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: 8312064
• 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/8312064
• 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. 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.

描述（由适用提供）：在2型糖尿病的自然病史中，患者胰岛素依赖性的进展与胰腺β细胞功能障碍，内质网应激（ER）胁迫以及最终的胰腺β细胞肿块有关。 ER是一个蛋白质折叠室，是分泌蛋白质合成的初始发射点，已经发现秘密蛋白质折叠与大约一百种不同疾病的发病机理有关。在β细胞中，蛋白酶合成最多可占细胞合成的总蛋白质的50％，因此即使是分数促硫素错误折叠也可以作为ER应力的主要来源。最近，在患者中因杂合蛋白突变体杂合子（预计将在ER中患有大约50％的错误折叠二硫素的50％），认为突变体Ins-Gene诱导的青年糖尿病（MIDY）的综合征被认为会触发糖尿病的糖尿病，实际上是100％的渗透性，即每位患者的糖尿病。我将这一观察结果付诸实践，这是一个起点，可以调查潜在的策略，以挽救因折叠蛋白错误而引起的表型。首先，错误折叠的促蛋白在其细胞内转运中被阻塞，无法退出ER，并且从这起点开始导致糖尿病发病机理。可以诱导这些错误折叠蛋白的ER出口，然后减少下游ER应力，预防β细胞功能障碍可能会成功。在此应用中，我描述了初步研究，其中我发现一种中硫酸突变体之一的分泌proinsulin-g（b23）V实际上是通过共表达的反应，而越来越浓度的野生型（WT）proinsulin-即使WT proinsulin越来越被浓度越来越多，因为越来越多的中型突变型突变型突变蛋白在艾伯中被抑制了！到目前为止，我的初步数据表明双向相互作用，我假设这是由于突变体和WT促硫素之间的尺寸引起的。尺寸的证明以及本地WT Proinsulin对错误折叠蛋白的潜在营救的机制是本提案的特定目的1的主题。其次，我发现对ER-OXIDUCTIN-1（ERO1¿）的表达水平的操纵也可以挽救Midy突变蛋白-G（B23）V的分泌。这种潜在救援的机制同样是我的特定目标2的主题。总而言之，我的初步发现和建议的实验都代表了一项关于胰腺β细胞中错误折叠的促二硫素的积累的作品，这可能是适当的生物医学问题 - 对哪些疾病的治疗可能是糖尿病的治疗 - 可能会舒张地降低beta betabet beter celbers dysfuntion dyysfunction。 公共卫生相关性：胰腺β细胞内质网（ER）中的秘书蛋白质错误折叠，与“ ER胁迫”现象有关，已在2型糖尿病的发病机理中实现。 β细胞ER中的主要秘密蛋白质是促蛋白，在此提出的提议中描述的目的两种新方法可以直接营救ER中的错误折叠的proinsulin。这是在该提案中概述的实验是针对保存糖尿病中胰腺β细胞功能的新疗法开发的初步概念验证研究。