Characterization of mammalian COG complex-interacting Golgi trafficking machinery

哺乳动物 COG 复杂相互作用的高尔基体运输机制的表征

• 批准号: 10658337
• 负责人: VLADIMIR V LUPASHIN
• 金额: $ 40.95万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10658337
• 关键词: Affect; Affinity Chromatography; Alzheimer' s Disease; Binding; Biochemical; Biotinylation; Categories; Cell Line; Cell Maintenance; Cell model; Cell physiology; Cells; Congenital disorders of glycosylation; Conserved Sequence; Cystic Fibrosis; Defect; Development; Diabetes Mellitus; Disease; Docking; Environment; Enzymes; Fibroblasts; Fluorescence Microscopy; Golgi Apparatus; Hermanski-Pudlak Syndrome; Human; Impairment; In Vitro; Intracellular Membranes; Kinetics; Label; Leg; Lipids; Liver; Malignant Neoplasms; Medial; Mediating; Membrane; Membrane Fusion; Microcephaly; Microscopic; Microscopy; Modeling; Molecular; Mutation; Nature; Neurologic; Neurons; Occupations; Pathway interactions; Patients; Phenotype; Physiological; Population; Post-Translational Protein Processing; Process; Protein Secretion; Protein Sortings; Proteins; Proteomics; Recycling; Regulation; SNAP receptor; Set protein; Signal Transduction; Symptoms; System; Testing; Tissues; Treatment Protocols; Vesicle; autosome; base; bone; flexibility; glycosylation; golgin; human disease; in vivo; induced pluripotent stem cell; novel; polarized cell; protein protein interaction; receptor; skeletal; stem cells; success; therapy development; tissue/cell culture; trafficking; ultra high resolution; vesicle transport

PROJECT SUMMARY/ABSTRACT Intracellular membrane trafficking mediates the intracellular delivery of proteins and lipids. The process is bidirectional and consists of the anterograde (secretory) and retrograde (endocytic) branches. Intracellular membrane trafficking is evolutionary conserved, and its machinery is modular, with functionally homologous components operating on different trafficking steps. Therefore, a detailed understanding of one trafficking step will help in understanding the entire intracellular membrane trafficking process. The Conserved Oligomeric Golgi (COG) complex operates as a vesicular tether for intra-Golgi trafficking. The Golgi is the central hub for protein posttranslational modifications, mostly glycosylation. Consequently, the primary job of the COG is to tether vesicles that recycle resident enzymes and cargo receptors. To achieve its function, the COG interacts with SNAREs, Rabs, and other tethers, but the detailed understanding of these interactions is an enigma that we propose to solve by pairwise probing of COG/partner interactions, their kinetics, and by defining their molecular environment through proximity-labeling studies. Depletion of COG causes accumulation of specific transport intermediates – COG complex dependent (CCD) vesicles that are likely to represent a major class of Golgi vesicles that recycle Golgi enzymes and cargo receptors. Mutations in COG subunits result in congenital disorders of glycosylation (CDG) type II category, which belong to a group of autosomal recessive multi-systemic disorders with several distinguishable symptoms that include global developmental defects and microcephaly. These deficits are often accompanied by neurological and liver impairment. COG-CDG defects are studied in patients’ fibroblasts, which do not represent the most affected tissues; a more flexible cell base model will benefit our progress in developing a cure for this disorder. We hypothesize that a revealing of the molecular basis of COG/partner interactions will help in deciphering the mechanisms of assembly/disassembly of vesicle docking platforms and that a detailed analysis of different populations of CCD vesicles will uncover their specific origin, budding and tethering machinery and a complete set of proteins that traffic in a COG-dependent manner. The development of cell-based models for COG-CDGs will allow us to test the effects of different COG mutations without the need for patient involvement and pave the way for the development of treatment protocols. To test this hypothesis, first, we will characterize in molecular interactions between COG and its key partner proteins (Aim1). Next, we will use a degrone-assisted COG depletion to accumulate, purify and characterize CCD vesicles (Aim2). Finally, we will develop and characterize a novel iPSC-based cellular model for COG CDGs (Aim 3). Success in accomplishing these aims will provide a mechanistic understanding of COG complex function, characterize COG-dependent trafficking intermediates, and create a set of isogenic stem cell lines bearing human COG mutations. Moreover, these results will be necessary for a functional understanding of Golgi dynamic and vesicular trafficking in general.

项目摘要/摘要 细胞内膜运输介导蛋白质和脂质的细胞内递送。这个过程是双向的 并且由顺行（分泌）和逆行（内吞）分支组成。细胞内膜运输是 进化保守，其机制是模块化的，功能同源的组件在不同的环境中运作。 贩运步骤。因此，详细了解贩运的一个步骤将有助于了解整个 细胞内膜运输过程。 保守寡聚高尔基体（COG）复合体作为高尔基体内运输的囊泡系链。高尔基体是 蛋白质翻译后修饰的中心枢纽，主要是糖基化。因此， COG是用来拴系回收驻留酶和货物受体的囊泡。为了实现其功能，COG与 与SNARE，Rabs和其他系绳，但这些相互作用的详细理解是一个谜，我们建议 通过成对探测COG/伴侣相互作用及其动力学并定义其分子环境来解决 通过邻近标记研究。COG的消耗导致特定转运中间体- COG的积累 复合物依赖性（CCD）囊泡，其可能代表回收高尔基体酶的主要类别的高尔基体囊泡 和货物受体。 COG亚基中的突变导致先天性糖基化障碍（CDG）II型，其属于先天性糖基化障碍。 一组常染色体隐性多系统性疾病，具有几种可区分的症状，包括全身性 发育缺陷和小头畸形。这些缺陷通常伴有神经和肝脏损伤。 在患者的成纤维细胞中研究了COG-CDG缺陷，这些成纤维细胞并不代表受影响最严重的组织;更灵活的 基于细胞的模型将有利于我们在开发这种疾病的治疗方法方面取得进展。 我们假设，揭示COG/伴侣相互作用的分子基础将有助于破译 组装/拆卸机制的囊泡对接平台，并详细分析了不同群体的 CCD囊泡将揭示其特定的起源，出芽和束缚机制以及一整套蛋白质， 以依赖于COG的方式进行通信。基于细胞的COG-CDG模型的开发将使我们能够测试 不同COG突变的影响，而不需要患者参与，并为发展铺平道路， 治疗方案。为了验证这一假设，首先，我们将描述COG及其关键分子之间的相互作用。 伴侣蛋白（Aim 1）。接下来，我们将使用一个degrone辅助COG耗尽积累，纯化和表征CCD 囊泡（Aim 2）。最后，我们将开发和表征一种新的基于iPSC的COG CDG细胞模型（目标3）。 成功地实现这些目标将提供一个机械的理解COG复杂的功能， COG依赖性运输中间体，并创建一组携带人COG突变的等基因干细胞系。 此外，这些结果将是必要的高尔基体动态和囊泡运输的功能性理解， 将军