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为两个阶段：快速但很小的第一阶段，以及延迟但持续的第二阶段。在第二阶段，从细胞的内部动员胰岛素将其分泌到血液中。微管，高度动态的结构细胞元素，对于这种动员是必需的，但我们不完全了解它们如何协调这一过程。具体而言，我们正在研究在微管末端结合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，并实际上将其绑在微管上来帮助胰岛素分泌。这样，胰岛素颗粒将优先将其在细胞边缘附近关联，在该细胞边缘将其固定为分泌。或者，微管末端结合蛋白可能会起作用以稳定微管，该微管经常自发拆卸。这样，胰岛素颗粒将从细胞内部具有稳定的路径。为了研究这些假设，我们将进行许多研究。首先，由于β细胞中的微管末端结合蛋白是完全未表征的，因此我们将首先确定在mRNA和蛋白质水平上表达哪些蛋白质。然后，我们将通过通过质谱分离和鉴定相互作用的蛋白质来确定末期结合蛋白如何与胰岛素颗粒相关联。接下来，使用孤立的β细胞，我们将询问末端结合蛋白及其相互作用者对胰岛素分泌的靶向耗竭的影响，这两者都通过：测量分泌的胰岛素量；通过显微镜从β细胞的内部募集到表面，通过显微镜跟踪胰岛素颗粒。最后，为了对此过程进行正式解剖，我们将使用纯化的蛋白质，微管和孤立的胰岛素颗粒重新构建胰岛素分泌。这些研究有可能帮助我们了解普通人的胰岛素分泌功能以及糖尿病患者的出问题。糖尿病从根本上是一种胰岛素分泌的疾病。随着时间的流逝，人体对胰岛素的敏感程度降低，但是只有在β细胞的分泌不再补偿时，该疾病的症状才能表现出来。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年内患有糖尿病，而成本将三倍至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。Hou，J.C.，Min，L。＆Pessin，J。E.胰岛素和IGFS（ed。），第473-506页（学术出版社，2009年）。 2。Wang，Z。＆Thurmond，D。C.双相胰岛素颗粒胞吐的机理 - 细胞骨架，小GTPase和Snare蛋白的作用。 J Cell Sci 122，893-903（2009）。 3。Huang，E。S.，Basu，A.，O'Grady，M。＆Capretta，J.C。预测未来糖尿病的人口规模和美国糖尿病护理的相关成本32，2225-2229（2009）。 公共卫生相关性：胰岛素通常从胰腺中的专用细胞内有效地运输到血液中。当此过程失败时，会发生糖尿病。我们正在研究这种运输过程的细节，以更好地了解正常人中这种功能的功能，这可能揭示了疾病环境中的失败。