Experimental and Computational Modeling of ERAD Substrate Retrotranslocation

描述（由申请人提供）：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在调节血压中起重要作用，如ENaC中导致Liddle综合征（高血压）和假性醛固酮减少症1型（低血压）的致病突变所证明的。最近的数据表明，ENaC编码基因的多态性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。调节ENaC的主要途径是称为内质网相关降解（ERAD）的过程。在ERAD过程中，错误折叠的底物被分子伴侣识别，被多泛素化，并从ER膜逆转位，由细胞质蛋白酶体降解。ERAD对人类健康的重要性通过发现约70种被ERAD降解的疾病相关蛋白而凸显，其中许多是整合的膜蛋白。然而，对多通道膜蛋白的逆易位知之甚少，因为从能量上讲，将疏水性跨膜（TM）结构域去除到细胞质的水性环境中是不利的。不同的TM域如何影响ERAD的速率/效率？为了解决这个问题，遗传，生物化学和计算的方法将被用来确定TM疏水性的贡献。该提案的总体假设是，逆易位效率将间接地与底物的TM的疏水性相关。该授权的具体目标是：（1）使用酿酒酵母（面包酵母）模型系统，通过体外提取测定来测量几种工程化ERAD底物的提取速率。这些底物仅在其TM的疏水性方面有所不同（2）生成计算模型以计算逆易位所需的自由能，并使用该模型预测酵母中表达的ENaC的提取特性。(3)测试如何抑制逆易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一个很好的模型系统。这些研究将共同推动未来关于如何通过靶向ERAD底物的逆转位来治疗性改变蛋白质水平的研究。 Guerriero博士的职业目标是获得独立调查员的职位。为了实现这一目标，Guerriero博士将获得多学科的职业培训：（1）使用 Michael Grabe博士和Markus Benno博士通过计算机驱动的模拟来预测ENaC提取特性，以及（2）使用电生理学技术将他的研究扩展到非洲爪蟾模型系统中，托马斯克莱曼博士。Guerriero博士未来的研究将调查更复杂的疾病相关ERAD底物的提取过程。

DESCRIPTION (provided by applicant): 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.