Proteostatic regulation of disease-causing polycystin 2 variants

致病多囊蛋白 2 变体的蛋白抑制调节

• 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
• 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

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控制疾病水平的质量控制机制- 导致肾脏中的蛋白质。我最近的项目集中在多囊蛋白2(PC2)上。PC2扮演着至关重要的角色 但编码PC2的PKD2基因突变可能导致常染色体 显性多囊肾病(ADPKD)。ADPKD的特征是形成大的充满液体的囊肿。 在肾脏中，从而降低肾功能。这种疾病没有治愈的办法。然而，更深一层的 了解突变如何改变PC2蛋白的命运将为新的治疗方法提供洞察力 ADPKD.有趣的是，PC2的大部分存在于内质网(ER)中，但也有一部分蛋白质 通过分泌途径进入质膜。控制蛋白质命运的一条途径 内质网相关降解(ERAD)。在ERAD过程中，错误折叠的蛋白质 由分子伴侣识别，多泛素化，并从内质网膜反向移位 胞质蛋白酶体的降解。ERAD对人类健康的重要性通过以下方面得到强调 发现靶向这一途径的&gt；70疾病相关蛋白，其中许多是通道和 传送者。事实上，我的初步数据第一次表明，ERAD在监管方面发挥着核心作用 选择致病的PC2错义突变体的生物发生。为了阐明这些蛋白质是如何 作为毁灭的目标，我将使用基因、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，在PC2中选择错义突变 通过分子伴侣靶向ERAD途径，目前正在对其进行治疗 由细胞中的“蛋白质质量控制”机制的其他组件开发。为了测试 这个假设，这个建议的具体目的是：(1)建立酵母PC2表达系统，该系统 请允许我选择简便的遗传方法，然后定义如何将PC2错义突变作为ERAD的靶点。 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；以及(2)开发一种 新的酵母筛选，在其中可以发现PC2蛋白周转的新的遗传修饰物，并且在其中 可以进行基因组分析。这个屏幕上的点击将在未来的细胞培养和 啮齿动物模型。该项目将确定导致ADPKD患者发生ADPKD的分子机制 明确的PC2突变组，并从长远来看，发现一系列潜在的治疗方法。