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

项目摘要 许多全球蛋白质组学研究都集中在蛋白质丰度的测量和后处理上。 翻译修饰状态作为细胞功能的量度。这些方法虽然信息量很大， 不足以作为独立的方法来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 蛋白质生物物理变化的表征作为错义突变的结果与 人类遗传疾病最初的工作将集中在TPP数据集分析的优化， 开发标准化方法、曲线拟合和确定关键质量控制截止值， 与人类遗传疾病有关的应用。与此同时，我们将进行突变的TPP分析人类 在RNA-DNA解旋酶Senataxin和多种遗传疾病相关蛋白序列变异中， 人RNA外泌体的亚基。Senataxin和RNA外泌体亚基的大量突变 临床上与罕见的神经退行性疾病有关：共济失调眼失用症2 （AOA2）、肌萎缩侧索硬化4（ALS 4）和脑桥小脑发育不全（PCH）。除了这些 在临床特征突变中，已经鉴定了许多未知临床特征突变的额外变体。 意义我们建议突变的TPP可以用来确定这些未表征的序列 变体相对于已知的致病变体具有相似或独特的热分布。除了 突变的TPP分析，我们将进行RNA测序和染色质免疫沉淀分析的一个 突变体的选择，以进一步深入研究突变蛋白表达的功能后果。变化 在致病突变体的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进行探索。等位基因特异性热 不可能通过非基于质谱的方法如ELISA进行分析，因为 将需要蛋白质序列来区分蛋白质序列中的微小变化。长期目标 包括开发突变TPP分析，以便于测量 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。

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.