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，患者进展为胰岛素依赖与胰腺β细胞功能障碍、内质网（ER）应激以及最终胰腺β细胞群丢失相关。ER是蛋白质折叠区室，其充当分泌蛋白质合成的初始启动点，并且已经发现分泌蛋白质错误折叠与大约一百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的总蛋白质的高达50%，因此即使是部分胰岛素原错误折叠也可作为ER应激的主要来源。最近，在错误折叠的胰岛素原突变体杂合子的患者中（预期这样的患者在ER中具有大约50%的错误折叠的胰岛素原），突变的胰岛素基因诱导的青年糖尿病（MIDY）综合征被认为以几乎100%的突变率触发糖尿病，即，每个病人都有一个MIDY突变等位基因我利用这一观察结果作为出发点，使调查的潜在战略，以挽救表型所造成的错误折叠胰岛素原。首先，错误折叠的胰岛素原在其细胞内转运中被阻断，不能离开ER，并且正是从这个起点，它们导致糖尿病发病机制。如果可以诱导这些错误折叠的胰岛素原的ER退出，则可以实现下游ER应激的减少和β细胞功能障碍的预防。在本申请中，我描述了初步研究，其中我发现MIDY突变体之一胰岛素原-G（B23）V的分泌实际上通过与浓度增加的野生型（WT）胰岛素原共表达而被拯救-即使WT胰岛素原在ER中被浓度增加的错误折叠MIDY突变体胰岛素原阻断！迄今为止，我的初步数据表明了一种双向相互作用，我假设这是由突变型和野生型胰岛素原之间的二聚化引起的。二聚化的证明和天然WT胰岛素原对错误折叠的胰岛素原的潜在拯救机制是本提案的具体目标1的主题。其次，我发现，ER-氧化还原蛋白-1（Ero 1 <$）表达水平的调控也可以拯救MIDY突变体胰岛素原-G（B23）V的分泌。这种潜在拯救的机制类似于我的具体目标2的主题。总之，我的初步发现和我提出的实验都代表了一个假设，即错误折叠的胰岛素原在胰腺β细胞内质网中的积累可能是一个可以解决的生物医学问题-治疗可能会改善糖尿病中的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.