Regulatory Networks and Biomarkers of Beta-cell Dysfunction and Apoptosis

描述(申请人提供)：在1型糖尿病(T1D)的研究中，仍然迫切需要对早期自身免疫介导的分子机制进行系统水平的了解，这些机制触发了胰腺β细胞的特异性破坏。因此，这项应用的总体目标是发现新的体内信号通路和调控网络，有助于早期β细胞应激和死亡，并确定潜在的干预治疗靶点和早期诊断生物标志物。这些目标将通过将使能蛋白质组学技术应用于对独特的人类胰岛样本进行翻译后蛋白质修饰(PTM)来实现。具体地说，我们假设翻译后调控，包括磷酸化、S-亚硝化和S-谷胱甘肽基化，代表了在显性T1D之前β细胞功能障碍的基本触发机制。为了解决与研究动态信号网络的临床样本相关的局限性，我们的计划使用了三个互补的模型系统：a)通过激光捕获显微切割从临床人类胰腺组织分离的人胰岛，b)体外用细胞因子处理的人胰岛，以及c)将人胰岛移植到“人化”小鼠中，以概括体内胰岛功能障碍的情况。AIM 1中的研究将利用全球PTM重点蛋白质组技术确定参与早期β细胞功能障碍和凋亡的信号通路和网络。在目标2中，我们将应用一种有针对性的量化方法来验证特定的调控网络和感兴趣的PTM，使用来自个别患者的样本以及来自“人源化”小鼠的时间进程胰岛样本。在目标3中，我们将评估在前两个目标中发现的蛋白质靶点，确定它们作为β细胞特异性标记物用于血清中早期T1D诊断的可能性，以及它们通过额外的 培养的胰岛研究。综上所述，我们预计该项目将展示β细胞功能障碍和凋亡的翻译后调控的系统水平研究的新范式，并将为潜在触发β细胞凋亡和T1D的早期调控网络提供一个新的综合视角。这项工作还将为β细胞死亡和存活联盟(CBDS)、人类胰岛研究网络(HIRN)和糖尿病社区生成新的功能研究假说提供丰富的分子数据资源。

DESCRIPTION (provided by applicant): In type 1 diabetes (T1D) research there remains a critical need for pursuing a systems level understanding of early stage autoimmune-mediated molecular mechanisms which trigger the specific destruction of pancreatic β cells. Thus, the overall objectives of this application are to discover novel in vivo signaling pathways and regulatory networks that contribute to early stage β cell stress and death, and to identify potential therapeutic targets for intervention and early diagnostic biomarkers. These objectives will be pursued by applying enabling proteomics technologies focusing on posttranslational protein modifications (PTMs) to unique sets of human islet samples. Specifically, we hypothesize that posttranslational regulation, involving phosphorylation, S- nitrosylation, and S-glutathionylation, represents a fundamental triggering mechanism of β cell dysfunction preceding overt T1D. To address the limitations associated with clinical samples for studying dynamic signaling networks, our plan utilizes three complementary model systems: a) human islets isolated by laser-capture microdissection from clinical human pancreatic tissues, b) human islets treated with cytokines in vitro, and c) human islets transplanted in 'humanized' mice for recapitulating in vivo islet dysfunction. Studies in Aim 1 will identify signaling pathway and networks involved in early stage β cell dysfunction and apoptosis using global PTM focused proteomic technologies. In Aim 2, we will apply a targeted quantification approach to verify specific regulatory networks and PTMs of interest using samples from individual patients as well as time- course islet samples from 'humanized' mice. In Aim 3, we will evaluate protein targets discovered in the first two aims, determining their potential as β cell specific markers for early T1D diagnosis in serum, and their potential functional roles in β cell apoptosis through additional cultured islet studies. Together, we anticipate that this project will demonstrate a new paradigm of systems level study of posttranslational regulation of β cell dysfunction and apoptosis, and will provide a novel integrative view of the early stage regulatory networks that potentially trigger β cell apoptosis and T1D. This work will also provide a rich molecular data resource for the consortium on beta-cell death and survival (CBDS), the human islet research network (HIRN), and the diabetes community in generating new hypotheses for functional studies.