Rare Human Missense Variants can affect the Function of Disease-Relevant Proteins by Loss and Gain of Peroxisomal Targeting Motifs.

Cheng-Shoong Chong,Sebastian Maurer-Stroh,Johannes Berger,Bernhard Hochreiter,Markus Kunze,Martin Krenn

doi:10.3390/ijms20184609

Abstract

Single nucleotide variants (SNVs) resulting in amino acid substitutions (i.e., missense variants) can affect protein localization by changing or creating new targeting signals. Here, we studied the potential of naturally occurring SNVs from the Genome Aggregation Database (gnomAD) to result in the loss of an existing peroxisomal targeting signal 1 (PTS1) or gain of a novel PTS1 leading to mistargeting of cytosolic proteins to peroxisomes. Filtering down from 32,985 SNVs resulting in missense mutations within the C-terminal tripeptide of 23,064 human proteins, based on gene annotation data and computational prediction, we selected six SNVs for experimental testing of loss of function (LoF) of the PTS1 motif and five SNVs in cytosolic proteins for gain in PTS1-mediated peroxisome import (GoF). Experimental verification by immunofluorescence microscopy for subcellular localization and FRET affinity measurements for interaction with the receptor PEX5 demonstrated that five of the six predicted LoF SNVs resulted in loss of the PTS1 motif while three of five predicted GoF SNVs resulted in de novo PTS1 generation. Overall, we showed that a complementary approach incorporating bioinformatics methods and experimental testing was successful in identifying SNVs capable of altering peroxisome protein import, which may have implications in human disease.

Highlights

Inter-individual DNA sequence variations in the human genome provoke diverse consequences ranging from physical trait differences to altered disease susceptibility and even drug therapy failure [1,2,3]
We chose to focus on single nucleotide variants (SNVs) lying in the last three codons of each transcript as they can potentially generate or abolish a peroxisomal targeting signal 1 (PTS1) mediating the interaction with the PTS1 receptor (PEX5) by mutating the C-terminal tripeptide of the analyzed proteins
We examined the possibility of naturally occurring SNVs creating or abolishing a PTS1 through a non-synonymous change in the primary sequence of a protein

Summary

Introduction

Inter-individual DNA sequence variations in the human genome provoke diverse consequences ranging from physical trait differences to altered disease susceptibility and even drug therapy failure [1,2,3]. They have been found to cause protein instability, change protein flexibility, abrogate protein–macromolecular and protein–chemical interactions, reduce enzyme activity, and even modify protein function [7,8,9]. Such SNVs can lead to aberrant protein localization in the cell by inactivating targeting signals [10,11], such effects have hardly been investigated since most targeting signals initiating these translocation processes are rather robust against individual mutations

Methods

Results

Conclusion