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

GET 通路的特征及其在心脏发育中的潜在作用

• 批准号: 8686615
• 负责人: BRITTNEY A MANVILLA
• 金额: $ 5.6万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8686615
• 关键词: ATP phosphohydrolase; Accounting; Address; Anabolism; Apoptosis; Architecture; Cardiac; Cardiac Myocytes; Cell physiology; Chemicals; Classification; Complex; Confocal Microscopy; Congenital Heart Defects; Cysteine; Dependence; Development; Diabetes Mellitus; Disease; Down Syndrome; Electron Spin Resonance Spectroscopy; Embryo; Endoplasmic Reticulum; Environment; Fetal Heart; Fluorescence Microscopy; Fluorescence Spectroscopy; Gene Expression Regulation; Genetic Transcription; Goals; Heart Diseases; Human; In Vitro; Integral Membrane Protein; Kidney; Label; Link; Lipid Bilayers; Liver; Lung; Malignant Neoplasms; Membrane; Membrane Proteins; Methodology; Molecular; Molecular Chaperones; Monitor; Orthologous Gene; Pathway interactions; Patients; Physiological; Play; Production; Protein Binding; Proteins; RNA Interference; Recombinants; Recruitment Activity; Role; Sarcoplasmic Reticulum; Series; Structure; System; Tail; Techniques; Technology; Therapeutic; Translations; Transmembrane Domain; Virus Diseases; Yeasts; biophysical techniques; cardiogenesis; congenital heart disorder; crosslink; emerin; interdisciplinary approach; junctophilin; membrane biogenesis; milligram; mutant; phospholamban; public health relevance; receptor; receptor binding; reconstitution; research study; single-molecule FRET; stoichiometry; tool; trafficking

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.

描述（由申请人提供）：尾锚定（TA）蛋白占所有完整膜蛋白的5%，其特征在于单个羧基跨膜结构域（TMD）和面向细胞溶质的N-末端。TA蛋白在许多细胞途径如膜生物发生、囊泡运输和细胞凋亡中起重要作用。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。与大多数膜蛋白相反，TA蛋白通过最近发现的翻译后插入GET（TA蛋白的引导进入）途径插入内质网（ER）膜中。在GET途径中，与Get 3（ATP酶伴侣）结合的TA蛋白被募集到ER中的Get 1/2受体复合物。该建议的目标是确定TA蛋白插入所需的Get 1/2受体的生理化学计量和动力学。为此，将使用荧光和EPR光谱技术来确定受体复合物的寡聚状态。此外，这些方法还将用于监测Get 1/2综合体在各种环境中的结构动力学。这些实验将显著有助于阐明TA蛋白通过GET插入途径插入的机制。有趣的是，GET途径的治疗相关性得到了最近将CHD 5（先天性心脏病5）鉴定为Get 1的人类直系同源物的进一步支持。CHD 5的功能以前是未知的，它在人类胎儿心脏、肾脏、肺和肝脏中大量表达，并且与唐氏综合征患者先天性心脏病的进展有关。由于CHD 5被分类为GET途径的一个组成部分，并且在肌浆网（SR）中存在许多心脏TA蛋白，因此本提案的第二个目的是使用RNAi技术，细胞技术和共聚焦显微镜来探索CHD 5在心脏TA蛋白插入SR/ER中的作用。总之，这些实验将建立GET途径可能在心脏发育和/或疾病中发挥的潜在作用。