Characterization of the GET Pathway and its Potential Role in Heart Development

描述（由申请人提供）：尾锚定（TA）蛋白占所有完整膜蛋白的5%，其特征是单个羧基跨膜结构域（TMD）和面向细胞质的n端。TA蛋白在许多细胞通路中发挥重要作用，如膜生物发生、囊泡运输和细胞凋亡。此外，它们与许多疾病有关，包括癌症、心脏病、糖尿病以及病毒感染。与大多数膜蛋白不同，TA蛋白通过最近发现的翻译后插入GET（引导进入TA蛋白）途径插入内质网（ER）膜。在GET途径中，与Get3（一种atp酶伴侣）结合的TA蛋白被募集到内质网中的Get1/2受体复合体中。本研究的目的是确定TA蛋白插入所需的Get1/2受体的生理化学计量学和动力学。为此，荧光和EPR光谱技术将用于确定受体复合物的低聚状态。此外，这些方法还将用于监测Get1/2复合物在各种环境中的结构动力学。这些实验将有助于阐明TA蛋白通过GET插入途径插入的机制。有趣的是，最近发现CHD5（先天性心脏病5）是Get1的人类同源基因，这进一步支持了GET通路的治疗相关性。CHD5在人类胎儿的心脏、肾脏、肺和肝脏中大量表达，其功能以前是未知的，并且与唐氏综合征患者先天性心脏病的进展有关。由于CHD5被分类为GET通路的一个组成部分，并且在肌浆网（SR）中存在许多心脏TA蛋白，本提议的第二个目的是利用RNAi技术、细胞技术和共聚焦显微镜探索CHD5在心脏TA蛋白插入SR/ER中的作用。总之，这些实验将确定GET通路在心脏发育和/或疾病中可能发挥的潜在作用。

DESCRIPTION (provided by applicant): Tail-anchored (TA) proteins account for 5% of all integral membrane proteins and are characterized by a single carboxyl transmembrane domain (TMD) and a cytosolic-facing N-terminus. TA proteins play essential roles in numerous cellular pathways such as membrane biogenesis, vesicular trafficking, and apoptosis. Moreover, they are implicated in many diseases including cancer, heart disease, diabetes, as well as viral infections. In contrast to most membrane proteins, TA proteins are inserted into the endoplasmic reticulum (ER) membrane through the recently discovered, post- translational insertion GET (guided entry of TA proteins) pathway. In the GET pathway, TA proteins bound to Get3 (an ATPase chaperone), are recruited to a Get1/2 receptor complex in the ER. The goals of this proposal are to determine the physiological stoichiometry and dynamics of the Get1/2 receptor required for TA protein insertion. To this end, fluorescence and EPR spectroscopy techniques will be used to determine the oligomeric state of the receptor complex. Additionally, these methodologies will also be used to monitor the structural dynamics of the Get1/2 complex in various environments. These experiments will significantly aid in elucidating the mechanism of TA protein insertion via the GET insertion pathway. Interestingly, the therapeutic relevance of the GET pathway is further supported by the recent identification of CHD5 (congenital heart disease 5) as the human ortholog of Get1. CHD5, which function was previously unknown, is abundantly expressed in human fetal heart, kidney, lungs, and liver and has been linked to the progression of congenital heart disease in Down Syndrome patients. Due to the classification of CHD5 as a component of the GET pathway and the presence of numerous cardiac TA proteins in the sarcoplasmic reticulum (SR), the second aim of this proposal is to explore the role of CHD5 in cardiac TA protein insertion into the SR/ER using RNAi technology, cellular techniques, and confocal microscopy. Altogether, these experiments will establish the potential roles that the GET pathway may play in heart development and/or disease.