A Stress-Induced Vicious Cycle In The Development of T1D

（前）胰岛素原介导的ER应激、泛素连接酶激活 和胰腺β细胞中的钙离子处理紊乱 本提案是为了响应发现早期1型糖尿病的申请而提交的 人类胰腺的疾病过程（包括研究信号/处理的可能性） 在T1 D的无症状阶段期间在应激β细胞中失调的途径）。我们 由三位具有不同专业知识的胰腺β细胞生物学家组成的小组（Arvan博士，Soleimanpour博士， Satin）提出了一个关于T1 D中启动β细胞事件的新假设，并认识到， 这些错综复杂的问题超出了任何一个实验室自己能够穷尽研究的范围。具体来说，三 合作的P.I.s提出了一系列β细胞缺陷，这些缺陷可以由亲- 炎症刺激，但随后通过细胞器间支持的失败而进一步传播，包括 内质网（ER）和线粒体。众所周知，ER应激信号转导的激活与细胞凋亡有关。 ER稳态的扰动（由多个潜在的引发剂，包括促炎性 细胞因子）触发包括（但不限于）ER膜蛋白的应激激酶的活化， 福利。新的证据表明，ER应激激酶活性导致磷酸化（从而 激活）位于ER-线粒体接触位点（称为MAM）的一种或多种E3泛素（Ub）连接酶。 我们证实，ER应激相关的E3 Ub连接酶激活刺激泛素化和蛋白酶体周转。 许多MAM常驻蛋白，包括公认的线粒体底物，mitofusin-2，但也 ER向线粒体Ca 2+转移的关键组分，包括IP 3R 1等。ER应激诱发 MAM蛋白组分的降解可以通过Western进行生化和定量评估 印迹;但我们超越这一点，通过测量受损的ER- 线粒体Ca ~（2+）转运我们认为，这种钙失衡进一步损害线粒体功能 包括（但不限于）受损的ATP生成，这加剧了ER稳态的扰动 伴随着ER应激。简而言之，我们认为T1 D的促炎触发因子可以启动一种新的炎症反应。 恶性循环导致β细胞衰竭这一假设将在体外人胰岛以及在体外人胰岛中进行测试。 涉及移植的人胰岛的体内模型。最后，我们建议探测某些触发点， 打破恶性循环，试图挽救β细胞的存活和功能， T1 D发作和进展。 1

A VICIOUS CYCLE OF (PRE)PROINSULIN-MEDIATED ER STRESS, UBIQUITIN-LIGASE ACTIVATION, and DISORDERED Ca2+-HANDLING IN PANCREATIC BETA CELLS This proposal is submitted in response to a request-for-applications for Discovery of Early Type 1 Diabetes Disease Processes in the Human Pancreas (including the possibility of studies of signaling/processing pathways that are dysregulated in stressed beta cells during the asymptomatic phase of T1D). Our group of three pancreatic beta cell biologists with distinct expertise (Drs. Arvan, Soleimanpour, and Satin) brings forward a novel hypothesis about the initiating beta cell events in T1D, with the realization that these intricacies exceed what any one laboratory could exhaustively study on their own. Specifically, the three collaborating P.I.s propose a chain of beta cell defects that can be initiated and exacerbated by pro- inflammatory stimuli but are then further propagated by a failure of inter-organellar support, including the endoplasmic reticulum (ER) and mitochondria. It is well known that activation of ER stress signaling upon perturbation of ER homeostasis (launched from multiple potential initiators, including proinflammatory cytokines) triggers activation of stress kinases that include (but are not limited to) the ER membrane protein, PERK. New evidence suggests that ER stress kinase activity results in phosphorylation (and thereby activation) of one or more E3 ubiquitin (Ub) ligases residing at ER-mitochondrial contact sites known as MAMs. We posit that ER stress-related E3 Ub-ligase activation stimulates ubiquitylation and proteasomal turnover of a number of MAM resident proteins including the well-recognized mitochondrial substrate, mitofusin-2, but also key components of ER-to-mitochondrial Ca2+ transfer including IP3R1 and others. ER stress-provoked degradation of MAM protein components can be evaluated biochemically and quantitatively by Western blotting; but we go beyond this to examine the functional consequences via measurements of impaired ER-to- mitochondrial Ca2+ transfer. We propose that such calcium imbalance further impairs mitochondrial function including (but not limited to) impaired ATP generation, which exacerbates the perturbation of ER homeostasis with concomitant ER stress. In short, we propose that pro-inflammatory triggers of T1D can initiate a vicious cycle leading to beta cell failure. This hypothesis will be tested in human islets in vitro as well as in an in vivo model involving transplanted human islets. Finally, we propose to probe certain trigger points to break the vicious cycle, in an attempt to rescue beta cell survival and function, with the intention of preventing T1D onset and progression. 1