Preproinsulin Translocation and Processing at the ER Membrane: a link to Diabetes

描述（由申请人提供）：大约2360万美国人（占人口的7.8%）患有糖尿病-由于胰岛素抵抗，大多数人需要产生更多的胰岛素，但几乎所有人都患有胰腺β细胞功能障碍。在胰岛β细胞中，胰岛素合成始于前体前胰岛素原，其必须经历共翻译易位进入内质网（ER）、信号肽（SP）切割和下游胰岛素原折叠。这些最早期的事件对胰岛素生物合成至关重要，但相对而言研究不足。在过去的三年中，四个前胰岛素原SP突变已被报道导致人类糖尿病，使调查这些最早的事件特别及时。虽然已知胰岛素单倍不足不会引起糖尿病，但具有前胰岛素原SP突变的患者是杂合子，表明突变体以显性阴性方式起作用。由这些突变体引起的细胞衰竭的分子机制仍然未知。有趣的是，与SP突变体相关的糖尿病表型范围从由A（SP24）D引起的严重的糖尿病发病到与R（SP 6）C或H相关的轻度成人发病糖尿病。我推测这两类SP突变体通过两种不同的机制导致β细胞衰竭。在一种情况下，我提出，无效的共翻译易位[R（SP 6）C]导致未易位突变体的胞质积累，这对β细胞是缓慢有毒的，导致成人发病的糖尿病，这可能类似于阿尔茨海默氏症和其他一些神经退行性疾病的发病机制。在另一种情况下，我建议失败的SP切割[A（SP24）D]干扰下游胰岛素原折叠，导致突变体的ER保留，与共表达的野生型（WT）胰岛素原异常相互作用，并阻止其ER退出反式，减少胰岛素的产生，并在生命早期引发严重的胰岛素缺乏型糖尿病。该建议旨在更好地理解胰岛素生物合成的最早事件的协调，并定义由这些事件的缺陷引起的胰岛细胞衰竭的分子机制。最终目标是开发新的策略来预防由错误折叠（前）胰岛素原引起的糖尿病的发展。提出了三个具体目的：1）检查前胰岛素原共翻译易位、SP切割和下游胰岛素原折叠的协调; 2）定义将允许WT胰岛素原从由突变体（前）胰岛素原引起的阻断中逃逸的遗传操作; 3）鉴定可以防止由突变体前胰岛素原引起的胰岛细胞衰竭的小分子。越来越多的证据表明，ER应激和胰岛素原错误折叠在最常见形式的糖尿病（2型）的发病机制中起作用，其不涉及任何前胰岛素原编码序列突变。这些新的致糖尿病突变体，其中潜在的（前）胰岛素原错误处理-错误折叠是明确的，是理解胰岛细胞衰竭的分子机制和测试旨在预防糖尿病的实验疗法的理想模型。

DESCRIPTION (provided by applicant): Approximately 23.6 million Americans (7.8% of the population) have diabetes - most needing to produce more insulin because of insulin resistance, yet nearly all with pancreatic ß-cell dysfunction. In ß-cells, insulin synthesis begins with the precursor, preproinsulin, which must undergo co-translational translocation into the endoplasmic reticulum (ER), signal peptide (SP) cleavage, and downstream proinsulin folding. These earliest events are critical to insulin biosynthesis, but they are relatively understudied. Over the past three years, four preproinsulin SP mutations have been reported to cause human diabetes that make investigation of these earliest events especially timely. While it is known that insulin haploinsufficiency does not cause diabetes, patients with preproinsulin SP mutations are heterozygotes, suggesting that mutants act in a dominant- negative fashion. The molecular mechanisms of ß-cell failure caused by these mutants remain unknown. Interestingly, diabetes phenotypes associated with the SP mutants ranges from severe neonatal-onset diabetes caused by A(SP24)D, to mild adult-onset diabetes associated with R(SP6)C or H. I hypothesize that these two classes of SP mutants cause ß-cell failure through two distinct mechanisms. In one case, I propose that inefficient co-translational translocation [of R(SP6)C] causes cytosolic accumulation of untranslocated mutant that is slowly toxic to ß-cells, leading to adult-onset diabetes that may be akin to the pathogenesis of Alzheimer's and some other neurodegenerative diseases. In the other case, I propose that failed SP cleavage [of A(SP24)D] disturbs downstream proinsulin folding, causing ER retention of the mutant that abnormally interacts with co-expressed wild-type (WT) proinsulin and blocks its ER exit in trans, decreasing insulin production and initiating severe insulin-deficient diabetes in early life. This proposal aims to better understand the coordination of the earliest events of insulin biosynthesis and define the molecular mechanisms of ß-cell failure caused by defects of those events. The ultimate goal is to develop novel strategies to prevent development of diabetes caused by misfolded (pre)proinsulin. Three Specific Aims are proposed: 1) To examine coordination of preproinsulin co-translational translocation, SP cleavage, and downstream proinsulin folding; 2) To define genetic manipulations that would allow WT proinsulin to escape from blockade caused by mutant (pre)proinsulins; 3) To identify small molecules that could prevent ß-cell failure caused by mutant preproinsulins. Accumulating evidence suggests that ER stress and proinsulin misfolding plays a role in the pathogenesis of the most common form of diabetes (type 2) which does not involve any preproinsulin coding sequence mutations. These new diabetogenic mutants, in which underlying (pre)proinsulin mishandling-misfolding is unequivocal, are ideal models for understanding molecular mechanisms of ß-cell failure, and for testing experimental therapies aiming at preventing diabetes.