Zellweger spectrum disorder patient–derived fibroblasts with the PEX1‐Gly843Asp allele recover peroxisome functions in response to flavonoids

Abstract

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5′,7′-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of importrescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843Dassociated ZSD. K E Y W O R D SAAA ATPase, betaine, chaperone therapy, diosmetin, peroxisome biogenesis disorder, PEX1 J Cell Biochem. 2019;120:3243-3258.wileyonlinelibrary.com/journal/jcb

The Nitric Oxide Donor, S-Nitrosoglutathione, Rescues Peroxisome Number and Activity Defects in PEX1G843D Mild Zellweger Syndrome Fibroblasts

“…To do the HTS, PEX1G843D fibroblasts and WT fibroblasts were treated either with 25 μM of each compound from the library or with 0.4% vehicle (DMSO). As a positive control, fibroblasts were treated with 100 mM betaine, a compound previously reported to increase peroxisome number in PEX1G843D fibroblasts ( MacLean et al, 2019 ). After 3 days, we performed indirect immunofluorescence (IF) microscopy using an antibody against the C-terminal Peroxisome Targeting Sequence Type 1 Ser-Lys-Leu (SKL), the canonical marker for peroxisomal matrix proteins ( Szilard et al, 1995 ).…”

A Chinese newborn with Zellweger syndrome and compound heterozygous mutations novel in the PEX1 gene: a case report and literature review

“…Furthermore, there is currently no known effective treatment for ZSDs, the value of genetic diagnosis is limited to prognostic estimation and prenatal genetic diagnosis for future pregnancies in the same family (19). However, the identification of temperature-sensitive peroxisomal defects in p.G843D fibroblasts has suggested that increased protein instability is among the underlying mechanisms for ZSD (20), and indeed there are evolving in vitro evidence showing that ZSDs could benefit from chaperone therapy that rescues molecular misfolding (11,21). It was proposed that ZSDs due to mutated PEX1 might benefit differently from molecular stabilizing treatment due to variations in each mutation's actual functional impairment (12).…”

The Peroxisomal PTS1-Import Defect of PEX1- Deficient Cells Is Independent of Pexophagy in Saccharomyces cerevisiae

“…It displays a rather mild clinical phenotype, which might be caused by a partial misfolding of the protein [20,21]. Therefore, it has been demonstrated before that Pex1(G843D) cells can recover Pex1(G843D)-, Pex6-and Pex5-protein levels when they are treated with chaperone-like small molecules or by lowering the incubation temperature [21][22][23]. However, because of the residual activity of the point mutant Pex1(G843D), which has been estimated to achieve ca.…”