Recovery of PEX1-Gly843Asp peroxisome dysfunction by small-molecule compounds

Abstract

Zellweger spectrum disorder (ZSD) is a heterogeneous group of diseases with high morbidity and mortality caused by failure to assemble normal peroxisomes. There is no therapy for ZSD, but management is supportive. Nevertheless, one-half of the patients have a phenotype milder than classic Zellweger syndrome and exhibit a progressive disease course. Thus, patients would benefit if therapies became available and were instituted early. Recent reports indicate several interventions that result in partial peroxisome recovery in ZSD fibroblasts. To identify drugs that recover peroxisome functions, we expressed a GFP-peroxisome targeting signal 1 reporter in fibroblasts containing the common disease allele, PEX1-p.Gly843Asp. The GFP reporter remained cytosolic at baseline, and improvement in peroxisome functions was detected by the redistribution of the GFP reporter from the cytosol to the peroxisome. We established a high-content screening assay based on this phenotype assay and evaluated 2,080 small molecules. The cells were cultured in chemical for 2 days and then, were fixed and imaged by epifluorescent microscopy on a high-content imaging platform. We identified four compounds that partially recover matrix protein import, and we confirmed three using independent assays. Our results suggest that PEX1-p.G843D is a misfolded protein amenable to chaperone therapy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Response to nonspecific chemical chaperones in PEX1-G843D-PTS1 cells. Cells were cultured for 2 days at 30 °C or 37 °C with chemical and imaged live (30 °C; glycerol and DMSO) or after fixation (the remainder). Note the redistribution of GFP-PTS1 from the cytosol to the peroxisome in the treated groups; results were similar for 300 mM proline. In the lower right two images, cells cultured in TMAO were permeabilized and incubated with PEX14 antiserum and Texas Red conjugated secondary antibody. Colocalization of PEX14 and GFP is shown in the last image.

Fig. 2.

Chemical treatment rescues PTS2 processing in PEX1- and PEX6-deficient primary fibroblasts. (A) Recovery of 42-kDa thiolase in two PEX1-G843D homozygous cell lines. (B) Recovery of 42-kDa thiolase in PEX1-G843D vs. PEX1 null cells is shown on the left, and PEX6 missense vs. null cells are shown on the right. (C) Recovery of 28 kDa PhyH in PEX1-G843D, PEX6 missense, and PEX1 null cells. (D) Recovery of 66 kDa AGPS in PEX1-G843D cells. Lane C, control; lane 1, untreated; lane 2, 200 mM TMAO; lane 3, 10 μM AD; lane 4, 10 μM Epicholestanol; lane 5, GF109203x 3 μM; lane 6, Ro31-8220 0.5 μM; lane 7, 5% glycerol; lane 8, 100 mM betaine; lane R, RCDP1 cells that were unable to import AGPS, which highlights the absence of the band corresponding to 66 kDa AGPS. Genotypes: PEX1 null-I700fs/I700fs, PEX6 null-802_815del/802_815del, and PEX6 missense-R60Q/R812W.

Fig. 3.

One section of each montage, obtained in the high-throughput screen, is shown in the GFP channel. Compared with the cells in 0.3% DMSO solvent, there is a higher proportion of cells with punctate structures, representing importing peroxisomes, in the hit compounds and TMAO.

Fig. 4.

PEX1 and PEX6 protein levels in PEX1-214 cells homozygous for G843D that do not recover with chemical treatment. Lanes C, control; lane 1, untreated; lane 2, 200 mM TMAO; lane 3, 3% DMSO; lane 4, 10 μM AD; lane 5, 10 μM Epicholestanol; lane 6, GF109203x 3 μM; lane 7, Ro31-8220 0.5 μM; lane 8, 5% glycerol, 37 °C and 30 °C. Note that PEX1 and PEX6 levels increase slightly at 30 °C. GAPDH was used to evaluate protein loading.