RESUMO
ß-dystroglycan (ß-DG) assembles with lamins A/C and B1 and emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of ß-DG, we characterized the interaction between ß-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery-Dreifuss muscular dystrophy (EDMD). Using truncated variants of ß-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the ß-DG-emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to ß-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of ß-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. ß-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that ß-DG plays a role as an emerin interacting partner modulating its stability and function.
Assuntos
Distroglicanas/metabolismo , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linfócitos B/metabolismo , Sítios de Ligação , Linhagem Celular , Células Cultivadas , Distroglicanas/química , Distroglicanas/genética , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação ProteicaRESUMO
ß-dystroglycan (ß-DG) is a key component of multiprotein complexes in the plasma membrane and nuclear envelope. In addition, ß-DG undergoes two successive proteolytic cleavages that result in the liberation of its intracellular domain (ICD) into the cytosol and nucleus. However, stimuli-inducing ICD cleavage and the physiological relevance of this proteolytic fragment are largely unknown. In this study we show for the first time that ß-DG ICD is targeted to the nucleolus where it interacts with the nuclear proteins B23 and UBF (central factor of Pol I-mediated rRNA gene transcription) and binds to rDNA promoter regions. Interestingly DG silencing results in reduced B23 and UBF levels and aberrant nucleolar morphology. Furthermore, ß-DG ICD cleavage is induced by different nucleolar stressors, including oxidative stress, acidosis, and UV irradiation, which implies its participation in the response to nucleolar stress. Consistent with this idea, overexpression of ß-DG elicited mislocalization and decreased levels of UBF and suppression of rRNA expression, which in turn provoked altered ribosome profiling and decreased cell growth. Collectively our data reveal that ß-DG ICD acts as negative regulator of rDNA transcription by impeding the transcriptional activity of UBF, as a part of the protective mechanism activated in response to nucleolar stress.
Assuntos
Nucléolo Celular/metabolismo , Distroglicanas/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Ribossômico/biossíntese , Animais , Proliferação de Células/genética , Citoplasma/metabolismo , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Distroglicanas/antagonistas & inibidores , Distroglicanas/genética , Camundongos , Mioblastos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Estresse Oxidativo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Domínios Proteicos/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Transcrição Gênica , Regulação para Cima/genéticaRESUMO
A quantitative study of toxicity levels of the San Pedro River and its main tributaries around the city of Aguascalientes, Mexico was conducted. Our study determined individual CL(50) values for each sampling point at 3 different times of the year corresponding to the main seasons of the year in terms of the hydrological cycle (dry, low rain and high rain season). Those LC(50) values were used to calculate the acute. Toxicity Units (aTU) that allowed us to compare levels of toxicity along the San Pedro River and two of its main tributaries. The sample that showed highest toxicity was IPIVA. This is due to the large quantity of industrial discharges that receives. Its effluent was responsible for the largest contribution of toxicity to the San Pedro River over the three rounds of sampling of this study. Our study classified an important portion of the San Pedro River and two of its main tributaries in toxic, moderately toxic and lightly toxic. No portion of the river studied was free of toxicity, either acute or sublethal. This study demonstrated that in spite of the operation of several water treatment plants along the San Pedro River, for the most part, the water quality of the river is still unacceptable.
