Uncovering Protein Interactions and Membrane Phase Preferences that alter the Plasma Membrane trafficking of Peripheral Myelin Protein 22

项目摘要：作为一名独立研究人员，我职业生涯的一个主要目标是了解 膜蛋白在内质网（ER）的拥挤环境中折叠。破坏了这个 这一过程导致许多人类疾病，包括阿尔茨海默氏症、帕金森氏症和腓骨肌萎缩症 （CMTD）。通过更好地了解这一过程，我希望最终确定新的治疗策略， 患有这些疾病的患者。CMTD是周围神经系统（PNS）最常见的神经病变， 每2,500人中就有1人患病超过80%的患有这种疾病的患者在蛋白质编码中有突变， PMP 22基因。PMP 22是一种四聚糖整合膜蛋白，其高度表达于小鼠的质膜（PM）， PNS的髓鞘化雪旺细胞。CMTD患者中PMP 22的不适当基因剂量导致过多或 许旺细胞PM处的PMP 22过少。据信，PM处的PMP 22的量的改变导致髓磷脂 在这些患者中观察到的异常。目前没有治疗CMTD的方法。PMP 22在ER中折叠， 对ER质量控制（QC）系统的监督。该系统既帮助PMP 22折叠，又介导 最终决定是将PMP 22向前运输到PM还是将蛋白质保留在ER中并最终靶向 这是为了退化。本提案的目标是了解调解这一决策过程的因素。先前 结果显示野生型（WT）PMP 22和PMP 22疾病突变体在ER QC中具有不同的蛋白质相互作用 系统导致不同的PM表达。初步数据显示，选择性调节ER中的蛋白质表达， 可以改变PMP 22及其疾病突变体的细胞表面运输。此外，PMP 22与胆固醇相关 在PM处有丰富的膜域。已经显示PNS的另一种膜蛋白P0与 ER中的胆固醇是细胞表面运输所必需的。来源于PMP 22-/-小鼠的雪旺细胞显示 与WT雪旺细胞相比，迁移和粘附能力降低，PM胆固醇含量降低。这 表明PMP 22与富含胆固醇的结构域的结合对于雪旺细胞功能是重要的。这是我 假设PMP 22细胞表面运输可以通过改变其蛋白质相互作用网络来调节， ER和通过改变其与富含胆固醇的膜结构域结合的能力。为了解决这个问题，我 将：（1）揭示ER中调节其细胞表面运输的PMP 22相互作用物组的突变特异性变化 以及（2）检查导致PMP 22与富含胆固醇的膜结构域结合的因素并评估其影响 胆固醇与PMP 22运输的关系。这项提议的结果将揭示新的蛋白质相互作用， 影响PMP 22运输和胆固醇关联的因素。这些信息将为科学家们开辟新的途径 试图为患有CMTD的患者开发治疗方法。

PROJECT SUMMARY: A major goal of my career as an independent researcher is to understand how integral membrane proteins fold in the crowded environment of the endoplasmic reticulum (ER). Disruptions in the fidelity of this process lead to a number of human diseases including Alzheimer’s, Parkinson’s and Charcot-Marie-Tooth Disease (CMTD). By better understanding this process, I hope to eventually identify novel therapeutic strategies for treating patients afflicted with these diseases. CMTD is the most common neuropathy of the peripheral nervous system (PNS) and afflicts 1 in 2,500 individuals. Over 80% of patients afflicted with this disease have a mutation in the protein encoding PMP22 gene. PMP22 is a tetraspan integral membrane protein that is highly expressed at the plasma membrane (PM) of myelinating Schwann cells of the PNS. Improper gene dosage of PMP22 in CMTD patients results in either too much or too little PMP22 at the PM of Schwann cells. The altered amount of PMP22 at the PM is believed to cause the myelin abnormalities observed in these patients. There are currently no treatments for CMTD. PMP22 folds in the ER under the surveillance of the ER quality control (QC) system. This system both helps PMP22 to fold as well as mediates the ultimate decision whether to traffic PMP22 forward towards the PM or to retain the protein in the ER and ultimately target it for degradation. The goals of this proposal are to understand the factors that mediate this decision process. Previous results show that wild type (WT) PMP22 and PMP22 disease mutants have different protein interactions in the ER QC system leading to varying PM expression. Preliminary data shows that selective modulation of protein expression in ER can alter the cell surface trafficking of PMP22 and its disease mutants. Additionally, PMP22 associates with cholesterol rich membrane domains at the PM. It has been shown for another membrane protein of the PNS, P0, that association with cholesterol in the ER is necessary for cell surface trafficking. Schwann cells derived from PMP22 -/- mice show decreased migration and adhesion capabilities and lower PM cholesterol content compared to WT Schwann cells. This indicates that PMP22 association with cholesterol rich domains is important for Schwann cell function. It is my hypothesis that PMP22 cell surface trafficking can be modulated by both changing its protein interaction network in the ER and through altering its ability to associate with cholesterol rich membrane domains. To address this hypothesis, I will: (1) uncover mutation specific changes to the PMP22 interactome in the ER that modulate its cell surface trafficking and (2) examine the factors that cause PMP22 to associate with cholesterol rich membrane domains and assess the effects of cholesterol association on PMP22 trafficking. The results from this proposal will uncover novel protein interactions and factors that affect PMP22 trafficking and cholesterol association. This information will open novel avenues for scientists attempting to develop therapeutics for patients who suffer from CMTD.