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

项目总结 胰腺中产生胰岛素的β细胞的丧失导致对注射胰岛素的绝对需求， 导致外周低血糖死亡和糖尿病并发症发病率的重大风险 神经、视网膜、心脏和肾脏。治疗T1D的一个关键目标是阻止这种细胞攻击， 通过阻止对贝塔细胞的免疫错误识别，或者通过保护贝塔细胞免受细胞死亡。然而，一个关键的 在这一领域取得进展的障碍是对小岛中的细胞事件缺乏全面的了解 与β细胞质量的丧失有关。通过表型筛选的方法，我们发现了BRD0476，一种化合物 这是选择性地有效地对抗细胞因子介导的细胞凋亡。对这种化合物的进一步研究表明，它 结合脱泛素酶USP9X，以阻止JAK2和STAT1信号对干扰素γ的反应。我们决定 JAK2可以通过泛素化而使信号失灵，而通过调节USP9X，我们可以使 平衡降低JAK2激酶活性，即使在干扰素γ存在的情况下也是如此。这些结果表明，一个正在出现的 泛素化在调节T1D中的β细胞凋亡中的作用，并提示对此有更好的理解 在T1D发育的早期阶段的过程(及其潜在的失调)可能导致1)能力 识别高危个体，以及2)在T1D早期保留β细胞团的新治疗策略。 使用我们的探针BRD0476和以前没有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USP9X在体外和体内β细胞存活中的作用：在目标1中， 我们将表征USP9X在人类胰岛中抑制JAK2的模式。在目标2中，我们将评估 抑制USP9X-JAK2(含BRD0476)对小鼠自身免疫性糖尿病发生发展的影响 1型糖尿病模型。在目标3中，我们将分析脱泛素酶(DUB)在人类胰岛中的表达和活性 在T1D早期发育期间，使用基于活动的蛋白质图谱(ABPP)和全球泛素组 测量。这项建议的成功成果是：1)更好地理解 促进T1D早期的β细胞存活，以及2)提供翻译概念验证的化学探针。这 该项目将为开发早期T1D开发的生物标记物以及高级 预防早期T1D中β细胞凋亡的治疗策略，具有潜在的疗效 接近。

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.