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

点击翻译按钮获取中文摘要

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.