Rescued Secretion of Misfolded Mutant Proinsulin

拯救错误折叠的突变胰岛素原的分泌

• 批准号: 8458637
• 负责人: Jordan James Wright
• 金额: $ 3.88万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8458637
• 关键词: Accounting; Alleles; Apoptosis; Beta Cell; Cell Death; Cell Line; Cell Survival; Cell physiology; Cells; Co-Immunoprecipitations; Code; Data; Dependency; Deterioration; Development; Diabetes Mellitus; Dimerization; Disease; Endoplasmic Reticulum; Failure; Functional disorder; Genes; Human; Insulin; Intracellular Transport; Investigation; Kinetics; Lead; Link; Measures; Methods; Modeling; Molecular Chaperones; NPM1 gene; Natural History; Non-Insulin-Dependent Diabetes Mellitus; Oxidative Stress; Oxidoreductase; Pancreas; Pathogenesis; Patients; Penetrance; Phenotype; Physiological; Plasmids; Prevention; Process; Proinsulin; Proteins; Quality Control; Radioimmunoassay; Source; Stress; Structure of beta Cell of islet; Syndrome; Testing; Translations; Work; Youth; biological adaptation to stress; endoplasmic reticulum stress; improved; mutant; novel; novel strategies; protein folding; protein misfolding; research study; response; secretory protein

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.

描述（由申请人提供）：在 2 型糖尿病的自然史中，患者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。内质网是一个蛋白质折叠区室，充当分泌蛋白合成的初始启动点，并且已经发现分泌蛋白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总蛋白的 50%，因此即使是部分胰岛素原错误折叠也可能成为 ER 应激的主要来源。最近，在错误折叠胰岛素原突变体杂合的患者中（预计此类患者在 ER 中大约有 50% 错误折叠胰岛素原），突变 Ins 基因诱导的青少年糖尿病 (MIDY) 综合征被认为会以几乎 100% 的外显率引发糖尿病，即每个患者都获得一个 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，错误折叠的胰岛素原在细胞内运输中被阻断，无法离开内质网，并且正是从这个起点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退出内质网，那么下游内质网应激的减少和β细胞功能障碍的预防可能是可以实现的。在本申请中，我描述了初步研究，其中我发现一种 MIDY 突变体胰岛素原-G(B23)V 的分泌实际上可以通过与浓度不断增加的野生型 (WT) 胰岛素原共表达来挽救 - 即使 WT 胰岛素原在 ER 中被浓度增加的错误折叠 MIDY 突变体胰岛素原所阻断！到目前为止，我的初步数据表明存在双向相互作用，我假设这是由突变体和 WT 胰岛素原之间的二聚化引起的。二聚化的论证，以及天然 WT 胰岛素原对错误折叠的胰岛素原的潜在拯救机制，是本提案具体目标 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。这种潜在的拯救机制同样是我的具体目标 2 的主题。总之，我的初步发现和我提出的实验都代表了基于以下假设的大量工作：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解决的生物医学问题 - 对其进行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。