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蛋白质组学后 翻译修饰状态作为细胞功能的衡量标准。这些方法虽然信息量很大， 作为单独的方法来解决遗传变异对人类蛋白质功能的作用是不够的 以高吞吐量的方式。我们的初步发现表明，热蛋白质组图谱(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 蛋白质相互作用(PPI)的变化。我们假设TPP将足以提供分子 错义突变导致的蛋白质生物物理变化的特征 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 制定归一化方法、曲线拟合和确定关键质量控制下限 与人类遗传病相关的应用。同时，我们将对人类的突变TPP进行分析 遗传病相关蛋白序列变异体在RNA-DNA解旋酶中的表达 人类RNA外切体的亚基。感觉神经毒素和RNA外切体亚单位的大量突变 临床上与罕见的神经退行性疾病有关：共济失调、动眼运动性失用2 AOA2、肌萎缩侧索硬化症4(ALS4)和桥小脑发育不全(PCH)。除了这些之外， 临床特征的突变，一些未知的临床变异已经被确定 意义。我们认为突变的TPP可以用来确定这些未表征的序列是否 与已知的致病变异体相比，变异体具有相似或独特的热分布。除了……之外 突变的TPP分析，我们将进行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谱分析方法进行了探索。等位基因特异性热效应 不可能通过非基于质谱学的方法，如酶联免疫吸附试验，因为确定 需要蛋白质序列才能区分蛋白质序列的细微变化。长期目标 包括开发突变TPP分析以便于测量 有害等位基因、蛋白质-蛋白质相互作用网络的改变以及蛋白质水平变异的影响 关于信使核糖核酸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.