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: 7783389
• 负责人: JEFFREY E. PESSIN
• 金额: $ 41.5万
• 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 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783389
• 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. PUBLIC HEALTH RELEVANCE: Diabetes, obesity and insulin resistant states can all be characterized as defects in the body's ability to adjust for differences in energy consumption and output. One of the key biological responses to insulin action is the translocation and fusion of the facilitative GLUT4 glucose transporter isoform from intracellular storage sites to the plasma membrane in skeletal muscle and adipose tissue. This process results in an approximate 10-fold increase in the number of cell surface GLUT4 molecules that accounts for the post-prandial increase in peripheral tissue glucose uptake. Thus, understanding the mechanism for this translocation process and the ability of insulin to increase glucose uptake are essentially process for the maintenance of normal glucose homeostasis. 1

描述（由申请人提供）：本研究计划的总体目标是进一步了解细胞内运输、胰岛素刺激的易位和胰岛素响应性葡萄糖转运蛋白（GLUT4）从细胞内储存位点到脂肪细胞质膜融合的基本分子机制。尽管在负责 GLUT4 易位的胰岛素信号通路方面已经取得了重大进展，但有关特定细胞内运输步骤中涉及的调控事件和蛋白质的信息很少。此外，该系统或任何其他系统的生物膜融合的基本生物物理机制尚未阐明。最近，我们发现新合成的GLUT4蛋白通过高尔基复合体直接进入胰岛素响应的储存室，而无需首先穿过质膜并进行内吞作用。基于 GLUT4 生物合成和细胞内分选的时间特性，再加上 siRNA 基因沉默的使用，我们设计了一种新的范例，用于检查在 GLUT4 生命周期中不同膜运输步骤中发挥作用的靶向机制和囊泡运输事件。基于这些数据，我们建议使用 siRNA 与 GLUT4 报告构建体的时间表达相结合，从功能上将参与生物合成分选的蛋白质映射到胰岛素反应区室（1 类），从该区室退出所需的蛋白（2 类）、内吞作用所需的蛋白（3 类）和再循环回胰岛素反应区室所需的蛋白（1 类） 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 SNARE 蛋白家族的具体要求。与此同时，我们已经开始使用 GLUT4 运输细胞生物学方法与磷脂-SNARE 蛋白相互作用的生物物理分析相结合来揭示 SNARE 依赖性融合过程本身的生化性质。我们的初步数据表明，通过与酸性磷脂的静电相互作用，质膜突触蛋白 (Stx) 和 VAMP 的近膜结构域 (JMD) 是该过程所必需的。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和串联 VAMP2 色氨酸在这一关键生物过程中的作用。 公共健康相关性：糖尿病、肥胖和胰岛素抵抗状态都可以被描述为身体适应能量消耗和输出差异的能力缺陷。对胰岛素作用的关键生物反应之一是易位 GLUT4 葡萄糖转运蛋白亚型从细胞内储存位点易位和融合到骨骼肌和脂肪组织的质膜。这一过程导致细胞表面 GLUT4 分子数量增加约 10 倍，从而导致餐后外周组织葡萄糖摄取增加。因此，了解这种易位过程的机制和胰岛素增加葡萄糖摄取的能力是维持正常葡萄糖稳态的本质过程。 1