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：
10349591
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/10349591
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 RNA immunoprecipitation sequencing Rare Diseases Reproducibility Research Role Screening procedure Signal Transduction Specificity Speed Spinocerebellar Ataxias Statistical Data Interpretation System Testing The Cancer Genome Atlas Therapeutic Variant Work base 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.

项目摘要许多全球蛋白质组学研究都集中于蛋白质丰度和蛋白质后的测量翻译修饰状态作为细胞功能的度量。这些方法虽然很有帮助，但不足以解决遗传变异对人类蛋白质功能的作用的独立方法以高通量方式。我们的初步发现表明，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相互作用（PPI）的变化。我们假设TPP足以提供分子蛋白质生物物理变化的表征是由于与人遗传疾病。最初的工作将重点介绍通过TPP数据集分析的优化开发标准化方法，曲线拟合以及确定关键质量控制截止与人类遗传疾病有关的应用。同时，我们将对人类进行突变TPP分析 RNA-DNA解旋酶鼻动蛋白的遗传性疾病相关蛋白序列变异，在多个人RNA外泌体的亚基。 RNA外泌体的Senataxin和亚基中的许多突变在临床上与罕见的神经退行性疾病有关：动脉秀动作失调2 （AOA2），肌萎缩性外侧硬化症4（ALS4）和pontocerebellar发育不全（PCH）。除了这些临床表征突变，已经确定了许多其他变体意义。我们建议可以使用突变体TPP来确定这些未表征的序列是否相对于已知疾病的变异，变体具有相似或独特的热谱。此外突变TPP分析，我们将对A进行RNA测序和染色质免疫沉淀分析选择突变体以进一步研究突变蛋白表达的功能后果。更改在引起突变体的疾病基因剂量和基因剂量对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探索。等位基因特异性热通过基于非质谱的方法，例如ELISA，无法进行分析将需要蛋白质序列来区分蛋白质序列的轻微变化。长期目标包括开发突变体TPP分析以促进测量的蛋白质表达缓冲有害等位基因，蛋白质 - 蛋白质相互作用网络的变化以及变化变化蛋白水平的影响关于mRNA和蛋白质丰度水平之间的相关性。