Thermal proteome profiling for analysis of protein sequence variants in human genetic disease

用于分析人类遗传疾病中蛋白质序列变异的热蛋白质组分析

基本信息

批准号：
10560577
负责人：
AMBER L. MOSLEY
金额：
$ 38.64万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-15 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10560577
关键词：
Address Algorithms Alleles Amino Acid Sequence Amyotrophic Lateral Sclerosis Apraxias Area Ataxia Axonal Neuropathy Biochemical Biological Models Biophysics Buffers Cell physiology Cells Characteristics Clinical Complementary RNA Complex Computer Analysis Computing Methodologies Data Data Analyses Data Set Databases Defect Detection Development Disease Drug Targeting Enzyme-Linked Immunosorbent Assay Eukaryota Follow-Up Studies Funding Gene Dosage Genetic Genetic Diseases Genetic Heterogeneity Genetic Variation Genome Goals Heterozygote Human Human Genetics Incidence Individual Investigation Label Lead Measurement Measures Messenger RNA Methods Missense Mutation Modeling Molecular Mutation Neurodegenerative Disorders Nucleic acid sequencing Outcome Pathology Patients Penetrance Peptide Sequence Determination Phosphorylation Pontocerebellar hypoplasia Population Post-Translational Protein Processing Process Protein Complex Subunit Protein Sequence Analysis Proteins Proteome Proteomics Quality Control RNA RNA Degradation Rare Diseases Reproducibility Research Role Screening procedure Signal Transduction Specificity Spectrometry Speed Spinocerebellar Ataxias Statistical Data Interpretation System Testing The Cancer Genome Atlas Therapeutic Variant Work candidate identification chromatin immunoprecipitation clinically significant computational chemistry computerized tools curve fitting exosome experimental analysis experimental study genetic variant genome sequencing helicase high throughput screening in vivo insight interest molecular pathology multidisciplinary mutant oculomotor patient screening protein expression protein function protein profiling protein protein interaction rare genetic disorder tool transcriptome sequencing

项目摘要

Project Summary Many global proteomics studies have focused on the measurement of protein abundance and post- translational modification status as measures of cellular function. These approaches, while highly informative, are not sufficient as standalone approaches to address the role of genetic variation on human protein function in a high-throughput manner. Our preliminary findings show that thermal proteome profiling (TPP) can measure changes in missense mutant protein stability as well as the impacts of mutant protein stability changes on protein-protein interactions (PPIs). We hypothesize that TPP will be sufficient to provide molecular characterization of protein biophysical changes as a consequence of missense mutations associated with human genetic disease. The initial work will focus on the optimization of TPP dataset analysis through development of normalization approaches, curve fitting, and determination of key quality control cutoffs for applications related to human genetic diseases. In parallel, we will perform mutant TPP analysis of human genetic disease-associated protein sequence variants in the RNA-DNA helicase Senataxin and in multiple subunits of the human RNA exosome. Numerous mutations in Senataxin and subunits of the RNA exosome have been clinically associated with the rare neurodegenerative diseases: Ataxia Oculomotor Apraxia 2 (AOA2), Amyotrophic Lateral Sclerosis 4 (ALS4), and PontoCerebellar Hypoplasia (PCH). In addition to these clinically characterized mutations, a number of additional variants have been identified of unknown clinical significance. We propose that mutant TPP could be used to determine if these uncharacterized sequence variants have similar or unique thermal profiles relative to known disease- causing variants. In addition to mutant TPP analyses, we will perform RNA-Sequencing and chromatin immunoprecipitation analysis of a selection of mutants to further delve into the functional consequences of mutant protein expression. Changes in gene dosage for disease causing mutants and the impact of gene dosage on TPP outcomes will also be explored through our recently developed approach for allele-specific thermal profiling. Allele-specific thermal profiling is not possible through non-mass spectrometry-based methods such as ELISA since determination of protein sequence will be required to differentiate the slight changes in protein sequence. Long-term goals include development of mutant TPP analysis to facilitate measurement of protein expression buffering of deleterious alleles, protein-protein interaction network changes, and the impact of altered variant protein levels on the correlation between mRNA and protein abundance levels.

项目概要许多全球蛋白质组学研究都集中在蛋白质丰度和后蛋白质的测量上。翻译修饰状态作为细胞功能的测量。这些方法虽然信息丰富，作为独立方法不足以解决遗传变异对人类蛋白质功能的作用以高通量的方式。我们的初步研究结果表明，热蛋白质组分析 (TPP) 可以测量错义突变蛋白稳定性的变化以及突变蛋白稳定性的影响蛋白质-蛋白质相互作用（PPI）的变化。我们假设 TPP 将足以提供分子与相关的错义突变导致的蛋白质生物物理变化的表征人类遗传病。初步工作将侧重于通过以下方式优化 TPP 数据集分析：标准化方法的开发、曲线拟合以及关键质量控制临界值的确定与人类遗传疾病相关的应用。与此同时，我们将对人类进行突变 TPP 分析。 RNA-DNA 解旋酶 Senataxin 和多种中与遗传病相关的蛋白质序列变异人类RNA外泌体的亚基。 Senataxin 和 RNA 外泌体亚基中存在大量突变临床上与罕见的神经退行性疾病相关：共济失调动眼神经失用症 2 (AOA2)、肌萎缩侧索硬化症 4 (ALS4) 和桥脑小脑发育不全 (PCH)。除了这些临床特征突变，已鉴定出许多未知临床的其他变异意义。我们建议突变的 TPP 可用于确定这些未表征的序列是否相对于已知的致病变异，变异具有相似或独特的热分布。此外突变 TPP 分析，我们将对一个突变体进行 RNA 测序和染色质免疫沉淀分析选择突变体以进一步深入研究突变蛋白表达的功能后果。变化致病突变体的基因剂量以及基因剂量对 TPP 结果的影响也将是通过我们最近开发的等位基因特异性热分析方法进行探索。等位基因特异性热无法通过基于非质谱的方法（例如 ELISA）进行分析，因为需要蛋白质序列来区分蛋白质序列的细微变化。长期目标包括开发突变 TPP 分析，以促进蛋白质表达缓冲的测量有害等位基因、蛋白质-蛋白质相互作用网络变化以及变异蛋白质水平改变的影响 mRNA 和蛋白质丰度水平之间的相关性。