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 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. In this way, our results link thermodynamic stability and evolutionary conservation to experimental studies of different properties of protein fitness landscapes.

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers

“…As an alternative approach to analyzing the consequences of PTEN mutations in the CRC cohort, we leveraged two published datasets probing PTEN lipid phosphatase activity (LPA) and protein abundance, in an approach similar to ref. 52 . Data from the extensive analysis of LPA in yeast 37 , 45 captures ~95% of the non-frameshift mutations from the CRC cohort.…”

Discovering functionally important sites in proteins

“…In a few, favourable cases, multiplexed assays of variant effects (MAVEs) have been used to probe the consequence of almost all individual substitutions using both a functional readout and a readout that probes cellular abundance. Analysis of such data have been used to shed light on the molecular mechanisms underlying perturbed function, and more specifically to pinpoint which functional properties an amino acid residue contributes to ( Jepsen et al, 2020 ; Cagiada et al, 2020 ; Chiasson et al, 2020 ; Faure et al, 2022 ). For example, variants that lose function together with loss of abundance are likely to be caused by perturbations to the overall protein fold and stability, whereas variants that lose function while retaining wild-type-like abundance in the cell are likely to be caused by perturbing sites that directly play a role in function ( Jepsen et al, 2020 ; Cagiada et al, 2020 ; Faure et al, 2022 ).…”

Towards Generalizable Predictions for the Effects of Mutations on G-Protein Coupled Receptor Expression

“…This is consistent with a recent analysis of DMS measurements on the effects of missense variants in PTEN and NUDT15, which shows that evolutionary rates are more closely associated with activity measurements whereas Rosetta ddG values correlate more closely with variant abundance. 33 Our PME measurements of β 2 AR variants include one notable variant (N322L) that illustrates how evolutionary data may obscure the proteostatic effects of certain mutants (Figure 5 A & C). This mutation is heavily penalized due to the extreme conservation of N322, which forms part of the NPXXY motif.…”