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。转录的 足细胞疾病的调控还不是很清楚。我们的长期目标是定义准确的 探讨蛋白尿和肾小球疾病的发病机制，为今后开发新的治疗方法提供新的思路。目标是 这一建议的目的是了解转录因子锌指和同源框3(ZHX3)在 肾小球疾病的发病机制。在具体目标1中，翻译后卵裂和 ZHX3在迁移到核中之前的磷酸化将被研究。然后我们将研究是否 阻断ZHX3核进出口导致ZHX3靶基因表达谱改变 基因。在特定目标2中，两个ZHX3蛋白片段对基因表达的单独影响 培养细胞的图谱将通过实时PCR进行研究。下一步，与其他已知和推定的 相互作用的蛋白质将被评估。影响ZHX3与COL4A3结合的特定因素 启动子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，ZHX3和相互作用伙伴对选定基因启动子活性的影响将是 评估过了。特异靶3中的间接蛋白质：ZHX3与氨基肽酶A的蛋白质相互作用 将对非核舱室中的(APA)进行详细探索。在抗APA诱导蛋白尿过程中 在小鼠的抗体中，ZHX3迁移到足细胞核的时间早于显性 蛋白尿。与APA和ZHX3相互作用的蛋白质将使用组合 抗APA和抗ZHX3免疫沉淀物的LC/MS分析以及正在进行的酵母双杂交研究。 与ZHX3和APA相关的蛋白质将在培养的GEC中被击倒，而这一影响 对抗APA抗体诱导的培养的GEC的基因表达谱的敲除将被评估。 最后，将连接APA和ZHX3的蛋白的表达构建体共转染到非上皮组织中 建立ZHX3与APA免疫共沉淀的细胞系。 研究啮齿类动物肾脏疾病的发生发展和尿蛋白渗漏将有助于我们 更好地了解人类肾脏疾病的过程。这将导致开发适当的 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.