Mutant thermal proteome profiling for characterization of missense protein variants and their associated phenotypes within the proteome [Protein Structure and Folding]

This study characterized global impacts of temperature sensitivity–inducing missense mutations in two different subunits of the 26S proteasome. The majority of alterations identified by RNA-Seq and global proteomics were similar between the mutants, which could suggest that a similar functional disruption is occurring in both missense variants. Results from mTPP, however, provide unique insights into the mechanisms that contribute to the TS phenotype in each mutant, revealing distinct changes that were not obtained using only steady-state transcriptome and proteome analyses. Computationally, multisite λ-dynamics simulations add clear support for mTPP experimental findings. This work shows that mTPP is a precise approach to measure changes in missense mutant–containing proteomes without the requirement for large amounts of starting material, specific antibodies against proteins of interest, and/or genetic manipulation of the biological system. Although experiments were performed under permissive conditions, mTPP provided insights into the underlying protein stability changes that cause dramatic cellular phenotypes observed at nonpermissive temperatures. Overall, mTPP provides unique mechanistic insights into missense mutation dysfunction and connection of genotype to phenotype in a rapid, nonbiased fashion.

Source: Journal of Biological Chemistry - November 27, 2020 Category: Chemistry Authors: Sarah A. Peck Justice, Monica P. Barron, Guihong D. Qi, H. R. Sagara Wiȷeratne, Jose F. Victorino, Ed R. Simpson, Jonah Z. Vilseck, Aruna B. Wiȷeratne, Amber L. Mosley Tags: Genomics and Proteomics Source Type: research