Transcriptional regulation of proteinuria

描述（由申请人提供）：肾衰竭是美国和全球发病率和死亡率的主要原因。足细胞疾病的转录调控尚未得到很好的理解。我们的长期目标是明确蛋白尿和肾小球疾病的确切机制，以便在未来开发新的治疗方法。本研究的目的是了解转录因子锌指和同源异型盒3（ZHX 3）在肾小球疾病发病机制中的作用。在特定目的1中，将研究ZHX 3在迁移到细胞核中之前的翻译后切割和磷酸化。然后，我们将研究阻断ZHX 3的核输入或输出是否会导致ZHX 3靶基因的基因表达谱改变。在特定目的2中，将通过真实的时间PCR研究两种ZHX 3蛋白片段对培养细胞中基因表达谱的个体影响。接下来，将评估与其他已知和假定的相互作用蛋白的相互作用。将通过染色质免疫沉淀和定点诱变的组合来研究影响ZHX 3与COL 4A 3启动子结合的特定因素。接下来，将评估ZHX 3和相互作用伴侣对所选基因的启动子活性的影响。在具体目标3中，将详细探索ZHX 3与非核区室中的氨肽酶A（阿帕）之间的间接蛋白质：蛋白质相互作用。在小鼠中用抗APA抗体诱导蛋白尿期间，ZHX 3迁移到足细胞核中发生在明显蛋白尿发作之前很久。与阿帕和ZHX 3相互作用的蛋白质将使用抗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的组合单独鉴定。在培养的GEC中敲低与ZHX 3和阿帕两者缔合的蛋白质，并且将评估这种敲低对抗APA抗体诱导的培养的GEC中的基因表达谱的影响。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系中共转染以再现ZHX 3和阿帕的免疫共沉淀。研究啮齿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。这将导致在未来制定适当的治疗策略。

DESCRIPTION (provided by applicant): 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.