few genes have been demonstrated to play tissue specific roles in virulence or carriage. Using signature-tagged mutagenesis to screen 6149 mariner -transposon insertion strains, we identified 387 mutants attenuated for infection in a murine model of pneumonia. Among these mutants are ones with disruptions in a number of putative tissue-specific transcriptional regulators, surface proteins, metabolic proteins and proteins of unknown function, most of which had not previously been associated with virulence. A subset of these, including most of those with insertions in putative transcriptional regulators, was examined for phenotypes in murine models of bacteraemia and nasopharyngeal carriage. Four classes of mutants defective in infection models of the: (I) lung, (II) lung and blood, (III) lung and nasopharynx, and (IV) all three tissues were identified, thus demonstrating the ex-istence of tissue-specific pneumococcal virulence factors. Included in these strains were two with disruptions in a genetic locus that putatively codes for a transcriptional regulator, three surface proteins and three sortase homologues. Mutation analysis revealed that three of the seven genes in this locus are virulence factors that are specific to mucosal surfaces. [source] The oligomerization of CynR in Escherichia coliPROTEIN SCIENCE, Issue 11 2009Gwendowlyn S. Knapp Abstract Deletion analysis and alanine-scanning based on a homology-based interaction model were used to identify determinants of oligomerization in the transcriptional regulator CynR, a member of the LysR-type transcriptional regulator (LTTR) family. Deletion analysis confirmed that the putative regulatory domain of CynR was essential for driving the oligomerization of , repressor-CynR fusion proteins. The interaction surface of a different LTTR and OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface, which were targeted for alanine-scanning mutagenesis. Oligomerization was assayed by the ability of mutant , repressor-CynR fusions to assemble in E. coli revealing interesting similarities and differences between OxyR and CynR. [source] Crystal structure of Mycobacterium tuberculosis LrpA, a leucine-responsive global regulator associated with starvation responsePROTEIN SCIENCE, Issue 1 2008Manchi C.M. Reddy Abstract The bacterial leucine-responsive regulatory protein (Lrp) is a global transcriptional regulator that controls the expression of many genes during starvation and the transition to stationary phase. The Mycobacterium tuberculosis gene Rv3291c encodes a 150-amino acid protein (designated here as Mtb LrpA) with homology with Escherichia coli Lrp. The crystal structure of the native form of Mtb LrpA was solved at 2.1 Å. The Mtb LrpA structure shows an N-terminal DNA-binding domain with a helix-turn-helix (HTH) motif, and a C-terminal regulatory domain. In comparison to the complex of E. coli AsnC with asparagine, the effector-binding pocket (including loop 100,106) in LrpA appears to be largely preserved, with hydrophobic substitutions consistent with its specificity for leucine. The effector-binding pocket is formed at the interface between adjacent dimers, with an opening to the core of the octamer as in AsnC, and an additional substrate-access channel opening to the outer surface of the octamer. Using electrophoretic mobility shift assays, purified Mtb LrpA protein was shown to form a protein,DNA complex with the lat promoter, demonstrating that the lat operon is a direct target of LrpA. Using computational analysis, a putative motif is identified in this region that is also present upstream of other operons differentially regulated under starvation. This study provides insights into the potential role of LrpA as a global regulator in the transition of M. tuberculosis to a persistent state. [source] The solution structure of the methylated form of the N-terminal 16-kDa domain of Escherichia coli Ada proteinPROTEIN SCIENCE, Issue 3 2006Hiroto Takinowaki N-Ada16k, the N-terminal 16-kDa domain of the Ada protein; meC38 N-Ada16k, the Cys38 methylated form of N-Ada16k; MTase, methyltransferase; HTH, helix-turn-helix; NMR, nuclear magnetic resonance; MALDI-TOF MS, matrix assisted laser desorption/ionization time of flight mass spectrometry; MNU, methylnitrosourea Abstract The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 103 -fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution structure of the methylated form of N-Ada16k by using NMR and mass spectrometry. The results of a 13C-filtered 1H- 13C HMBC experiment and MALDI-TOF MS and MS/MS experiments clearly showed that the methyl acceptor residue is Cys38. The solution structure revealed that it has two distinct subdomains connected by a flexible linker loop: the methyltransferase (MTase) subdomain with the zinc,thiolate center, and the helical subdomain with a helix-turn-helix motif. Interestingly, there is no potential hydrogen bond donor around Cys38, whereas the other three cysteine residues coordinated to a zinc ion have potential donors. Hence, Cys38 could retain its inherent nucleophilicity and react with a methyl phosphotriester. Furthermore, the structure comparison shows that there is no indication of a remarkable conformational change occurring upon the methylation. This implies that the electrostatic repulsion between the negatively charged DNA and the zinc,thiolate center may avoid the contact between the MTase subdomain and the DNA in the nonmethylated form. Thus, after the Cys38 methylation, the MTase subdomain can bind the cognate DNA because the negative charge of the zinc,thiolate center is