Regulatory Networks and Biomarkers of Beta-cell Dysfunction and Apoptosis

描述（由申请人提供）：在1型糖尿病（T1 D）研究中，仍然迫切需要对触发胰腺β细胞特异性破坏的早期自身免疫介导的分子机制进行系统水平的理解。因此，本申请的总体目标是发现有助于早期β细胞应激和死亡的新型体内信号传导途径和调控网络，并鉴定用于干预的潜在治疗靶标和早期诊断生物标志物。这些目标将通过应用蛋白质组学技术来实现，该技术专注于翻译后蛋白质修饰（PTM），以独特的人类胰岛样本集。具体而言，我们假设，涉及磷酸化、S-亚硝基化和S-谷胱甘肽化的翻译后调节代表了明显T1 D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品相关的限制，我们的计划利用三个互补的模型系统：a）通过激光捕获显微切割从临床人胰腺组织分离的人胰岛，B）体外用细胞因子处理的人胰岛，和c）移植到“人源化”小鼠中的人胰岛用于重现体内胰岛功能障碍。目标1中的研究将使用全球PTM聚焦蛋白质组学技术鉴定参与早期β细胞功能障碍和凋亡的信号通路和网络。在目标2中，我们将应用靶向定量方法，使用来自个体患者的样品以及来自“人源化”小鼠的时程胰岛样品来验证感兴趣的特定调控网络和PTM。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T1 D诊断的潜力，以及它们通过额外的细胞凋亡在β细胞凋亡中的潜在功能作用。 培养的胰岛研究。总之，我们预计，该项目将展示一个新的模式，系统水平的研究β细胞功能障碍和凋亡的翻译后调控，并将提供一个新的综合性的观点，早期阶段的调控网络，可能触发β细胞凋亡和T1 D。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）和糖尿病社区提供丰富的分子数据资源，以产生新的功能研究假设。

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.