RESUMO
Hereditary hearing loss (HHL) is an extremely heterogeneous disorder with autosomal dominant, recessive, and X-linked forms. Here, we described an Italian pedigree affected by HHL but also prostate hyperplasia and increased ratio of the free/total PSA levels, with the unusual and extremely rare Y-linked pattern of inheritance. Using exome sequencing we found a missense variant (r.206A>T leading to p.Asp69Val) in the TBL1Y gene. TBL1Y is homologous of TBL1X, whose partial deletion has described to be involved in X-linked hearing loss. Here, we demonstrate that it has a restricted expression in adult human cochlea and prostate and the variant identified induces a lower protein stability caused by misfolded mutated protein that impairs its cellular function. These findings indicate that TBL1Y could be considered a novel candidate for HHL.
Assuntos
Doenças Genéticas Ligadas ao Cromossomo Y/genética , Perda Auditiva/genética , Hiperplasia Prostática/genética , Transducina/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cóclea/metabolismo , Feminino , Doenças Genéticas Ligadas ao Cromossomo Y/patologia , Perda Auditiva/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Próstata/metabolismo , Hiperplasia Prostática/patologia , Estabilidade Proteica , Síndrome , Transducina/metabolismoRESUMO
The tyrosine kinase receptor vascular endothelial growth factor receptor 2 (VEGFR2) is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetylases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In particular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phosphorylation and intracellular signaling upon prolonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetylation is a critical mechanism that directly affects VEGFR2 function.
Assuntos
Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Proteína p300 Associada a E1A/metabolismo , Células HEK293 , Histona Desacetilases/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Lisina/metabolismo , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Estabilidade Proteica , Estrutura Secundária de Proteína , Sus scrofa , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/químicaRESUMO
The genus Burkholderia includes over 60 species isolated from a wide range of environmental niches and can be tentatively divided into two major species clusters. The first cluster includes pathogens such as Burkholderia glumae, B. pseudomallei, and B. mallei and 17 well-studied species of the Burkholderia cepacia complex. The other recently established cluster comprises at least 29 nonpathogenic species, which in most cases have been found to be associated with plants. It was previously established that Burkholderia kururiensis, a member of the latter cluster, possesses an N-acyl homoserine lactone (AHL) quorum-sensing (QS) system designated "BraI/R," which is found in all species of the plant-associated cluster. In the present study, two other BraI/R-like systems were characterized in B. xenovorans and B. unamae and were designated the BraI/R(XEN) and BraI/R(UNA) systems, respectively. Several phenotypes were analyzed, and it was determined that exopolysaccharide was positively regulated by the BraIR-like system in the species B. kururiensis, B. unamae, and B. xenovorans, highlighting commonality in targets. However, the three BraIR-like systems also revealed differences in targets since biofilm formation and plant colonization were differentially regulated. In addition, a second AHL QS system designated XenI2/R2 and an unpaired LuxR solo protein designated BxeR solo were also identified and characterized in B. xenovorans LB400(T). The two AHL QS systems of B. xenovorans are not transcriptionally regulating each other, whereas BxeR solo negatively regulated xenI2. The XenI2/R2 and BxeR solo proteins are not widespread in the Burkholderia species cluster. In conclusion, the present study represents an extensive analysis of AHL QS in the Burkholderia plant-associated cluster demonstrating both commonalities and differences, probably reflecting environmental adaptations of the various species.
Assuntos
Acil-Butirolactonas , Proteínas de Bactérias , Burkholderia/genética , Regulação Bacteriana da Expressão Gênica , Oryza/microbiologia , Percepção de Quorum , Acil-Butirolactonas/metabolismo , Acil-Butirolactonas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Burkholderia/classificação , Burkholderia/crescimento & desenvolvimento , Burkholderia/metabolismo , Dados de Sequência Molecular , Percepção de Quorum/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.
Assuntos
Nuclease do Micrococo/isolamento & purificação , Nuclease do Micrococo/metabolismo , Interferência de RNA , Processamento Pós-Transcricional do RNA , Complexo de Inativação Induzido por RNA/química , Animais , Sítios de Ligação , Caenorhabditis elegans/enzimologia , Drosophila melanogaster/enzimologia , Substâncias Macromoleculares , Nuclease do Micrococo/química , Estrutura Terciária de Proteína , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
RNA interference (RNAi) is a flexible gene silencing mechanism that responds to double-stranded RNA by suppressing homologous genes. Here, we report the characterization of RNAi effector complexes (RISCs) that contain small interfering RNAs and microRNAs (miRNAs). We identify two putative RNA-binding proteins, the Drosophila homolog of the fragile X mental retardation protein (FMRP), dFXR, and VIG (Vasa intronic gene), through their association with RISC. FMRP, the product of the human fragile X locus, regulates the expression of numerous mRNAs via an unknown mechanism. The possibility that dFXR, and potentially FMRP, use, at least in part, an RNAi-related mechanism for target recognition suggests a potentially important link between RNAi and human disease.