RESUMO
Genetic factors strongly contribute to the pathogenesis of sporadic Alzheimer's disease (AD). Nevertheless, genome-wide association studies only yielded single nucleotide polymorphism loci of moderate importance. In contrast, microsatellite repeats are functionally less characterized structures within our genomes. Previous work has shown that endothelin-converting enzyme-1 (ECE-1) is able to reduce amyloid ß content. Here we demonstrate that a CpG-CA repeat within the human ECE-1c promoter is highly polymorphic, harbors transcriptional start sites, is able to recruit the transcription factors poly(ADP-ribose) polymerase-1 and splicing factor proline and glutamine-rich, and is functional regarding haplotype-specific promoter activity. Furthermore, genotyping of 403 AD patients and 444 controls for CpG-CA repeat length indicated shifted allelic frequency distributions. Sequencing of 245 haplotype clones demonstrated that the overall CpG-CA repeat composition of AD patients and controls is distinct. Finally, we show that human and chimpanzee [CpG](m)-[CA](n) ECE-1c promoter repeats are genetically and functionally distinct. Our data indicate that a short genomic repeat structure constitutes a novel core promoter element, coincides with human evolution, and contributes to the pathogenesis of AD.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Ácido Aspártico Endopeptidases/genética , Evolução Biológica , Metaloendopeptidases/genética , Repetições de Microssatélites/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Animais , Western Blotting , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Cromatografia em Gel , DNA/genética , DNA/isolamento & purificação , Ensaio de Desvio de Mobilidade Eletroforética , Enzimas Conversoras de Endotelina , Genótipo , Humanos , Ensaios de Proteção de Nucleases , Pan troglodytes , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND/AIMS: Putative in vitro-in vivo correlations of pharmacokinetic (PK) parameters are regarded as a prerequisite to filter hits derived from high-throughput screening (HTS) approaches for subsequent murine in vivo PK studies. METHODS: In this study, we assessed stabilities in rat and human microsomes of 121 compounds from an early, academic drug discovery programme targeting the (pro)renin receptor and correlated the respective data with single-dose, in vivo PK parameters of 22 hits administered intravenously in rats. RESULTS: After transformation of in vitro half-lives to predicted in vivo hepatic clearances, r(2) regarding in vitro-in vivo clearance correlations were 0.31 and 0.27 for the rat and human species, respectively. CONCLUSIONS: Our data concerning structurally diverse real-world compounds indicate that microsomal stability testing is not a tool to triage early compounds for in vivo PK testing.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/metabolismo , Farmacocinética , Alternativas aos Testes com Animais/métodos , Animais , Células Cultivadas , Meia-Vida , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.
Assuntos
Doenças dos Bovinos/microbiologia , Escherichia coli Êntero-Hemorrágica/classificação , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Infecções por Escherichia coli/veterinária , Animais , Técnicas de Tipagem Bacteriana , Bovinos , Análise por Conglomerados , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Fatores de Virulência/genéticaRESUMO
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.
Assuntos
Genisteína/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Macrolídeos/farmacologia , Receptores de Superfície Celular/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Glucose/farmacologia , Glicosilação/efeitos dos fármacos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , Modelos Biológicos , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Regiões Promotoras Genéticas/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Receptores de Superfície Celular/genética , Transdução de Sinais/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
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)RRs signal transduction pathways in cardiovascular disease and tumorigenesis.
Assuntos
Doenças Cardiovasculares/genética , Transformação Celular Neoplásica/genética , Fatores de Transcrição Kruppel-Like/genética , Receptores de Superfície Celular/genética , Transcriptoma , ATPases Vacuolares Próton-Translocadoras/genética , Doenças Cardiovasculares/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/metabolismo , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/metabolismo , Macrolídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteína com Dedos de Zinco da Leucemia Promielocítica , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Receptor de Pró-ReninaRESUMO
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 ß-catenin. These repressive effects are mediated by Dvl but are independent of the mutation status of ß-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.
Assuntos
Receptores de Superfície Celular/fisiologia , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Genisteína/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Macrolídeos/farmacologia , Camundongos , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Pró-ReninaRESUMO
Stroke is one of the major medical burdens in industrialized countries. Animal experiments indicate that blockade of the angiotensin AT1 receptor (AT1R) improves neurological outcome after cerebral ischemia. These protective effects are partially mediated by the angiotensin AT2 receptor (AT2R). The transcription factor promyelocytic leukemia zinc finger (PLZF) was identified as a direct adapter protein of the AT2R. Furthermore, our group was able to demonstrate that PLZF also directly binds and mediates the effects of the human (pro)renin receptor [(P)RR] which is involved in brain development. Therefore, we hypothesized that PLZF is involved in neuroprotection. Here we show that PLZF and its receptors (P)RR and AT2R exhibited an ubiquitous expression pattern in different brain regions. Furthermore, stable PLZF overexpression in human neuronal cells was able to mediate neuroprotection in a glutamate toxicity model in vitro. Consistently, PLZF mRNA and protein were downregulated on the ipsilateral side in a stroke model in vivo, whereas the neurodetrimental PLZF target genes cyclin A2 and BID were upregulated under this condition. Further analyses indicated that the neuroprotective AT2R is upregulated upon stable PLZF overexpression in cultured neuronal cells. Finally, reporter gene assays demonstrated the functionality of (P)RR promoter polymorphisms regarding basal and PLZF-induced activity.
Assuntos
Córtex Cerebral/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Neurônios/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Dedos de Zinco/genética , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/patologia , Ciclina A2/genética , Ciclina A2/metabolismo , Regulação para Baixo/genética , Humanos , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Fatores de Transcrição Kruppel-Like/genética , Imageamento por Ressonância Magnética , Masculino , Neurônios/patologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor de Pró-ReninaRESUMO
The renin-angiotensin system (RAS) plays a crucial role in cardiovascular and neuronal (patho-)physiology. The angiotensin AT2 receptor (AT2R) seems to counteract the proinflammatory, prohypertrophic and profibrotic actions of the AT1 receptor. Recently, we identified a novel protein, termed "AT2R binding protein" (ATBP/ATIP) which seems essential for AT2R-mediated growth inhibition. Poly(ADP-ribose) polymerase-1 (PARP-1) can act as a nuclear integrator of angiotensin II-mediated cell signalling, and has been implicated in the pathogenesis of cardiovascular and neuronal disease. In this study, promoters of human AT2R and ATIP1 were cloned and two transcriptional start sites in the ATIP1 promoter were identified whereas only one was detected in the AT2R promoter. Promoter assays indicated that the exon 1-intron 1 region of AT2R is necessary and sufficient for AT2R promoter activity. Inverse cloning experiments indicated that this regulatory region is a promoter but not an enhancer element implicating (a) further start site(s) in this region. Consistently, the exon 1-intron 1 region of AT2R was shown to tether the basal transcriptional machinery. Overexpression, pharmacological inhibition and ablation of PARP demonstrated that PARP-1 activates the ATIP1 gene but represses the AT2R on promoter and mRNA levels in vitro, and in brain tissue in vivo. Additional experiments indicated that AT2R activation does not modulate PARP-1 transcript levels but increases AT2R promoter activity, thereby creating a positive feedback mechanism. Our results demonstrate that PARP-1 acts as novel node within the RAS network based on its ability to regulate downstream targets such as AT2R and its adapter protein ATBP.