Experimental and Computational Modeling of ERAD Substrate Retrotranslocation

ERAD 底物逆转位的实验和计算模型

• 批准号: 8677120
• 负责人: Christopher James Guerriero
• 金额: $ 10.57万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8677120
• 关键词: ATP phosphohydrolase; Accounting; Address; Apical; Biochemical; Biological Assay; Biological Models; Blood Pressure; Cardiovascular Diseases; Cells; Chimera organism; Complex; Computer Simulation; Computers; Cytoplasm; Data; Degradation Pathway; Dependence; Diabetes Mellitus; Disease; Electrodes; Endoplasmic Reticulum; Engineering; Environment; Epithelial; Equilibrium; Experimental Models; Free Energy; Future; Genes; Genetic; Genetic Polymorphism; Goals; Grant; Health; Homeostasis; Homologous Gene; Human; Hydrophobicity; Hypertension; Hypotension; In Vitro; Individual; Integral Membrane Protein; Ions; Kidney; Kidney Diseases; Kinetics; Link; Lipid Bilayers; Lipids; Measurement; Measures; Mechanics; Membrane; Membrane Proteins; Modeling; Molecular Chaperones; Monitor; Nephrogenic Diabetes Insipidus; Organism; Pathway interactions; Play; Polycystic Kidney Diseases; Positioning Attribute; Process; Property; Proteins; Pseudohypoaldosteronism; Publishing; Quality Control; Relative (related person); Renal function; Renal tubular acidosis; Renal tubule structure; Reporter; Research; Research Personnel; Role; Saccharomyces cerevisiae; Sodium Channel; Sodium Chloride; Staging; Surface; Syndrome; System; Techniques; Testing; Toxic effect; Training; Transmembrane Domain; Variant; Water; Work; Xenopus; Xenopus oocyte; Yeast Model System; Yeasts; aquaporin-2; aqueous; career; design; disease-causing mutation; epithelial Na+ channel; human disease; in vitro Assay; insight; multicatalytic endopeptidase complex; novel; protein degradation; protein folding; protein function; protein misfolding; public health relevance; salt balance; simulation; ubiquitin ligase; voltage

7. Project Summary/Abstract My long-term focus is to investigate the quality control mechanisms that regulate protein levels, such as for the trimeric epithelial sodium channel (ENaC). In the kidney, ENaC plays an important role in regulating blood pressure as evidenced by disease-causing mutations in ENaC which result in Liddle Syndrome (hypertension) and pseudohypoaldosteronism type 1 (hypotension). Recent data indicate that polymorphisms in the genes encoding ENaC may also predispose individuals to high blood pressure. Therefore, a better understanding of the mechanisms that regulate ENaC levels can provide new insights into a way to alter blood pressure. A major pathway that regulates ENaC is a process known as endoplasmic reticulum-associated degradation (ERAD). During ERAD, misfolded substrates 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 degraded by ERAD, many of which are integral membrane proteins. However, the retrotranslocation of multi-pass membrane proteins is poorly understood, as it is energetically unfavorable to remove hydrophobic transmembrane (TM) domains into the aqueous environment of the cytoplasm. How do different TM domains impact the rate/efficiency of ERAD? To address this question, genetic, biochemical, and computational approaches will be used to determine the contribution of TM hydrophobicity to retrotranslocation. The overall hypothesis of this proposal is that retrotranslocation efficiency will indirectly correlate with the hydrophobicity of a substrate's TM. The specific aims for this grant are to: (1) Measure the rate of extraction for several engineered ERAD substrates with an in vitro extraction assay using the Saccharomyces cerevisiae (Baker's Yeast) model system. These substrates differ only in the hydrophobicity of their TMs (2) Generate a computational model to calculate the free energy required for retrotranslocation and use this model to predict the extraction properties of ENaC expressed in yeast. (3) Test how inhibiting the retrotranslocation process alters ENaC function in Xenopus oocytes, an excellent model system for studying channel function. Together these studies will drive future research on how to therapeutically alter protein levels by targeting the retrotranslocation of ERAD substrates. Dr. Guerriero's career goal is to obtain a position as an independent investigator. To facilitate this goal, Dr. Guerriero will obtain multi-disciplinary career training in: (1) using computer-driven simulations to predict ENaC extraction properties with Drs. Michael Grabe and Markus Deserno, and (2) using electrophysiological techniques to extend his research into the Xenopus model system with Dr. Thomas Kleyman. Dr. Guerriero's future research will investigate the extraction process for more complex disease-relevant ERAD substrates.

7. 项目总结/摘要 我的长期重点是研究调节蛋白质水平的质量控制机制，例如 三聚体上皮钠通道 (ENaC)。在肾脏中，ENaC 在调节血液中发挥着重要作用 ENaC 中的致病突变可导致压力，从而导致 Liddle 综合征（高血压） 和 1 型假性醛固酮减少症（低血压）。最近的数据表明，基因多态性 编码 ENaC 也可能使个体易患高血压。因此，更好地了解 调节 ENaC 水平的机制可以为改变血压的方法提供新的见解。一个 调节 ENaC 的主要途径是一个称为内质网相关降解的过程 （ERAD）。在 ERAD 过程中，错误折叠的底物被分子伴侣识别，多聚泛素化，并且 从 ER 膜逆向转位，被细胞质蛋白酶体降解。的重要性 发现约 70 种被降解的疾病相关蛋白凸显了 ERAD 对人类健康的影响 通过 ERAD，其中许多是完整的膜蛋白。然而，多通道逆转位 人们对膜蛋白知之甚少，因为它在很大程度上不利于去除疏水性 跨膜（TM）结构域进入细胞质的水环境。不同的TM域名有何不同 影响 ERAD 的速率/效率？为了解决这个问题，遗传学、生物化学和计算 方法将用于确定 TM 疏水性对逆向易位的贡献。整体 该提议的假设是逆转录效率将与 底物TM的疏水性。这笔赠款的具体目的是： (1) 衡量提取率 使用酿酒酵母进行体外提取测定，用于多种工程化 ERAD 底物 （面包酵母）模型系统。这些底物的区别仅在于其 TM 的疏水性 (2) 生成 计算模型来计算逆转位所需的自由能并使用该模型进行预测 酵母中表达的 ENaC 的提取特性。 (3) 测试如何抑制逆转录转位过程 改变非洲爪蟾卵母细胞中的 ENaC 功能，这是研究通道功能的优秀模型系统。一起 这些研究将推动未来关于如何通过靶向治疗性改变蛋白质水平的研究 ERAD 底物的逆转位。 Guerriero 博士的职业目标是获得独立调查员的职位。为了促进这一目标，博士。 Guerriero 将获得以下方面的多学科职业培训：(1) 使用计算机驱动的模拟来预测 ENaC Drs 的提取特性。 Michael Grabe 和 Markus Deserno，以及 (2) 使用电生理学 与 Thomas Kleyman 博士一起将他的研究扩展到非洲爪蟾模型系统。格雷罗博士的 未来的研究将研究更复杂的疾病相关 ERAD 底物的提取过程。