Rescued Secretion of Misfolded Mutant Proinsulin

拯救错误折叠的突变胰岛素原的分泌

基本信息

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
项目类别：
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

项目摘要

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。是一种蛋白质折叠区室，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β细胞中大约一百种不同疾病的发病机制有关。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，在错误折叠的胰岛素原突变体杂合的患者中（预计此类患者大约有 10%）。 ER 中 50% 错误折叠的胰岛素原），突变 Ins 基因诱发的青少年糖尿病 (MIDY) 综合征被认为可引发糖尿病100% 外显率，即在每位患者中获得一个 MIDY 突变等位基因，我利用这一观察结果来研究挽救由错误折叠的胰岛素原引起的表型的潜在策略。它们的细胞内运输，无法离开内质网，正是从这个起点，它们导致了糖尿病的发病机制。如果可以诱导这些错误折叠的胰岛素原的内质网离开，那么减少下游 ER 应激和预防 β 细胞功能障碍可能是可以实现的。在本申请中，我描述了初步研究，其中我发现 MIDY 突变体之一胰岛素原-G(B23)V 的分泌实际上是由 co 拯救的。 -随着野生型（WT）胰岛素原浓度的增加而表达 - 即使 WT 胰岛素原在 ER 中被错误折叠的 MIDY 突变胰岛素原浓度增加所阻断！我加强的双向相互作用是由突变体和 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 ER-氧化还原素-1 (Ero1¿ ）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代表了一系列工作。该假设认为，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。公共健康相关性：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。β 细胞中的主要分泌蛋白。 ER 是胰岛素原，本文描述的目的提出了两种直接拯救 ER 中错误折叠的胰岛素原的新方法。因此，本提案中概述的实验作为初步实验。开发旨在保护糖尿病胰腺β细胞功能的新疗法的概念验证研究。