Transcriptional regulation of proteinuria

描述(申请人提供)：肾衰竭是美国和世界范围内发病率和死亡率的主要原因。足细胞疾病的转录调控还不是很清楚。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。这项建议的目的是了解转录因子锌指和同源盒3(ZHX3)在肾小球疾病发病机制中的作用。在具体目标1中，将研究ZHX3在迁移到核之前的翻译后切割和磷酸化。然后，我们将研究阻止ZHX3的核进出口是否会导致ZHX3靶基因的基因表达谱发生变化。在特定的目的2中，将用实时定量聚合酶链式反应研究ZHX3蛋白片段对培养细胞基因表达谱的单独影响。下一步，将评估与其他已知和推定的相互作用蛋白的相互作用。影响ZHX3与COL4A3启动子结合的特定因素将通过染色质免疫沉淀和定点突变相结合的方法进行研究。接下来，我们将评估ZHX3及其相互作用伙伴对所选基因启动子活性的影响。在具体目标3中，将详细探讨ZHX3与非核间氨基肽酶A(APA)之间的间接蛋白质相互作用。在用抗APA抗体诱导小鼠蛋白尿的过程中，ZHX3在显性蛋白尿开始之前很久就迁移到足细胞核中。与APA和ZHX3相互作用的蛋白质将通过结合抗APA和抗ZHX3免疫沉淀物的LC/MS分析和正在进行的酵母双杂交研究来单独鉴定。与ZHX3和APA相关的蛋白质将在培养的GEC中被击倒，这种敲除对抗APA抗体诱导的GEC基因表达谱的影响将被评估。最后，将连接APA和ZHX3的蛋白的表达构建体共转染到非上皮细胞系中，以复制ZHX3和APA的免疫共沉淀。研究啮齿类动物肾脏疾病的发生发展和尿中蛋白质的渗漏将有助于我们更好地了解人类肾脏疾病的发生发展过程。这将导致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.