Microtubule end-binding proteins in insulin secretion

描述(申请人提供)：在健康的个体中，进食后，β细胞的胰岛素分泌经历了两个阶段：快速但很小的第一阶段，和延迟但持续的第二阶段。在第二阶段，胰岛素从细胞内部被动员起来，分泌到血液中。微管是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是如何协调这一过程的。具体地说，我们正在研究微管末端结合的一系列蛋白质的作用。我们认为，这些微管末端结合蛋白通过结合到胰岛素颗粒表面，并有效地将其拴在微管上，帮助协调胰岛素的分泌。通过这种方式，胰岛素颗粒将优先结合在细胞边缘附近，在那里它将处于分泌状态。或者，微管末端结合蛋白可能会起到稳定微管的作用，而微管经常自发解体。这样，胰岛素颗粒从细胞内部的路径就会更稳定。为了研究这些假设，我们将进行一些研究。首先，由于β细胞中的微管末端结合蛋白完全没有特征，我们将首先确定哪些蛋白在mRNA和蛋白质水平上表达。然后，我们将通过质谱学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相关联。接下来，利用分离的β细胞，我们将通过测量胰岛素的分泌量，以及通过显微镜跟踪胰岛素颗粒从β细胞内部招募到表面，来询问有针对性地耗尽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个体的胰岛素分泌功能，以及糖尿病患者的胰岛素分泌是如何出错的。从根本上讲，糖尿病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，超过4400万美国人将在25年内患上糖尿病，而这一成本将增加两倍，达到3000亿美元以上。通过研究这些基本的细胞过程，我们寻求减轻这一迫在眉睫的负担。参考文献列表1.Hou，J.C.，Min，L.&Pessin，J.E.Insulin and IGFS(编辑)，pp.473-506(学术出版社，2009)。2.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》122,893-903(2009)。3.Huang，E.S.，Basu，A.，O‘Grady，M.和Capretta，J.C.预测美国糖尿病护理32,2225-2229(2009)的未来糖尿病人口规模和相关成本。 与公共健康相关：胰岛素通常能有效地从胰腺内的特殊细胞输送到血液中。当这一过程失败时，就会发生糖尿病。我们正在研究这一运输过程的细节，以更好地了解这一过程在正常人中是如何发挥作用的，这可能揭示在疾病背景下什么是失败的。

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.