Experimental and Computational Modeling of ERAD Substrate Retrotranslocation

7.项目摘要/摘要 我的长期目标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在调节血液方面起着重要作用 导致利德尔综合征(高血压)的ENaC致病突变证明了压力 假性低醛固酮增多症1型(低血压)。最近的数据表明，基因的多态 编码ENaC也可能使个人容易患高血压。因此，更好地理解 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的主要途径是内质网相关的降解过程 (Erad)。在ERAD过程中，错误折叠的底物被分子伴侣、多泛素化和 从内质网转位到细胞质蛋白酶体降解。重要的是 Erad对人类健康的突出表现是发现了~70种可降解的疾病相关蛋白 ERAD，其中许多是完整的膜蛋白。然而，多通道的逆转位 膜蛋白知之甚少，因为它在能量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域如何 影响ERAD的速度/效率？为了解决这个问题，遗传、生化和计算 将使用方法来确定TM疏水性对逆转录易位的贡献。整体而言 这一建议的假设是，回溯易位效率将间接与 底物TM的疏水性。这笔赠款的具体目的是：(1)测量萃取率 几种工程ERAD底物的酿酒酵母体外提取试验 (面包酵母)模型系统。这些底物的不同之处仅在于它们的TM(2)产生的疏水性 计算逆转位所需自由能的计算模型，并用该模型预测 酵母表达的ENaC的提取性质。(3)测试如何抑制逆转位过程 改变非洲爪哇卵母细胞的ENaC功能，这是研究通道功能的一个很好的模型系统。同舟共济 这些研究将推动未来关于如何通过靶向改变蛋白质水平的研究 ERAD底物的逆转位。 格雷罗博士的职业目标是获得一份独立调查员的工作。为了促进这一目标，Dr。 Guerriero将在以下方面获得多学科职业培训：(1)使用计算机驱动的模拟来预测ENaC 迈克尔·格拉贝博士和马库斯·沙诺博士的提取性质，以及(2)电生理 与托马斯·克利曼博士一起将他的研究扩展到非洲爪哇模型系统的技术。格列罗医生的 未来的研究将探索更复杂的与疾病相关的ERAD底物的提取过程。

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.