Functional mapping of SNARE-dependent trafficking and fusion

SNARE 依赖性运输和融合的功能图谱

• 批准号: 8436135
• 负责人: JEFFREY E. PESSIN
• 金额: $ 32.91万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436135
• 关键词: Accounting; Address; Adipocytes; Adipose tissue; Affect; Amino Acids; Anabolism; Antibodies; Back; Binding; Biochemical; Biological; Biological Process; Cell membrane; Cell surface; Cells; Cellular biology; Consumption; Coupled; Data; Defect; Diabetes Mellitus; Electrostatics; Endocytosis; Evaluation; Event; GLUT 4 protein; GLUT4 gene; Gene Silencing; Glucose Transporter; Golgi Apparatus; Insulin; Insulin Resistance; Insulin Signaling Pathway; Label; Maintenance; Maps; Mediating; Membrane; Membrane Fusion; Membrane Potentials; Molecular; Mutagenesis; Nature; Obesity; Output; Peripheral; Phospholipids; Process; Property; Protein Family; Protein Isoforms; Proteins; RNA Interference; Recycling; Regulation; Relative (related person); Reporter; Research Proposals; Role; SNAP receptor; Series; Site; Skeletal Muscle; Small Interfering RNA; Sorting - Cell Movement; Surface; System; Tissues; Transmembrane Transport; Transport Vesicles; Tryptophan; Vesicle; base; blood glucose regulation; cell type; glucose uptake; novel; protein transport; public health relevance; response; syntaxin; trafficking

DESCRIPTION (provided by applicant): The overall objective of this research proposal is to further our understanding of the basic molecular mechanisms accounting for the intracellular trafficking, insulin-stimulated translocation and fusion of the insulin-responsive glucose transporter (GLUT4) from intracellular storage sites to the plasma membrane in adipocytes. Although significant progress has been made in terms of the insulin signaling pathways responsible for GLUT4 translocation, there is little information with regard to the regulatory events and proteins involved in specific intracellular trafficking steps. Moreover, the basic biophysical mechanism of biological membrane fusion for this, or any other system, has not been elucidated. Recently, we have found that newly synthesized GLUT4 protein traffics through the Golgi complex and directly enters the insulin-responsive storage compartment without first transiting the plasma membrane and undergoing endocytosis. Based upon the temporal properties of GLUT4 biosynthesis and intracellular sorting coupled with the use of siRNA gene silencing, we have devised a novel paradigm for examining the targeting machinery and vesicle trafficking events that function at different membrane transport steps in the GLUT4 lifecycle. Based upon these data we propose to use siRNA coupled with the temporal expression of GLUT4 reporter constructs to functionally map proteins involved in the biosynthetic sorting to the insulin-responsive compartment (Class 1) from those required for the exit from this compartment (Class 2), those required for endocytosis (Class 3) and those required for recycling back to the insulin-responsive compartment (Class 4). We propose to use this approach to identify and classify the specific requirements for the SNARE family of proteins required for biological membrane fusion. In parallel, we have begun to unravel the biochemical nature of the SNARE-dependent fusion process itself using a combination of GLUT4 trafficking cell biological approach coupled with biophysical analysis of phospholipid-SNARE protein interactions. Our preliminary data has demonstrated that the juxtamembrane domain (JMD) of both plasma membrane Syntaxins (Stx) and VAMP are required for this process through electrostatic interactions with acidic phospholipids. To address the mechanism of fusion pore formation, we will use a series of biophysical and cell biological approaches to determine the role of membrane electrostatics and the tandem VAMP2 tryptophans in this key biological process.

描述（由申请人提供）：这项研究建议的总体目标是进一步理解我们对细胞内贩运，胰岛素刺激的易位和融合的基本分子机制的理解，以及胰岛素反应性葡萄糖转运蛋白（Glut4）的融合，这些葡萄糖转运蛋白（Glut4）是从细胞内存储位点到脂质膜中的血脑膜膜中的。尽管根据负责GLUT4易位的胰岛素信号传导途径取得了重大进展，但关于参与特定细胞内运输步骤的调节事件和蛋白质的信息很少。此外，尚未阐明生物膜融合的基本生物物理机制，或任何其他系统。最近，我们发现新合成的GLUT4蛋白运输通过高尔基体复合物，直接进入胰岛素反应性的储存室而不首先转移质膜和内吞作用。基于GLUT4生物合成和细胞内分类的时间特性，再加上siRNA基因沉默的使用，我们设计了一种新型的范式，用于检查靶向机械和囊泡运输事件，这些事件在Glut4 Lifecycle中在不同的膜运输步骤中起作用。基于这些数据，我们建议使用siRNA，再加上GLUT4报道构建体的时间表达在功能上绘制涉及生物合成分类的蛋白质与胰岛素响应性隔室（1类），从该隔室（类别2）中所需的蛋白质（1类）（第2类），这些组合（3级），以及reccyssoss（3） - 恢复性的固定（recive） - 恢复性（综述） - 4）。我们建议使用这种方法来识别和分类生物膜融合所需的蛋白质蛋白质家族的特定要求。同时，我们已经开始使用GLUT4运输细胞生物学方法以及对磷脂 - 螺旋蛋白相互作用的生物物理分析的结合来阐明依赖SNARE依赖性融合过程的生化性质。我们的初步数据表明，通过与酸性磷脂的静电相互作用，质膜语法（STX）（STX）（STX）和VAMP的近去膜结构域（JMD）都是必需的。为了解决融合孔形成的机制，我们将使用一系列生物物理和细胞生物学方法来确定膜静电剂和串联VAMP2色氨酸在此关键生物学过程中的作用。