Microtubule end-binding proteins in insulin secretion

胰岛素分泌中的微管末端结合蛋白

• 批准号: 8142910
• 负责人: Jacob Eric Lazarus
• 金额: $ 2.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8142910
• 关键词: Affect; Affinity Chromatography; American; Beryllium; Beta Cell; Binding; Binding Proteins; Biological Assay; Blood; Caring; Cause of Death; Cell membrane; Cell physiology; Cell secretion; Cell-Free System; Cells; Complement; Cytoskeleton; Development; Diabetes Mellitus; Disease; Dissection; Dominant-Negative Mutation; Eating; Exocytosis; Fluorescence Microscopy; Future; Health; Immunoblotting; Immunofluorescence Microscopy; Individual; Insulin; Insulin Resistance; Life; Mass Spectrum Analysis; Measures; Messenger RNA; Microtubule Proteins; Microtubules; Modeling; Monomeric GTP-Binding Proteins; Motor; Non-Insulin-Dependent Diabetes Mellitus; Organ; Pancreas; Peripheral; Phase; Plus End of the Microtubule; Polymers; Population Sizes; Process; Protein Family; Proteins; RNA Interference; Radioimmunoassay; Recruitment Activity; Reverse Transcriptase Polymerase Chain Reaction; Role; SNAP receptor; Specificity; Structure of beta Cell of islet; Surface; Symptoms; System; Testing; Time; Transport Process; United States; Vesicle; burden of illness; cell cortex; cell type; cost; insight; insulin granule; insulin secretion; reconstitution

DESCRIPTION (provided by applicant): In healthy individuals, after eating, insulin secretion from beta cells takes place in two phases: a rapid but small first phase, and a delayed but sustained second phase. During this second phase, insulin is mobilized from the interior of the cell for secretion into the blood1. Microtubules, highly dynamic structural cellular elements, are necessary for this mobilization, but we do not fully understand how they coordinate this process. Specifically, we are investigating the role of a family of proteins which bind at the end of the microtubule. We believe that these microtubule end-binding proteins help coordinate insulin secretion by binding to the surface of the insulin granule, and in effect, tethering it to the microtubule. In this way, the insulin granule would preferentially associate near the edge of the cell, where it would be poised for secretion. Alternatively, the microtubule end-binding proteins might instead be acting to stabilize the microtubule, which frequently spontaneously disassembles. In this way, the insulin granule would have a steadier path from the interior of the cell. To investigate these hypotheses, we will perform a number of studies. First, because the microtubule end- binding proteins in beta cells are completely uncharacterized, we will first determine which proteins are expressed, both at the level of mRNA and protein. We will then determine how the end-binding proteins become associated with the insulin granule by isolating and identifying interacting proteins through mass spectrometry. Next, using isolated beta cells, we will ask what effect targeted depletion of the end-binding proteins and their interactors has on insulin secretion, both by: measuring the amount of insulin that is secreted; and by microscopically tracking the insulin granules as they are recruited from the interior of the beta cell to the surface. Finally, to enable formal dissection of this process, we will reconstitute insulin secretion using purified proteins, microtubules, and isolated insulin granules. These studies have the potential to help us understand how insulin secretion functions in normal individuals, and how it goes wrong in individuals with diabetes. Diabetes is fundamentally a disease of insulin secretion. Over time, the body becomes less sensitive to insulin, but symptoms of the disease only manifest when secretion from beta cells can no longer compensate. This is a serious problem; a recent study estimated that more than 44 million Americans will have diabetes within 25 years and that costs will triple to more than $300 billion. By studying these fundamental cellular processes, we seek to alleviate this looming burden. Reference List 1. Hou, J. C., Min, L. & Pessin, J. E. Insulin and IGFs (ed.), pp. 473-506 (Academic Press,2009). 2. Wang, Z. & Thurmond, D. C. Mechanisms of biphasic insulin-granule exocytosis - roles of the cytoskeleton, small GTPases and SNARE proteins. J Cell Sci 122, 893-903 (2009). 3. Huang, E. S., Basu, A., O'Grady, M. & Capretta, J. C. Projecting the future diabetes population size and related costs for the U.S. Diabetes Care 32, 2225-2229 (2009). PUBLIC HEALTH RELEVANCE: Insulin is normally efficiently transported from within specialized cells in the pancreas into the blood. When this process fails, diabetes occurs. We are studying the specifics of this transport process to better understand how this functions in normal individuals, which may reveal what fails in the setting of disease.

描述（由申请人提供）：在健康个体中，进食后，β细胞的胰岛素分泌分两个阶段进行：快速但小的第一阶段，和延迟但持续的第二阶段。在该第二阶段，胰岛素从细胞内部动员，分泌到血液中1。微管是一种高度动态的细胞结构元件，对这种动员是必要的，但我们还不完全了解它们是如何协调这一过程的。具体地说，我们正在研究结合在微管末端的蛋白质家族的作用。我们认为这些微管末端结合蛋白通过与胰岛素颗粒表面结合，并有效地将其束缚在微管上，从而帮助协调胰岛素分泌。以这种方式，胰岛素颗粒将优先结合在细胞边缘附近，在那里它将处于分泌状态。或者，微管末端结合蛋白质可能起稳定微管的作用，而微管经常自发地解体。以这种方式，胰岛素颗粒将具有从细胞内部的更稳定的路径。为了研究这些假设，我们将进行大量的研究。首先，由于β细胞中的微管末端结合蛋白完全未被表征，我们将首先确定哪些蛋白被表达，无论是在mRNA水平还是在蛋白质水平。然后，我们将通过质谱分离和鉴定相互作用的蛋白质来确定末端结合蛋白质如何与胰岛素颗粒结合。接下来，我们将使用分离的β细胞，通过测量分泌的胰岛素量，并通过显微镜跟踪胰岛素颗粒从β细胞内部募集到表面的过程，来研究靶向去除末端结合蛋白及其相互作用物对胰岛素分泌的影响。最后，为了能够对这一过程进行正式的解剖，我们将使用纯化的蛋白质、微管和分离的胰岛素颗粒来重建胰岛素分泌。这些研究有可能帮助我们了解正常人的胰岛素分泌功能，以及糖尿病患者的胰岛素分泌是如何发生问题的。糖尿病从根本上说是一种胰岛素分泌疾病。随着时间的推移，身体对胰岛素的敏感性会降低，但只有当β细胞的分泌不再能够补偿时，疾病的症状才会显现。这是一个严重的问题;最近的一项研究估计，在25年内，将有超过4400万美国人患有糖尿病，其费用将增加两倍，超过3000亿美元。通过研究这些基本的细胞过程，我们试图减轻这种迫在眉睫的负担。参考列表1.侯继忠，字宗，敏湖，澳-地&佩辛，J. E.胰岛素和IGFs（编辑），pp. 473-506（学术出版社，2009年）。2. Wang， Z. &瑟蒙德，D。C.双相胰岛素颗粒胞吐机制-细胞骨架，小GTP酶和SNARE蛋白作用细胞科学杂志122，893-903（2009）。3. Huang，E.美国，巴苏，A.，奥格雷迪M。和卡普雷塔，J.C.美国糖尿病护理32，2225-2229（2009）预测未来糖尿病人群规模和相关成本。 公共卫生相关性：胰岛素通常从胰腺中的特化细胞内有效地转运到血液中。当这个过程失败时，就会发生糖尿病。我们正在研究这种转运过程的细节，以更好地了解这种转运过程在正常个体中是如何发挥作用的，这可能会揭示在疾病背景下是什么失败了。