Rescued Secretion of Misfolded Mutant Proinsulin

描述（由申请人提供）：在2型糖尿病的自然史中，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）应激以及最终胰腺β细胞质量的损失有关。内质网是一种蛋白质折叠区，是合成分泌蛋白的初始起点，而分泌蛋白的错误折叠已经被发现与大约100种不同疾病的发病机制有关。在β细胞中，胰岛素原合成可占细胞合成总蛋白的50%，因此即使是部分胰岛素原错误折叠也可能是内质网应激的主要来源。最近，在错误折叠的胰岛素原突变体杂合的患者中（这类患者预计内质网中有大约50%的胰岛素原折叠错误），突变型胰岛素基因诱导的青年糖尿病（MIDY）综合征被认为几乎100%外显率触发糖尿病，即每个患者获得一个MIDY突变等位基因。我利用这一观察结果作为起点，研究挽救由错误折叠的胰岛素原引起的表型的潜在策略。首先，错误折叠的原胰岛素在细胞内转运受阻，无法离开内质网，正是从这个起点导致糖尿病发病。如果可以诱导这些错误折叠的原胰岛素的内质网退出，那么减少下游内质网应激和预防β细胞功能障碍可能是可以实现的。在这个应用程序中，我描述了我已经发现的一个MIDY突变体的分泌，原胰岛素- g (B23)V，实际上是通过与野生型（WT）胰岛素原的浓度增加的共表达来拯救的——即使WT胰岛素原在ER中被错误折叠的MIDY突变体胰岛素原的浓度增加所阻断！到目前为止，我的初步数据表明双向相互作用，我假设这是由突变体和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原胰岛素对错误折叠的胰岛素原的潜在拯救机制，是本提案Specific Aim 1的主题。其次，我发现操纵er -氧化还原素-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。这种潜在救助的机制与我的具体目标2的主题类似。总之，我的初步发现和我提出的实验都代表了一个假设，即在胰腺β细胞内质网中错误折叠的胰岛素原的积累可能是一个可接近的生物医学问题，其治疗可能改善β细胞功能障碍和β细胞死亡。

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.