Understanding the origins of loss of protein function by analyzing the effects of thousands of variants on activity and abundance

Abstract

Understanding and predicting how amino acid substitutions affect proteins is key to practical uses of proteins, and to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analysed 6749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects, and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyse the structural and mechanistic origins of loss of function, and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance.

Predicting and interpreting large scale mutagenesis data using analyses of protein stability and conservation

“…The data generated by MAVEs have been shown to help predict the status of pathogenic and benign variants, while also serving as useful benchmarks for computational variant classification methods ( Livesey and Marsh, 2020; Frazer et al, 2020 ). More generally, the data can also provide detailed insight into general aspects of protein structure and function ( Gray et al, 2017; Ahler et al, 2019b; Dunham and Beltrao, 2020; Chiasson et al, 2020; Starr et al, 2020; Cagiada et al, 2021; Amorosi et al, 2021 ). The extensive coverage of variants measured in the same assay provides a rich source of data that may be used for protein design, structure prediction and identification of crucial regions related to function and stability ( Stein et al, 2019 ).…”

“…While the detailed relationship between protein stability and abundance is complicated and not fully understood ( Hingorani and Gierasch, 2014; Stein et al, 2019 ), we and others have shown that unstable proteins are often targeted for proteasomal degradation leading to lowered cellular abundance ( Nielsen et al, 2017; Chen et al, 2017; Nielsen et al, 2017; Scheller et al, 2019; Abildgaard et al, 2019; Nielsen et al, 2020 ). Thus, by combining the results from a VAMP-seq experiment with a MAVE probing protein activity it is possible to distinguish between variants that cause loss of function due to lowered abundance from those that change the intrinsic activity of a protein ( Jepsen et al, 2020; Chiasson et al, 2020; Cagiada et al, 2021; Amorosi et al, 2021 ). These experiments and analyses suggest that a relatively large fraction of variants that cause of loss-of-function are due to loss of stability and resulting degradation in the cell.…”

“…As evolution disfavours unstable proteins ( Bloom et al, 2006; Echave and Wilke, 2017 ), there is some correlation between the two approaches, however, there are also differences, e.g. for active sites, where variants are typically only identified as detrimental by evolutionary sequence analysis ( Cheng et al, 2005; Echave, 2019; Jepsen et al, 2020; Cagiada et al, 2021 ). We train a machine learning model that uses ΔΔG and ΔΔE as input to predict variant effects as probed by MAVE experiments.…”

Mapping the degradation pathway of a disease-linked aspartoacylase variant

“…We propose that Hsp110 should be further investigated as a potential therapeutic target to stabilize some ASPA variants and possibly other disease-linked missense protein variants. However, low abundance protein variants, including ASPA C152W, might also affect enzyme catalysis in addition to stability and/or folding [ 32 ], and therefore targeting molecular chaperones is not a universal therapeutic strategy.…”

“…Consequently, it might be possible to uncouple loss of protein stability from loss of function [32], if components of the PQC system required for a protein's degradation can be identified and targeted. Since loss of protein stability is proposed to be a central cause of human diseases [33][34][35][36], this strategy holds wide-ranging therapeutic potential.…”