Proteostatic regulation of disease-causing polycystin 2 variants

致病多囊蛋白 2 变体的蛋白抑制调节

基本信息

批准号：
10092157
负责人：
Christopher James Guerriero
金额：
$ 11.74万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10092157
关键词：
Address Affect Alleles Anabolism Animal Model Autosomal Dominant Polycystic Kidney Biochemical Biogenesis Biological Assay CCL13 gene Calcium Cell Culture System Cell Culture Techniques Cell Line Cell membrane Cells Chimeric Proteins Confocal Microscopy Cycloheximide Cyst Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Degradation Pathway Disease Drug Targeting End stage renal failure Endoplasmic Reticulum Epithelial Cells Future Genes Genetic Genetic Diseases Genomics Goals Health Heart Hereditary Disease Homeostasis Homologous Gene Human Integral Membrane Protein Investigation KCNJ1 gene Kidney Kidney Diseases Lead Liquid substance Liver Mammalian Cell Mammals Mediating Membrane Methods Missense Mutation Modeling Molecular Molecular Chaperones Mutation Nature PKD2 gene PKD2 protein Pancreas Pathway interactions Patients Physiological Play Polyubiquitination Process Proteasome Inhibitor Proteins Quality Control Regulation Renal function Ribosomes Rodent Model Role Saccharomyces cerevisiae Severities Site Sucrose Suggestion System Testing Therapeutic Time Travel Triage Ubiquitin Uracil Variant Yeasts auxotrophy base deletion library disease phenotype epithelial Na+ channel experience experimental study genetic approach glycosylation insight misfolded protein multicatalytic endopeptidase complex mutant novel overexpression polycystic kidney disease 1 protein protein degradation protein folding proteostasis renal epithelium therapeutic target therapy development tool

项目摘要

My long-term goal has been to investigate the quality control mechanisms that regulate the levels of disease- causing proteins in the kidney. My most recent project has focused on polycystin 2 (PC2). PC2 plays a vital role in regulating calcium homeostasis, but mutations in the PKD2 gene, which encodes PC2, can lead to autosomal dominant polycystic kidney disease (ADPKD). ADPKD is characterized by the formation of large fluid-filled cysts in the kidney, thereby decreasing kidney function. There is no cure for this disease. However, a deeper understanding of how mutations alter the fate of the PC2 protein will provide insight into novel treatments for ADPKD. Interestingly, the majority of PC2 is found in the endoplasmic reticulum (ER), but some of the protein travels through the secretory pathway to the plasma membrane. One pathway that controls the fate of proteins in the ER is endoplasmic reticulum-associated degradation (ERAD). During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated from the ER membrane for degradation by the cytoplasmic proteasome. The importance of ERAD to human health is highlighted by the discovery of >70 disease-associated proteins that are targeted to this pathway, many of which are channels and transporters. Indeed, my preliminary data suggest—for the first time—that ERAD plays a central role in regulating the biogenesis of select PC2 missense mutants that are disease-causing. To elucidate how these proteins are targeted for destruction, I will use a powerful combination of genetic, biochemical, and physiological methods and will develop new experimental tools. My overall hypothesis is that select missense mutations in PC2 are targeted to the ERAD pathway by molecular chaperones, for which therapeutics are currently being developed, as well as by other components of the “protein quality control” machinery in the cell. To test this hypothesis, the specific aims of this proposal are: (1) To establish a yeast PC2 expression system, which allow me to coopt facile genetic approaches and then define how PC2 missense mutants are targeted for ERAD. Discoveries from this attack will next be confirmed in renal epithelial cell culture systems; and (2) To develop a new yeast screen in which novel genetic modifiers of PC2 protein turnover can be identified and in which a whole genomic analysis can be undertaken. Hits from this screen will be evaluated in the future in cell culture and rodent models. This project will identify the molecular mechanisms that lead to ADPKD in patients who carry a defined group of PC2 mutations and, in the long-term, uncover a range of potential therapeutics.

我的长期目标是研究调节疾病水平的质量控制机制- 我最近的项目重点关注多囊蛋白 2 (PC2)。调节钙稳态，但编码 PC2 的 PKD2 基因突变可导致常染色体显性多囊肾病 (ADPKD) 的特点是形成大的充满液体的囊肿。肾脏，从而降低肾功能然而，这种疾病无法治愈。了解突变如何改变 PC2 蛋白的命运将为寻找新的治疗方法提供见解 ADPKD 表明，大部分 PC2 在内质网 (ER) 中发现，但也有一些蛋白质存在于内质网 (ER) 中。通过分泌途径到达质膜的一种控制蛋白质命运的途径。 ER 中的蛋白是内质网相关降解 (ERAD)。被分子伴侣识别，多聚泛素化，并从 ER 膜逆向转位细胞质蛋白酶体的降解强调了 ERAD 对人类健康的重要性。发现了超过 70 种针对该途径的疾病相关蛋白，其中许多是通道和事实上，我的初步数据首次表明 ERAD 在监管方面发挥着核心作用。特定 PC2 错义突变体的生物发生阐明这些蛋白质是如何致病的。针对破坏，我将使用遗传、生化和生理方法的强大组合并将开发新的实验工具我的总体假设是 PC2 中的选择性错义突变。通过分子伴侣靶向 ERAD 途径，目前正在针对该途径进行治疗以及细胞中“蛋白质质量控制”机制的其他组件进行测试。根据这一假设，本提案的具体目标是：（1）建立酵母PC2表达系统，该系统请允许我选择简单的遗传方法，然后定义 PC2 错义突变体如何针对 ERAD。这次攻击的发现接下来将在肾上皮细胞培养系统中得到证实；(2) 开发一种新的酵母筛选，其中可以鉴定 PC2 蛋白周转的新遗传修饰剂，并且其中整个将来可以在细胞培养和评估中进行基因组分析。该项目将确定携带 ADPKD 的患者发生 ADPKD 的分子机制。确定了一组 PC2 突变，并从长远来看，揭示了一系列潜在的治疗方法。