Experimental and Computational Modeling of ERAD Substrate Retrotranslocation

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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.