reduced. [source] On the use of DXMS to produce more crystallizable proteins: Structures of the T. maritima proteins TM0160 and TM1171PROTEIN SCIENCE, Issue 12 2004Glen Spraggon DXMS, deuterium exchange mass spectroscopy Abstract The structure of two Thermotoga maritima proteins, a conserved hypothetical protein (TM0160) and a transcriptional regulator (TM1171), have now been determined at 1.9 Å and 2.3 Å resolution, respectively, as part of a large-scale structural genomics project. Our first efforts to crystallize full-length versions of these targets were unsuccessful. However, analysis of the recombinant purified proteins using the technique of enhanced amide hydrogen/deuterium exchange mass spectroscopy (DXMS) revealed substantial regions of rapid amide deuterium hydrogen exchange, consistent with flexible regions of the structures. Based on these exchange data, truncations were designed to selectively remove the disordered C-terminal regions, and the resulting daughter proteins showed greatly enhanced crystallizability. Comparative DXMS analysis of full-length protein versus truncated forms demonstrated complete and exact preservation of the exchange rate profiles in the retained sequence, indicative of conservation of the native folded structure. This study presents the first structures produced with the aid of the DXMS method for salvaging intractable crystallization targets. The structure of TM0160 represents a new fold and highlights the use of this approach where any prior structural knowledge is absent. The structure of TM1171 represents an example where the lack of a substrate/cofactor may impair crystallization. The details of both structures are presented and discussed. [source] Impact of the transcriptional regulator, Ace2, on the Candida glabrata secretomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2010David A. Stead Abstract Candida glabrata is a major fungal pathogen of humans, and the virulence of C. glabrata is increased by inactivation of the transcription factor, Ace2. Our previous examination of the effects of Ace2 inactivation upon the intracellular proteome suggested that the hypervirulence of C. glabrata ace2 mutants might be caused by differences in the secretome. Therefore in this study we have characterised the C. glabrata secretome and examined the effects of Ace2 inactivation upon this extracellular proteome. We have identified 31 distinct proteins in the secretome of wild-type C. glabrata cells by MS/MS of proteins that were precipitated from the growth medium and enriched by affinity chromatography on concanavalin A. Most of these proteins are predicted to be cell wall proteins, cell wall modifying enzymes and aspartyl proteinases. The endochitinase Cts1 and the endoglucanase Egt2 were not detected in the C. glabrata secretome following Ace2 inactivation. This can account for the cell separation defect of C. glabrata ace2 cells. Ace2 inactivation also resulted in the detection of new proteins in the C. glabrata secretome. The release of such proteins might contribute to the hypervirulence of ace2 cells. [source] Identification of a novel bursicon-regulated transcriptional regulator, md13379, in the house fly Musca domestica,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Shiheng An Abstract Bursicon is a neuropeptide that regulates cuticle sclerotization (hardening and tanning) and wing expansion in insects via a G-protein coupled receptor. The hormone consists of , and , subunits. In the present study, we cloned bursicon , and , genes in the house fly Musca domestica using 3' and 5' RACE and expressed the recombinant bursicon (rbursicon) heterodimer in mammalian 293 cells and insect HighfiveTM cells. The rbursicon displayed a strong bursicon activity in the neck-ligated house fly assay. Using rbursicon, we identified and cloned a novel bursicon-regulated gene in M. domestica encoding a transcriptional regulator homologous to ataxin-7-like3 in human, CG13379 in Drosophila and sgf11 in yeast Saccharomyces cerevisiae. We named the gene md13379. Both ataxin-7-like3 and sgf11 are a novel subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex that is involved in regulation of gene transcription. Real-time PCR analysis of temporal response profile revealed that the level of md13379 transcript was up-regulated by 6.6 fold 1,h after rbursicon injection, which correlates well with the cuticle sclerotization process observed in the rbursicon-injected flies. The composite data suggest that md13379 plays a role in regulating the expression of bursicon-regulated genes involved in the cuticle sclerotization process. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley-Liss, Inc. [source] Structure of the transcription regulator CcpA from Lactococcus lactisACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2007Bernhard Loll Catabolite control protein A (CcpA) functions as master transcriptional regulator of carbon catabolism in Firmicutes. It belongs to the family of bacterial repressor/regulator proteins. Here, the crystal structure of the 76,kDa homodimeric CcpA protein from Lactococcus lactis subsp. lactis IL1403 is presented at 1.9,Å resolution in the absence of cognate DNA. The phases were derived by molecular replacement and the structure was refined to crystallographic R and Rfree factors of 0.177 and 0.211, respectively. The presence of a sulfate molecule in the direct vicinity of a putative effector-binding site in the monomer allowed the derivation of a model for the possible binding of small organic effector molecules. [source] | |||||||||||||||||||||||||||||||||||||