Improving Proinsulin Folding to Ameliorate Type II Diabetes

项目摘要 糖尿病是21世纪增长最快的健康挑战之一，影响着3000万人， ≥每年死亡80,000人，涉及美国国民医疗支出的约15%。2型糖尿病(T2D)是 最常见的糖尿病形式，与循环中胰岛素量不足有关，因为 身体对荷尔蒙不敏感。维持胰岛素储存池需要合成~6000 胰岛素原(PI)分子/细胞/秒，每个分子都被送到内质网(ER)进行折叠。连 在胰岛素抵抗状态下需要更多的分子。值得注意的是，我们发现PI进入异常状态 二硫键连接的分子间复合体，即使在健康的(人和小鼠)胰岛中也是如此。在符合以下条件的情况下 需求增加了胰岛素的产生(甚至糖尿病前期)，这些复合体急剧增加，从而限制了 胰岛素的产生。我们提供了新的关键证据，证明这些异常折叠的PI复合体可以分解为 内质网中的单体PI。我们最近阐明了第一个人类PI相互作用组识别PI的图谱 折叠修饰符。人类胰岛中最重要的PI相互作用因子是内质网伴侣蛋白Bip，我们现在 新的证据(包括功能的获得和功能的丧失)表明，这种相互作用，由BIP共同支持 伴侣是产生胰岛素原折叠(和限制错误折叠)所绝对需要的，导致 顺行运输成功。在我们的研究中，我们创造了一种新型的Bip标记小鼠，它可以用于 首次确定PI折叠的基本步骤，这些步骤对胰岛素的产生至关重要。此外，我们证明了 Bip及其辅助伴侣P58IPK的表达增加显著减少了高水平的 相对分子质量的PI络合物。因此，我们的发现开启了一种可能性，即药物干预可能 改善依赖伴侣的PI折叠，这可能会减弱T2D。当我们开始阐明人类的PI 在折叠途径方面，我们正在开发平行动物模型来确定PI是如何折叠/错折叠的。在这里我们 建议：1)机械地剖析内质网中BiP和其他PI交互作用如何协调成功的PI 折叠并确定PI折叠的哪一步(S)在T2D中出错；2)确定PI互动组如何变化 在人类T2D中；确定T2D患者胰岛中改变的PI相互作用的功能；并利用 新的方法来测量生产的PI折叠/运输在？细胞。我们将整合生理学研究 人类胰岛具有新的遗传和生化方法，以产生对 PI折叠动态平衡如何在健康和疾病中影响？细胞功能。我们认为这一假设是一种 对于NIDDK的使命至关重要的高影响力的想法，我们现在带来了工具、分析和方法 目前在其他地方是不可用的。

Project Summary Diabetes is among the fastest growing health challenges of the 21st century, affecting >30 million people, with ≥80,000 deaths annually, and involving ~15% of U.S. national health expenditures. Type 2 diabetes (T2D) is the most common form of diabetes, which is linked to an insufficient amount of circulating insulin because of the body’s insensitivity to the hormone. Maintenance of the insulin storage pool requires synthesis of ~6000 proinsulin (PI) molecules/ß-cell/second, each delivered to the endoplasmic reticulum (ER) for folding. Even more molecules are needed in states of insulin resistance. Significantly, we discovered that PI enters aberrant disulfide-linked intermolecular complexes, even in healthy (human and murine) islets. Under conditions that demand increased insulin production (even prediabetes), these complexes dramatically increase, thus limiting insulin production. We show new key evidence that these aberrantly folded PI complexes can be resolved to monomeric PI within the ER. We recently elucidated the first map of the human PI interactome identifying PI folding modifiers. The most significant PI interactor in human islets is the ER chaperone BiP and we present new evidence (both gain of function, and loss of function) that this interaction, supported by BiP co- chaperones, is absolutely required for productive proinsulin folding (and limiting misfolding), leading to successful anterograde transport. For our studies we generated a novel BiP-tagged mouse that can for the first time identify fundamental steps in PI folding essential for insulin production. Moreover, we show that increased expression of BiP and its co-chaperone P58IPK dramatically reduces accumulation of the high molecular weight PI complexes. Thus, our discoveries open the possibility that pharmacologic intervention may improve chaperone-dependent PI folding, and this may attenuate T2D. As we begin to elucidate the human PI folding pathway, we are developing parallel animal models to determine how PI folds/misfolds. Here we propose to: 1) Mechanistically dissect how BiP and additional PI interactors in the ER orchestrate successful PI folding and determine which step(s) of PI folding go awry in T2D; 2) Identify how the PI interactome changes in human T2D; determine the function of altered PI interactions in islets from patients with T2D; and utilize novel assays to measure productive PI folding/trafficking in ß-cells. We will integrate physiologic studies of human islets with novel genetic and biochemical approaches to generate a comprehensive understanding of how PI folding homeostasis impacts ß-cell function in health and disease. We believe that this hypothesis is a high-impact idea essential to the mission of the NIDDK, and we now bring tools, assays, and approaches that are not currently available anywhere else.