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

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.

Keywords: PEX5; disease; gnomAD; missense variant; mistargeting; peroxisomal targeting signal 1; peroxisome; protein transport.

Figure 1

Three-dimensional crystal structure of a PTS1 receptor-ligand complex from Reference [29] (pdb ID = 2c0l), illustrated using Yasara. The PTS1 tripeptide motif (yellow) is located at the extreme C-terminus of the protein (pale red) and is extended and bound to the tetratricopeptide repeat (TPR) region of the receptor (pale green) (C-terminus extension from ligand is highlighted in opaque colors of red and yellow).

Figure 2

Overall bioinformatics workflow for the mining of sequence variation data from gnomAD and the selection of SNVs for experimental testing. (A) 32,985 bi- or multi-allelic gnomAD SNVs capable of altering at least one amino acid in the tripeptide motifs of 23,064 proteins and generating over 7000 unique mutant (MT) tripeptide combinations from 5614 unique wild-type (WT) tripeptides attained (B) Prioritization of variants for testing from the SNVs retrieved from gnomAD. TMD, Transmembrane domain; a.a, amino acid.

Figure 3

Study of PTS1 loss of function mutations by SNV: (A–J) COS7 cells were transfected with expression plasmids for different EGFP-PTS1 variants, encoding the C-terminus of multifunctional protein (HSD17B4) (A) or the variants K760E (B) and A759P (C), of d-amino acid oxidase (DAO) (D), or the variant S345F (E), of acyl-CoA oxidase 2 (ACOX2) (F), or the variant K680T (G), and of EHHADH (H) or the variants S721G (I) or S721N (J). The subcellular localization was determined by fluorescence microscopy (EGFP, green) in combination with immunofluorescence microscopy of the peroxisomal marker PMP70 (red). I,J: white arrows indicate co-localization between PMP70 and a small fraction of EGFP. Scale bars indicate 20 µm and white squares define enlarged areas.

Figure 4

Study of PTS1 gain of function mutations by SNV: (A–J) COS7 cells were transfected with expression plasmids for different EGFP-PTS1 variants, encoding the C-terminus of PPP4R4 (A) or the variant P873L (B), of RFLNA (C) or the variant T134K (D), ARHGAP1 (E) or the variant G438R (F), of HPGDS (G) or the variant T197S (H), and of GLTP (I) or the variant Y207C (J). The subcellular localization was determined by fluorescence microscopy (EGFP, green) in combination with immunofluorescence microscopy of the peroxisomal marker PMP70 (red). Scale bars indicate 20 µm and white squares define enlarged areas.

Figure 5

FlowFRET analysis based measurement of affinity between the PTS1 receptor (PEX5) and diverse EGFP-PTS1 variants harboring either the native C-termini of peroxisomal proteins (blue) or LoF mutants thereof (orange) (A), or the C-termini of cytosolic proteins (blue) and variants (orange) thereof harboring SNVs (B); K_a^app: apparent interaction strength as a correlative measure of affinity obtained by fitting; blue: native C-terminus; orange: C-terminus harboring the SNV; n.a: not analyzed (the low affinity of the interaction partners does not allow fitting).

Figure 6

Correlation analysis of the experimental measure of affinity (log₁₀ K_a^app ratio) with the prediction values: (A) FoldX-computed median ddG_bind, and (B) PTS1 Score difference (MT-WT) upon introducing a single point mutation into the native protein’s tripeptide motif. Proteins and their mutants in green are associated with a GoF change from cytosolic to peroxisomal localization, while mutants in red are associated with a LoF in peroxisome localization. The rest had no change in subcellular localization compartments based on the IFA results. For each plot, we report the Pearson’s r values and its associated p-value and 95% confidence interval (in brackets).