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

项目概要 胰腺中产生胰岛素的β细胞的损失导致绝对需要注射胰岛素， 导致外周低血糖死亡和糖尿病并发症发病的重大风险 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β细胞的免疫错误识别或保护β细胞免于细胞死亡。然而，一个关键的 该领域取得进展的障碍是缺乏对胰岛细胞事件的完整了解，这些细胞事件有助于 导致β细胞质量的损失。使用表型筛选方法，我们发现了 BRD0476，一种化合物 它选择性地对抗细胞因子介导的细胞凋亡。对该化合物的进一步研究表明， 结合去泛素酶 USP9X 以停止响应 IFNγ 的 JAK2 和 STAT1 信号传导。我们确定 JAK2 可以通过泛素化而变得信号失灵，通过调节 USP9X，我们可以提示 即使存在 IFNγ，也能保持 JAK2 激酶活性降低的平衡。这些结果表明一个新兴的 泛素化在调节 T1D β 细胞凋亡中的作用，并表明对此有更深入的了解 T1D 发展早期阶段的过程（及其潜在的失调）可能导致 1) 识别高危个体，2) 在早期 T1D 中保留 β 细胞质量的新治疗策略。 使用我们的探针 BRD0476 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 通过以下目标了解 USP9X 在体外和体内 β 细胞存活中的作用： 在目标 1 中， 我们将描述 USP9X 在人胰岛中抑制 JAK2 的模式。在目标 2 中，我们将评估 抑制 USP9X-JAK2（与 BRD0476）对小鼠自身免疫性糖尿病的发生和进展 1型糖尿病模型。在目标 3 中，我们将分析人类胰岛中的去泛素酶 (DUB) 表达和活性 在 T1D 早期发展过程中，使用基于活性的蛋白质分析 (ABPP) 和全局泛素组 测量。该提案的成功成果是 1) 更好地理解了机制 促进早期 T1D 中 β 细胞的存活，2) 提供转化概念验证的化学探针。这个 该项目将为开发早期 T1D 发展以及高级阶段的生物标志物奠定基础 预防早期 T1D β 细胞凋亡的治疗策略，代表了一种潜在的治疗方法 方法。

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.