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.

我的长期目标是调查调节疾病水平的质量控制机制 - 在肾脏中引起蛋白质。我最近的项目集中在Polycystin 2（PC2）上。 PC2起着至关重要的作用在调节钙稳态，但编码PC2的PKD2基因中的突变会导致常染色体主要的多囊性肾脏疾病（ADPKD）。 ADPKD的特征是形成大液体囊肿在肾脏中，肾功能降低。无法治愈这种疾病。但是，更深了了解突变如何改变PC2蛋白的命运将为新疗法提供洞察 ADPKD。有趣的是，大多数PC2都在内质网（ER）中发现，但有些蛋白质穿过秘密途径到达质膜。控制蛋白质命运的一种途径在ER中是内质网相关降解（ERAD）。在Erad期间，错误折叠的蛋白质是通过分子伴侣识别，多泛素化并从ER膜转循环细胞质蛋白酶体降解。 Erad对人类健康的重要性由发现针对该途径的> 70种与疾病相关的蛋白质，其中许多是通道和运输商。实际上，我的初步数据首次表明，这首先在调节中发挥了核心作用疾病引起疾病的精选PC2错义突变体的生物发生。阐明这些蛋白质的方式针对破坏的目标，我将使用遗传，生化和物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中的某些错义突变由分子伴侣针对ERAD途径，目前正在治疗开发了细胞中“蛋白质质量控制”机制的其他组件。测试该假设，该提案的具体目的是：（1）建立酵母PC2表达系统，该系统请允许我使用便利的遗传方法，然后定义PC2错义突变体是如何针对ERAD的。接下来将在肾上皮细胞培养系统中确认这一攻击的发现。（2）开发新的酵母屏幕可以鉴定出新型PC2蛋白质周转的新型遗传修饰符，其中整个可以进行基因组分析。该屏幕的命中将来将在细胞培养中评估啮齿动物模型。该项目将确定导致携带A的患者ADPKD的分子机制定义的PC2突变组，从长远来看，揭示了一系列潜在的治疗剂。