Rescued Secretion of Misfolded Mutant Proinsulin

描述(申请人提供)：在2型糖尿病的自然病史中，患者向胰岛素依赖的进展与胰腺β细胞功能障碍、内质网(ER)压力，最终导致胰腺β细胞质量丧失有关。内质网是一个蛋白质折叠隔间，是合成分泌蛋白质的初始起点，分泌蛋白质的错误折叠已被发现与大约100种不同疾病的发病机制有关。在β细胞中，胰岛素原的合成可以占细胞合成的总蛋白质的50%，所以即使是部分胰岛素原的错误折叠也可以作为内质网应激的主要来源。最近，在错折叠胰岛素原突变杂合子的患者中(这类患者预计内质网中约50%的错误折叠胰岛素原突变)，突变INS基因诱导的青年糖尿病综合征(Midy)被认为以几乎100%的外显性触发糖尿病，即每个获得一个Midy突变等位基因的患者。我正在利用这一观察作为一个起点，以便能够研究可能的策略来挽救由错误折叠的胰岛素原引起的表型(S)。首先，错误折叠的胰岛素原阻止了它们的细胞内运输，无法退出内质网，正是从这个起点，它们导致了糖尿病的发病。如果能诱导这些错误折叠的胰岛素原的内质网退出，那么就有可能减轻下游的内质网应激，防止β细胞功能障碍。在本申请中，我描述了一些初步研究，其中我发现Midy突变体之一的胰岛素原G(B23)V的分泌实际上是通过与浓度增加的野生型(WT)胰岛素原共表达而被挽救的-即使野生型(WT)胰岛素原在内质网中被错误折叠的Midy突变原浓度增加所阻断！到目前为止，我的初步数据表明这是一种双向相互作用，我推测这是由突变体和WT胰岛素原之间的二聚化引起的。二聚化的证明，以及天然WT胰岛素原挽救错误折叠的胰岛素原的潜在机制，是本提案的特定目标1的主题。其次，我发现操纵内质网氧化还原蛋白-1(Ero1？)的表达水平也可以挽救Midy突变体胰岛素原-G(B23)V的分泌。这种潜在挽救的机制同样是我特定目标2的主题。总而言之，我的初步发现和我提出的实验代表了一系列工作，假设胰腺β细胞的内质网中错误折叠的胰岛素原积累可能是一个可接近的生物医学问题-治疗可能改善糖尿病患者的β细胞功能障碍和β细胞死亡。 公共卫生相关性：胰腺β细胞内质网(ER)中的分泌蛋白错误折叠，与“ER应激”现象有关，与2型糖尿病的发病机制有关。内质网中的主要分泌蛋白是胰岛素原，本研究的目的是提出两种新的方法来直接挽救内质网中错误折叠的胰岛素原。因此，本提案中概述的实验可作为开发新疗法的初步概念验证研究，目的是保护糖尿病患者的胰岛β细胞功能。

DESCRIPTION (provided by applicant): In the natural history of type 2 diabetes, progression of patients to insulin-dependency is linked to pancreatic beta cell dysfunction, endoplasmic reticulum (ER) stress, and ultimately, loss of pancreatic beta cell mass. The ER is a protein folding compartment that serves as the initial launch point in the synthesis of secreted proteins, and secretory protein misfolding has already been found to be linked to the pathogenesis of roughly a hundred distinct diseases. In beta cells, proinsulin synthesis can account for up to 50% of total protein synthesized by the cell, so even fractional proinsulin misfolding can serve as a dominant source of ER stress. Recently, in patients heterozygous for misfolded proinsulin mutants (such patients would be anticipated to have approximately 50% misfolded proinsulin in the ER), the syndrome of Mutant Ins-Gene Induced Diabetes of Youth (MIDY) is thought to trigger diabetes with virtually 100% penetrance, i.e., in every patient acquiring one MIDY mutant allele. I am capitalizing on this observation as a starting point to enable investigation of potential strategies to rescue the phenotype(s) caused by misfolded proinsulin. First, misfolded proinsulins are blocked in their intracellular transport, unable to exit the ER, and it is from thi starting point that they lead to diabetes pathogenesis. If ER exit of these misfolded proinsulins could be induced, then the reduction of downstream ER stress and prevention of beta cell dysfunction might be achievable. In this application, I describe preliminary studies in which I have found that secretion of one of the MIDY mutants, proinsulin-G(B23)V, is actually rescued by co-expression with an increasing concentration of wild- type (WT) proinsulin - even as WT proinsulin is blocked in the ER by an increasing concentration of misfolded MIDY mutant proinsulin! My preliminary data thus far suggest a bi-directional interaction, which I hypothesize is caused by dimerization between mutant and WT proinsulin. The demonstration of dimerization, and the mechanism of the potential rescue of misfolded proinsulin by native WT proinsulin, is the subject of Specific Aim 1 of this proposal. Secondly, I have found that manipulation of the expression level of the ER-oxidoreductin-1 (Ero1¿) can also rescue secretion of the MIDY mutant proinsulin-G(B23)V. The mechanism of this potential rescue is similarly the subject of my Specific Aim 2. In summary, both my preliminary findings and my proposed experiments represent a body of work on the hypothesis that accumulation of misfolded proinsulin in the ER of pancreatic beta cells may be an approachable biomedical problem - the treatment of which might ameliorate beta cell dysfunction and beta cell death in diabetes. PUBLIC HEALTH RELEVANCE: Secretory protein misfolding in the endoplasmic reticulum (ER) of pancreatic beta cells, which is linked to the phenomenon of "ER stress", has been implicated in the pathogenesis of type 2 diabetes. The major secretory protein in the beta cell ER is proinsulin, and the Aims described herein propose two new approaches to directly rescue misfolded proinsulin in the ER. Thus, the experiments outlined in this proposal serve as initial proof-of- concept studies in the development of novel therapies directed at preserving pancreatic beta cell function in diabetes.