A Stress-Induced Vicious Cycle In The Development of T1D

(前)胰岛素原介导的内质网应激、泛素连接酶激活、 和胰岛β细胞内钙离子处理的紊乱 本提案是应发现早期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T1D的无症状阶段，应激的β细胞中调控失调的通路)。我们的 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、Soleimanour博士和 Satin)提出了一种关于T1D中启动β细胞事件的新假说，认识到 这些错综复杂的情况超出了任何一个实验室能够自己详尽研究的范围。具体地说，三个 合作的P.I.s提出了一连串的β细胞缺陷，这些缺陷可以由亲- 炎性刺激，但随后由于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，内质网应激信号的激活在 内质网稳态的扰动(由多个潜在的启动者发起，包括促炎 细胞因子)触发包括(但不限于)ER膜蛋白的应激蛋白的激活， 额外津贴。新的证据表明，内质网应激激酶活性导致磷酸化(从而 激活)位于内质网线粒体接触部位的一个或多个E3泛素(Ub)连接酶，称为MAM。 我们假设内质网应激相关的E3Ub-连接酶激活刺激A细胞的泛素化和蛋白酶体周转。 MAM驻留蛋白的数量，包括公认的线粒体底物mitofusin-2，但也包括 内质网向线粒体钙离子转运的关键成分包括IP3R1等。急诊室应激 MAM蛋白质组分的降解可以用生化和定量的方法进行评价 Blotting；但我们超越了这一点，通过测量受损的内质网至内质网来检查功能后果。 线粒体钙离子转运。我们认为这种钙失衡会进一步损害线粒体的功能。 包括(但不限于)ATP生成受损，这加剧了内质网稳态的扰动 伴随着内质网压力。简而言之，我们认为T1D的促炎因子可以启动一种 导致β细胞衰竭的恶性循环。这一假说将在体外的人类胰岛以及在 移植人胰岛的活体模型。最后，我们建议探索某些触发点，以 打破恶性循环，试图挽救贝塔细胞的生存和功能，意在防止 T1D的发病和进展。 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