Interrogating the ubiquitin pathway to understand and treat cytokine-induced beta-cell death in type 1 diabetes

项目摘要 胰腺中产生胰岛素的β细胞的损失导致对注射胰岛素的绝对需求， 在外周血中引起低血糖死亡和糖尿病并发症发病的显著风险 神经视网膜心脏和肾脏治疗T1 D的一个关键目标是阻止这种细胞攻击， 通过阻止β细胞的免疫错误识别或通过保护β细胞免于细胞死亡。然而，一个关键的 该领域进展的障碍是缺乏对胰岛中细胞事件的完全理解， 与β细胞团的丧失有关使用表型筛选方法，我们发现了BRD 0476， 其选择性地对精氨酸介导的细胞凋亡具有活性。对这种化合物的进一步研究表明， 结合去泛素化酶USP 9 X以响应IFNγ而停止JAK 2和STAT 1信号传导。我们确定 JAK 2可以通过泛素化使其信号传导功能丧失，并且通过调节USP 9 X，我们可以使JAK 2的信号传导功能丧失。 平衡降低JAK 2激酶活性，即使在IFNγ存在下。这些结果表明， 泛素化在调节T1 D β细胞凋亡中的作用，并建议更好地理解这一点， 在T1 D发展的早期阶段的过程（及其潜在的失调）可能导致1） 鉴定处于风险中的个体，和2）在早期T1 D中保留β细胞群的新治疗策略。 使用我们的探针BRD 0476和以前未应用于胰岛生物学的化学生物学工具，我们将改进我们的 通过以下目的了解USP 9 X在体外和体内β-细胞存活中的作用：在目的1中， 我们将描述USP 9 X在人胰岛中抑制JAK 2的模式。在目标2中，我们将评估 在小鼠中抑制USP 9 X-JAK 2（与BRD 0476一起）对自身免疫性糖尿病的发展和进展的影响 1型糖尿病的发病机制在目标3中，我们将分析人胰岛中去泛素化酶（DUB）的表达和活性 在早期T1 D发展过程中，使用基于活性的蛋白质谱（ABPP）和全局泛素化 测量.这一建议的成功结果是：1）更好地理解了 促进早期T1 D中的β细胞存活，和2）提供翻译概念验证的化学探针。这 该项目将为开发早期T1 D发展的生物标志物以及高级生物标志物奠定基础。 在早期T1 D中预防β细胞凋亡的治疗策略，代表了一种潜在的治疗方法 approach.

PROJECT SUMMARY The loss of insulin-producing beta cells in the pancreas results in an absolute requirement for injected insulin, causing significant risks of mortality from hypoglycemia and morbidity from diabetic complications in peripheral nerves, the retina, the heart, and the kidney. A key goal of efforts to treat T1D is to stop this cellular attack, either by halting the immune mis-recognition of beta cells or by protecting beta cells from cell death. However, a critical barrier to progress in the field is a lack of complete understanding of the cellular events in the islet that contribute to the loss of beta-cell mass. Using a phenotypic screening approach, we discovered BRD0476, a compound that is selectively active against cytokine-mediated apoptosis. Further study of this compound revealed that it binds the deubiquitinase USP9X to halt JAK2 and STAT1 signaling in response to IFNγ. We determined that JAK2 can be rendered signaling incompetent by ubiquitination, and that by modulating USP9X, we can tip the balance toward reduced JAK2 kinase activity, even in the presence of IFNγ. These results point to an emerging role for ubiquitination in regulating beta-cell apoptosis in T1D, and suggest that a greater understanding of this process (and its potential dysregulation) in the early stages of T1D development could lead to 1) the ability to identify at-risk individuals, and 2) novel therapeutic strategies to preserve beta-cell mass in early-stage T1D. Using our probe BRD0476 and chemical biology tools not previously applied to islet biology, we will improve our understanding of the role of USP9X in beta-cell survival in vitro and in vivo through the following aims: In Aim 1, we will characterize the mode of JAK2 inhibition by USP9X in human islets. In Aim 2, we will assess effects of inhibiting USP9X-JAK2 (with BRD0476) on development and progression of autoimmune diabetes in a mouse model of type 1 diabetes. In Aim 3, we will profile deubiquitinase (DUB) expression and activity in human islets during early T1D development, using activity-based protein profiling (ABPP) and global ubiquitome measurements. The successful outcomes of this proposal are 1) a greater understanding of mechanisms to promote beta-cell survival in early T1D, and 2) a chemical probe to provide translational proof-of-concept. This project will set the stage for developing a biomarker of early-stage T1D development, as well as advanced therapeutic strategies for preventing beta-cell apoptosis in early-stage T1D, representing a potentially curative approach.