Secretory Pathway Protein Degradation Maintains Insulin Biogenesis + Secretion

胰岛β细胞会合成大量的胰岛素。越来越多的证据表明，胰岛素生物合成的任何缺陷(遗传的或后天的)都会导致糖尿病。我们知道胰岛素的生物合成始于胰岛素原的翻译。这种短暂的前体必须移位到内质网(ER)，去除信号肽，正确折叠胰岛素原，才能成功地从内质网输出到远端分泌途径，最终发生胰岛素原向胰岛素的加工和分泌颗粒中的胰岛素储存。相反，不成功的分子甚至可能在转运到内质网之前就被降解了，或者可能被抑制了从内质网顺行输出--事实上，强有力的证据表明，错误折叠的胰岛素原分子是被降解的目标。分泌途径蛋白的降解还涉及其他内源性底物，这些底物有助于分化的胰岛β细胞表型。这种多P.I.R01的竞争性延续将有助于阐明分泌途径蛋白质处理的三个主要机制--转位前降解、内质网相关降解(ERAD)和自噬--都是如何对正常的β细胞功能至关重要的。这项提议延续了三位密切合作的研究人员(齐、蔡、阿文)的长期合作关系，他们正是这些过程的专家：胰岛素原转位到内质网管腔，随后胰岛素原折叠/错折叠，通过ERAD的分泌途径蛋白质降解，以及我们认为主要是ER自噬(ER-吞噬)的ER到溶酶体的降解途径。我们有充分的理由相信，这些质量控制机制中的缺陷与胰岛素不足所致的2型糖尿病(T2D)有关，初步数据支持了这一信念。在这项研究中，我们试图研究与胰岛β细胞早期分泌途径相关的三个相互关联的区域。首先，我们将继续进行研究，在这些研究中，跨ER膜的胰岛素前原转位的不忠直接与胰岛素原和胰岛素生物合成缺陷有关，从而直接导致糖尿病。其次，我们将跟进一些引人注目的初步数据，这些数据表明，胰岛β细胞的去分化是由有效的ERAD功能丧失引发的，也直接导致胰岛素缺乏性糖尿病。最后，我们不仅深入研究了触发错误折叠的胰岛素原的内质网吞噬降解的内质网因素，而且还提出了对无效或不适当的内质网吞噬如何触发β细胞衰竭的更深层次的理解，这也直接导致胰岛素缺乏性糖尿病。这些新的研究方向引导我们寻求一种新的治疗方法来治疗β细胞分泌途径功能障碍，重点是刺激细胞内蛋白质清除机制，以预防糖尿病的发生和/或限制其进展。

Pancreatic beta-cells synthesize large quantities of insulin. Growing evidence indicates that any of a number of deficiencies in insulin biosynthesis (genetic, or acquired) can lead to diabetes. We know that insulin biosynthesis begins with translation of preproinsulin. This short-lived precursor must be translocated into the endoplasmic reticulum (ER), signal peptide excised, and proinsulin properly folded in order to undergo successful export from the ER for delivery to the distal secretory pathway in which proinsulin-to-insulin processing and insulin storage in secretory granules finally occurs. In contrast, unsuccessful molecules may be degraded before they are even translocated into the ER, or may be restrained from anterograde export from the ER — indeed, strong evidence indicates that misfolded proinsulin molecules are targeted for degradation. Secretory pathway protein degradation also involves other endogenous substrates that contribute to the differentiated pancreatic beta cell phenotype. The competing continuation of this multi-P.I. R01 will help clarify how three major mechanisms of secretory pathway protein disposal – pre-translocation degradation; ER-Associated Degradation (ERAD); and Autophagy – are all critical for proper beta-cell function. This proposal continues the longstanding association of three tightly collaborative investigators (Qi, Tsai, Arvan) that are experts in exactly these processes: preproinsulin translocation into the ER lumen with the subsequent folding/misfolding of proinsulin, secretory pathway protein degradation via ERAD, and an ER-to-lysosome degradative pathway that we believe is primarily ER-autophagy (ER-phagy). We have strong reason to believe that defects in these quality control mechanisms are linked to type 2 diabetes (T2D) as a result of insulin insufficiency, and this belief is supported by preliminary data. In this proposal, we seek to examine three interlinked areas related to the early secretory pathway of pancreatic beta-cells. For one, we will pursue studies in which infidelity of preproinsulin translocation across the ER membrane is directly linked to deficient proinsulin and insulin biosynthesis, leading directly to diabetes. Second, we will follow-up on some remarkable preliminary data demonstrating that de-differentiation of pancreatic beta-cells is triggered by a loss of efficient ERAD function, also leading directly to insulin-deficient diabetes. Finally, we not only delve deeply into the ER factors that trigger ER-phagic degradation of misfolded proinsulin, but we also propose a deeper understanding of how ineffective or improper ER-phagy can trigger beta cell failure, which also leads directly to insulin-deficient diabetes. These new research directions lead us to pursue a novel therapeutic approach to beta-cell secretory pathway dysfunction focused on stimulating intracellular protein clearance mechanisms, in order to prevent diabetes onset and/or limit its progression.