Microtubule end-binding proteins in insulin secretion: enhanced efficiency of pol

胰岛素分泌中的微管末端结合蛋白：增强pol的效率

• 批准号: 8003659
• 负责人: Jacob Eric Lazarus
• 金额: $ 4.21万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003659
• 关键词: American; Beta Cell; Binding; Binding Proteins; Blood; Caring; Cell physiology; Cell secretion; Cells; Cytoskeleton; Diabetes Mellitus; Disease; Dissection; Eating; Elements; Exocytosis; Future; Individual; Insulin; Mass Spectrum Analysis; Measures; Messenger RNA; Microtubule Proteins; Microtubules; Monomeric GTP-Binding Proteins; Pancreas; Phase; Population Sizes; Process; Protein Family; Proteins; Recruitment Activity; Role; SNAP receptor; Surface; Symptoms; Time; Transport Process; cost; insulin granule; insulin secretion; public health relevance; 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：快速但较小的第一阶段，以及延迟但持续的第二阶段。在第二阶段，胰岛素从细胞内部动员并分泌到血液中1。微管，高度动态的结构细胞元件，对于这种动员是必要的，但我们并不完全了解它们如何协调这个过程。具体来说，我们正在研究结合在微管末端的一系列蛋白质的作用。我们相信这些微管末端结合蛋白通过结合到胰岛素颗粒的表面来帮助协调胰岛素分泌，并且实际上将其束缚到微管上。通过这种方式，胰岛素颗粒将优先结合在细胞边缘附近，在那里准备分泌。或者，微管末端结合蛋白可能会起到稳定微管的作用，而微管经常会自发分解。这样，胰岛素颗粒就能从细胞内部获得更稳定的路径。为了调查这些假设，我们将进行大量研究。首先，由于 β 细胞中的微管末端结合蛋白完全未表征，因此我们将首先确定哪些蛋白质在 mRNA 和蛋白质水平上表达。然后，我们将通过质谱分离和鉴定相互作用的蛋白质，确定末端结合蛋白如何与胰岛素颗粒结合。接下来，使用分离的 β 细胞，我们将通过以下方法来探究末端结合蛋白及其相互作用物的靶向消耗对胰岛素分泌有何影响：测量分泌的胰岛素量；并通过显微镜追踪胰岛素颗粒从β细胞内部募集到表面的过程。最后，为了能够正式剖析这一过程，我们将使用纯化的蛋白质、微管和分离的胰岛素颗粒重建胰岛素分泌。这些研究有可能帮助我们了解正常人的胰岛素分泌功能如何，以及糖尿病患者的胰岛素分泌如何出错。糖尿病从根本上来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度会降低，但只有当β细胞的分泌物不再能够补偿时，疾病的症状才会显现出来。这是一个严重的问题；最近的一项研究估计，超过 4400 万美国人将在 25 年内患上糖尿病，费用将增加两倍，达到超过 3000 亿美元。通过研究这些基本的细胞过程，我们寻求减轻这种迫在眉睫的负担。参考文献列表 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. 双相胰岛素颗粒胞吐作用的机制 - 细胞骨架、小 GTP 酶和 SNARE 蛋白的作用。细胞科学杂志 122, 893-903 (2009)。 3. Huang, E. S., Basu, A., O'Grady, M. & Capretta, J. C. 预测美国糖尿病护理的未来糖尿病人口规模和相关成本 32, 2225-2229 (2009)。 公共健康相关性：胰岛素通常从胰腺的特殊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。我们正在研究这种运输过程的细节，以更好地了解其在正常个体中的功能，这可能会揭示在疾病情况下失败的原因。