Transcriptional regulation of proteinuria

肾衰竭是美国和世界范围内发病率和死亡率的主要原因。转录 足细胞疾病的调节还没有被很好地理解。我们的长期目标是精确定义 蛋白尿和肾小球疾病的机制，以便在未来开发新的治疗方法。目标 本研究的目的是了解转录因子锌指和同源异型盒3（ZHX 3）在 肾小球疾病的发病机制。在特异性目标1中，翻译后切割和 将研究ZHX 3在迁移到细胞核中之前的磷酸化。然后我们将研究是否 阻断ZHX 3的核输入或输出导致ZHX 3靶基因表达谱的改变 基因.在特异性目的2中，两个ZHX 3蛋白片段对基因表达的个体影响 将通过真实的时间PCR研究培养细胞中的基因谱。 将评估相互作用的蛋白质。影响ZHX 3与COL 4A 3结合的特异性因素 将通过染色质免疫沉淀和定点诱变的组合来研究启动子。 接下来，将研究ZHX 3和相互作用伴侣对所选基因的启动子活性的影响。 评估。在特异性目的3中，ZHX 3和氨肽酶A之间的间接蛋白质：蛋白质相互作用 (APA)将详细探讨非核舱的情况。在用抗APA诱导蛋白尿期间 在小鼠中，ZHX 3迁移到足细胞核中发生在明显的免疫反应发生之前很久。 蛋白尿。与阿帕和ZHX 3相互作用的蛋白质将使用以下组合单独鉴定： 抗APA和抗ZHX 3免疫沉淀物的LC/MS分析和正在进行的酵母双杂交研究。 在培养的GEC中，与ZHX 3和阿帕两者结合的蛋白质将被敲低，并且这种敲低的作用是抑制细胞增殖。 将评估在培养的GEC中抗APA抗体诱导的基因表达谱的敲低。 最后，将连接阿帕和ZHX 3的蛋白质的表达构建体在非上皮细胞中共转染。 细胞系以再现ZHX 3和阿帕的免疫共沉淀。 研究啮齿类动物肾脏疾病的发展和尿液中蛋白质的泄漏将有助于我们 更好地了解人类肾脏疾病的过程。这将导致发展适当的 未来的治疗策略。

Kidney failure is a major cause of morbidity and mortality in the United States and worldwide. Transcriptional regulation of podocyte diseases is not as yet well understood. Our long term goal is to define the precise mechanisms of proteinuria and glomerular disease, so as to develop new treatments in the future. The goal of this proposal is to understand the role of transcriptional factor Zinc Fingers and Homeoboxes 3 (ZHX3) in the pathogenesis of glomerular disease. In Specific Aim 1, the post-translational cleavage and phosphorylation of ZHX3 prior to migration into the nucleus will be studied. We will then study whether blocking nuclear import or export of ZHX3 causes alterations in the gene expression profile of ZHX3 target genes. In Specific Aim 2, the individual effects of both ZHX3 protein fragments on the gene expression profile in cultured cells will be studied by real time PCR.Next, interaction with other known and putative interacting proteins will be assessed. Specific factors that influence the binding of ZHX3 to the COL4A3 promoter will be studied by a combination of chromatin immunoprecipitation and site directed mutagenesis. Next, the influence of ZHX3 and interacting partners on the promoter activity of selected genes will be assessed. In Specific Aim 3, the indirect protein : protein interaction between ZHX3 and aminopeptidase A (APA) in the non-nuclear compartment will be explored in detail. During induction of proteinuria with anti-APA antibodies in mice, migration of ZHX3 into the podocyte nucleus occurs long before the onset of overt proteinuria. Proteins that interact with APA and ZHX3 will be individually identified using a combination of LC/MS analysis of anti-APA and anti-ZHX3 immunoprecipitates and ongoing yeast two-hybrid studies. Proteins that associate with both ZHX3 and APA will be knocked down in cultured GEC, and the effect of this knockdown on the anti-APA antibody induced gene expression profile in cultured GECs will be assessed. Finally, expression constructs of proteins that link APA and ZHX3 will be co-transfected in a non-epithelial cell line to reproduce co-immunoprecipitation of ZHX3 and APA. Studying the development of kidney disease and the leakage of protein in the urine in rodents will help us better understand the process of kidney disease in humans. This will lead to the development of appropriate treatment strategies in the future.