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Unveiling the Proteostasis Network of Normal and Disease_Causing Collagen_I

揭示正常和疾病的蛋白质稳态网络_Causing Collagen_I

基本信息

批准号：
9118077
负责人：
Matthew Donald Shoulders
金额：
$ 7.8万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9118077
关键词：
Address Amyloidosis Antibodies Apoptosis Biogenesis Biological Assay Biological Models Cell Line Cell model Cells Cellular Stress Collagen Collagen Diseases Collagen Type I Data Defect Disease Ehlers-Danlos Syndrome Ensure Epitopes Extracellular Matrix Proteins Face Failure Foundations Future Genes Goals Grant Health Homeostasis Huntington Disease Lead Learning Link Literature Mass Spectrum Analysis Methods Modeling Molecular Molecular Chaperones Mutation Osteogenesis Imperfecta Outcome Pathologic Pathology Patients Phenotype Play Prealbumin Production Proteins Proteomics Quality Control RNA Interference Research Role Stem cells Structure System Testing Therapeutic Time Validation Variant Work base bone bone turnover comparative effective therapy fibrillogenesis fibrosarcoma gene therapy high throughput screening improved insight killings mutant new therapeutic target novel therapeutics prevent protein misfolding protein protein interaction protein transport repository skeletal targeted treatment trafficking triple helix

项目摘要

DESCRIPTION (provided by applicant): Autosomal dominant osteogenesis imperfecta (OI) is typically caused by mutations in collagen-I genes that engender brittle bones and other pathologic phenotypes. Severe OI pathology may be linked to the secretion of malformed, mutant strand-containing collagen-I triple helices or to cellular stress owing to misfolding collagen strands accumulating inside cells and ultimately causing apoptosis. Haploinsufficiency owing to reduced collagen-I secretion can also cause OI with moderate pathologic phenotypes. Targeting the cell's protein homeostasis (or proteostasis) network to resolve failures in collagen-I folding and quality control could one day lead to a new therapeutic paradigm for OI. Such a system-targeted therapeutic strategy could also prove valuable for other collagenopathies, such as Ehlers-Danlos Syndrome. However, we must first learn much more about how the cell solves the collagen-I folding problem and how the quality control machinery handles misfolding collagen-I. Here, we deploy quantitative mass spectrometry-based proteomics to identify the proteostasis network machinery responsible for (1) folding and secreting wild-type collagen-I strands, (2) folding and secreting the OI-causing, misfolding collagen-α1(I) Gly247Ser and Cys1299Trp variants, and (3) identifying and disposing of misfolding collagen-I strands. Interactomics studies have not been previously performed with collagen-I owing to the absence of a suitable collagen-I expressing cell model system. We recently overcame this critical roadblock by generating immortalized fibrosarcoma cells that inducibly express wild-type and OI-causing collagen-I tagged with distinct antibody epitopes. We can now selectively immunoprecipitate wild-type and misfolding collagens, along with their interacting partners, from these cells, making comparative interactomics studies possible for the first time. We shall carefully prioritize collagen-I interacting partners we identify on the basis of multiple parameter. Top hits will be validated using RNAi depletion and assays already established in our lab to elucidate how collagen-I homeostasis is influenced by those interacting partners. Our most important findings will eventually be validated in mutation-matched primary cell lines obtained from OI patients via the Coriell Cell Repository. In the longer term, we will extend these studies to other collagen-I variants, study the molecular mechanisms by which the cell solves the collagen-I folding and misfolding problem, develop high throughput assays for collagen folding and secretion, and establish new strategies that adapt the proteostasis network to enhance collagen-I homeostasis and/or prevent the secretion of misfolded collagen-I triple helices.