The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress

Characterization of the (pro)reninreceptor signal transduction regarding transcriptome and genome-wide protein-DNA-interactions

Die (Pro-)Reninrezeptor ((P)RR)-Signaltransduktion ist in verschiedene pathophysiologische Prozesse involviert, von kardiorenalen Endorganschäden und diabetischer Retinopathie bis hin zur Tumorgenese. Darüber hinaus konnte unsere Arbeitsgruppe in diesem Kontext den Transkriptionsfaktor PLZF als ein Adapterprotein des (P)RR identifizieren.\medskip\newline Mittels Mikroarray- und ChIP-chip-basierten Experimenten wurden in dieser Arbeit Transkriptionsprofile und Protein-DNA-Interaktionen zur Entschlüsselung von transkriptionellen Signalwegen downstream des (P)RR charakterisiert. Die Transkriptomanalysen erfolgten unter Verwendung von siRNA gegen den (P)RR, durch PLZF-Überexpression sowie durch Einsatz des Translokationsinhibitors Genistein und des spezifischen V-ATPase-Inhibitors Bafilomycin. Dabei wurden sowohl distinkte als auch überlappende Gensignaturen sowie neue Zielgene downstream der unterschiedlichen molekularen Funktionen des (P)RR identifiziert. Die funktionellen Analysen zeigten einen (P)RR-abhängigen Einfluss auf Gene, die bei dem Transport von Proteinen und bei der Signalweitergabe eine Rolle spielen. Des Weiteren bewirkte die Überexpression von PLZF eine veränderte Expression von Genen bezüglich des Zellzyklusarrests. Die Analyse der Transkriptionsprofile aller durchgeführten Behandlungen konnte die Beteiligung in entwicklungsbiologischen Prozessen bestätigen, welche mit den drei bisher publizierten knockout-Modelle des (P)RR übereinstimmen. Weiterhin wurde die Rolle der (P)RR-Signaltransduktionskaskade in Bezug auf kardiovaskuläre Erkrankungen und der Tumorgenese und damit seine Bedeutung als pharmakologisches Ziel bekräftigt. Mittels ChIP-chip-Analysen, basierend auf Polymerase II und PLZF-Antikörpern, konnten neue PLZF-Zielgene wie TUBE1 und SEC31a detektiert, und mittels quantitativer real time-PCR validiert werden. Darüber hinaus wiesen einige der hier identifizierten differenziell regulierten und validierten Transkripte wie TUBE1, FN1 und ID3, auf zellspezifische Genregulationsmechanismen hin. Die Ergebnisse dieser Arbeit stellen eine wichtige Grundlage zur Selektion von pharmakologischen Substanzen bezüglich der Entwicklung von (P)RR-Antagonisten sowie der Identifizierung von Biomarkern dar.(Pro)renin receptor ((P)RR) signalling is involved in a variety of pathophysiologic mechanisms spanning from cardiorenal end-organ damage to diabetic retinopathy and tumorigenesis. The (P)RR putatively mediates these biological effects dependently or independently of its ligands, renin and prorenin. In addition, our group has identified the transcription factor PLZF as an adaptor protein of (P)RR. In this study, a set of microarray and ChIP- chip experiments were performed to dissect transcriptional pathways downstream of the (P)RR using siRNA against (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin. Distinct and overlapping genetic signatures as well as novel target genes of the different molecular functions of the (P)RR were identified. Functional analysis revealed the relevance of the (P)RR in processes such as protein transport and the regulation of genes in signal transduction pathways. The PLZF overexpression influences genes involved in cell cycle arrest. Furthermore, all performed system manipulations were shown to be implicated in developmental processes, which is consistent with the three knockout models of the (P)RR. Additionally, the role of the (P)RR’s signal transduction pathway in cardiovascular diseases and tumorigenesis confirmed its relevance as a drug target. The ChIP-chip-experiments based on polymerase II and PLZF-antibodies revealed new target genes of PLZF, e.g. TUBE1 and SEC31a which were validated by quantitative real time-PCR. Finally, selected identified differentially regulated and validated transcripts such as TUBE1, FN1 and ID3 suggest cell specific mechanisms of gene regulation. The results of these studies provide an important insight for the selection of pharmacological compounds concerning the development of (P)RR antagonists

Distinct Signal Transduction Pathways Downstream of the (P)RR Revealed by Microarray and ChIP-chip Analyses

