Regulatory Networks and Biomarkers of Beta-cell Dysfunction and Apoptosis

描述（由申请人提供）：在1型糖尿病（T1D）的研究中，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，本应用的总体目标是发现有助于早期β细胞应激和死亡的新的体内信号通路和调节网络，并确定干预和早期诊断生物标志物的潜在治疗靶点。这些目标将通过应用蛋白质组学技术来实现，重点是翻译后蛋白质修饰（PTMs），以实现独特的人类胰岛样本集。具体来说，我们假设翻译后调控，包括磷酸化、S-亚硝基化和S-谷胱甘肽化，代表了显性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，我们的计划利用三种互补的模型系统：a)通过激光捕获显微解剖从临床人类胰腺组织分离的人类胰岛，b)体外细胞因子处理的人类胰岛，以及c)移植在“人源化”小鼠体内的人类胰岛功能障碍。Aim 1的研究将利用全球PTM聚焦的蛋白质组学技术，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，我们将采用一种有针对性的量化方法，使用来自个体患者的样本以及来自“人源化”小鼠的胰岛样本来验证特定的调控网络和感兴趣的ptm。在Aim 3中，我们将评估前两个目标中发现的蛋白靶点，确定它们作为血清中早期T1D诊断的β细胞特异性标记物的潜力，以及它们在β细胞凋亡中的潜在功能作用

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.