Inhibition of peroxisomal functions due to oxidative imbalance induced by mistargeting of catalase to cytoplasm is restored by vitamin E treatment in skin fibroblasts from Zellweger syndrome-like patients

Abstract

Many of the peroxisomal diseases exhibit excessive oxidative stress leading to neurological alterations and dysfunction. The role of peroxisomal oxidative stress in cellular function was highlighted by the loss of metabolic functions in peroxisomes of mutant cell lines, where catalase is mistargeted to the cytoplasm, but restored to peroxisomes by genetic manipulation (Sheikh et al. [Proc. Natl. Acad. Sci. USA 95 (1998) 2961)]. We report here that two human skin fibroblast cell lines from Zellweger syndrome-like patients are defective in the import of catalase into peroxisomes, causing impairment of metabolic function of this organelle. However, by lowering the cell culturing temperature (30 °C) the targeting of catalase to peroxisomes was restored, and with it the metabolic functions. Furthermore, mislocalization of catalase induces an oxidative imbalance in the cells which on treatment with a natural antioxidant, α-tocopherol (vitamin E), resulted in reduction of the oxidative levels and restoration of metabolic function (peroxisomal β-oxidation and levels of very long chain fatty acids and plasmalogen as well as α-oxidation of branched-chain fatty acids). However, restoration of peroxisomal functions was not associated with the targeting of catalase to peroxisomes. Therefore, our finding suggests that correction of mistargeted catalase to peroxisomes is a temperature sensitive event and supports the hypotheses that its location outside peroxisomes induces an oxidative imbalance that results in metabolic dysfunction. The imbalance can be reversed by treatment with vitamin E, leading to normalization of peroxisomal functions. These findings open a novel approach for therapeutic treatment of certain peroxisomal disorders where gene or hypothermic therapies are not an option.

Introduction

Peroxisomal disorders are a group of inherited metabolic diseases caused by either a defect in the biogenesis of peroxisomes or a deficiency of one or more peroxisomal enzyme activities. The combined incidence of peroxisomal disorders is estimated to be 1 in 20,000 or greater, with most of these disorders being lethal and/or resulting in severe neurological alterations. Peroxisomal diseases are divided into two groups based on single or multiple enzyme defects and the integrity of peroxisomes. Peroxisome biogenesis disorders are associated with defective biogenesis of peroxisomes which results in multiple developmental and metabolic alterations, including Zellweger syndrome (the most severe form), neonatal adrenoleukodystrophy, infantile Refsum disease (milder variants) and Rhizomelic chondrodysplasia punctate [1]. Single peroxisomal enzyme deficiencies present morphologically normal peroxisomes with defects in a single metabolic event such as that in X-linked adrenoleukodystrophy [2], [3].

Our laboratory described earlier that a human skin fibroblast cell lines obtained from a patient with Zellweger syndrome-like clinical features has multiple peroxisomal enzymatic deficiencies [4], as a result of mislocalization of catalase to the cytoplasm [5]. This cell line contains morphologically normal peroxisomes. The analysis of the proteins present in peroxisomes with antibodies against specific matrix proteins bearing peroxisomal-targeting sequence 1 (PTS1; i.e., acyl-CoA oxidase) or 2 (PTS2; i.e., keto acyl-CoA thiolase) showed no alteration in these two peroxisomal translocation pathways [4]. However, these cells lack the enzymatic activities involved in the β-oxidation of very long chain (VLC) fatty acids, α-oxidation of phytanic acid, and biosynthesis of plasmalogens [4], [5]. All these peroxisomal functions were restored when the cells were transfected with a vector carrying the catalase DNA sequence bearing PTS1 or PTS2 information at its carboxyl terminal end [5].

Interestingly, clinical phenotypic variations between the peroxisome biogenesis disorders (PBDs), mainly present in the milder forms (IRD), have been found to be due to temperature sensitive mutations in proteins, related to the transport of the proteins localized in the matrix of peroxisomes. These proteins contain PTS1, a carboxyl-terminal tripeptide (SKL or conservative variant consensus sequence) [6]. Earlier reports described the presence of abnormal membrane structures called peroxisomal ghosts in skin fibroblast from PBD patients [7], [8]. Initially, only a membrane protein of 70 kDa (PMP-70) was identified in these membranous structures. However, recent studies have identified partially functional “catalase-less peroxisomes,” present in the fibroblasts of several patients with milder forms of PBDs. These fibroblasts exhibit punctate pattern of distribution for the peroxisomal matrix enzymes acyl-CoA oxidase and thiolase that overlap that of PMP-70 [9], [10].

Catalase is an enzyme localized in the matrix of peroxisomes and functions in the removal of hydrogen peroxide (H₂O₂) produced during the degradation of VLC fatty acids by β-oxidation and by other oxidases. Abnormal targeting of catalase to peroxisomes is thought to produce excessive accumulation of H₂O₂ which results in excessive oxidative stress, concomitant inactivation of enzymes, and inactivation of catabolic functions and modification of membrane lipid components in this organelle. Peroxidation of membrane polyunsaturated fatty acids (PUFA) has been shown to produce very reactive species which can further chemically modify proteins and lipids [11], [12]. Based on the antioxidant properties of α-tocopherol (vitamin E), a lipid-soluble vitamin, and its demonstrated protective effect against free radicals on cellular membranes [13], [14], [15] we hypothesized that it may be beneficial in the treatment of cells that have oxidative imbalance as a result of mistargeting of catalase.

In this manuscript, we report that Rh and Sm fibroblasts are temperature sensitive cells in the targeting of catalase. Mistargeting of catalase results in cellular oxidative imbalance leading to multiple metabolic alterations of peroxisomal functions in these cells. However, treatment with a natural antioxidant such as vitamin E can normalize these metabolic alterations. Our findings support the hypothesis that the oxidative imbalance in cells containing catalase-negative peroxisomes can be reversed by treatment with vitamin E. This finding opens a novel approach for the pharmacological treatment of certain mild peroxisomal biogenesis disorders, where other therapies (gene or hypothermal therapies) are not available at present.