The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)R?s signal transduction pathways in cardiovascular disease and tumorigenesis

The (pro)renin receptor mediates constitutive PLZF-independent pro-proliferative effects which are inhibited by bafilomycin but not genistein

The (pro)renin receptor [(P)RR] is crucial for cardio-renal pathophysiology. The distinct molecular mechanisms of this receptor are still incompletely understood. The (P)RR is able to interact with different signalling proteins such as promyelocytic leukemia zinc finger protein (PLZF) and Wnt receptors. Moreover, domains of the (P)RR are essential for V-ATPase activity. V-ATPase- and Wnt-mediated effects imply constitutive, i.e., (pro)renin-independent functions of the (P)RR. Regarding ligand-dependent (P)RR signalling, the role of prorenin glycosylation is currently unknown. Therefore, the aim of this study was to analyse the contribution of constitutive (P)RR activity to its cellular effects and the relevance of prorenin glycosylation on its ligand activity. We were able to demonstrate that high glucose induces (P)RR signal transduction whereas deglycosylation of prorenin abolishes its intrinsic activity in neuronal and epithelial cells. By using siRNA against (P)RR or PLZF as well as the PLZF translocation blocker genistein and the specific V-ATPase inhibitor bafilomycin, we were able to dissect three distinct sub-pathways downstream of the (P)RR. The V-ATPase function is ligand-independently associated with strong pro-proliferative effects whereas prorenin causes moderate proliferation in vitro. In contrast, PLZF per se [i.e., in the absence of (pro)renin] does not interfere with cell number

The (pro)renin receptor ((P)RR) can act as a repressor of Wnt signalling

The (pro)renin receptor ((P)RR) and Wnt signalling are both involved in different diseases ranging from cardiac and renal end-organ damage to cancer. (P)RR function involves signalling via the transcription factor promyelocytic leukemia zinc finger protein (PLZF) as well as the furin-mediated generation of vacuolar proton-translocating ATPase (V-ATPase)-associated and soluble (P)RR isoforms. Recently, the (P)RR was described as adaptor protein of Wnt (co)receptors. The aim of this study was to analyse the contribution of these distinct (P)RR functions to Wnt signalling. Using Tcf/Lef reporter gene systems in HEK293T and HepG2 cells and quantification of endogenous axin2 mRNA and protein levels in HEK293T cells we were able to demonstrate that full-length (P)RR acts as a repressor of Wnt signalling in a system preactivated either by Wnt3a stimulation or by constitutively active ?-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of ?-catenin. Furthermore, the V-ATPase complex, but not PLZF translocation or renin enzymatic activity, is necessary for the induction of Tcf/Lef-responsive genes by Wnt3a. Our data indicate interference of (P)RR and Wnt cascades, a fact that has to be considered concerning pathophysiology of cardio-renal and oncological entities as well as in drug development programs targeting (P)RR or Wnt pathways

Distinct Signal Transduction Pathways Downstream of the (P)RR Revealed by Microarray and ChIP-chip Analyses

<div><p>The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RŔs signal transduction pathways in cardiovascular disease and tumorigenesis.</p> </div

Overlap of intervention-specific transcriptional signatures.

<p>Venn diagram displaying the intersections between genes identified as being differentially regulated by microarrays Ma(si(P)RR), Ma(bafi), Ma(PLZF; H) ∩ Ma(PLZF; K) and Ma(geni). Under- and overrepresented transcripts were used as input.</p

Signal transduction of the (P)RR.

<p>Schematic overview of the (P)RR-PLZF signal transduction pathway and the interventions performed (red) in this study. Furin and ADAM are capable of cleaving full-length (P)RR into the soluble (P)RR isoform and a V-ATPase-associated identity.</p

Validation of microarray- and ChIP-chip-derived results by real-time PCR.

<p>Total RNA used for microarray analyses was subjected to real-time PCR quantification. The standardised expression ratio (SER) indicates the expression normalised to 18S rRNA and standardised to control condition (scrambled siRNA or vehicle). A SER of 100% indicates no expression difference versus control. P-values were based on a two-tailed, unpaired t-test. SEM: standard error of the mean with respect to the intervention; n: number of single PCR measurements. Fold changes are given in percent relative to the control condition based on the microarray data. Validated Genes, i.e., those which showed significantly (p<0.05) altered mRNA levels as detected by real-time PCR and which additionally are concordantly regulated in microarray-analyses, are highlighted in bold.</p