Intraperoxisomal Localization of Very-Long-Chain Fatty Acyl-CoA Synthetase: Implication in X-Adrenoleukodystrophy
ABSTRACT X-Adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain (VLC) fatty acids (>C22:0) due to the impaired activity of VLC acyl-CoA synthetase (VLCAS). The gene responsible for X-ALD was found to code for a peroxisomal integral membrane protein (ALDP) that belongs to the ATP binding cassette superfamily of transporters. To understand the function of ALDP and how ALDP and VLCAS interrelate in the peroxisomal β-oxidation of VLC fatty acids we investigated the peroxisomal topology of VLCAS protein. Antibodies raised against a peptide toward the C-terminus of VLCAS as well as against the N-terminus were used to define the intraperoxisomal localization and orientation of VLCAS in peroxisomes. Indirect immunofluorescent and electron microscopic studies show that peroxisomal VLCAS is localized on the matrix side. This finding was supported by protease protection assays and Western blot analysis of isolated peroxisomes. To further address the membrane topology of VLCAS, Western blot analysis of total membranes or integral membranes prepared from microsomes and peroxisomes indicates that VLCAS is a peripheral membrane-associated protein in peroxisomes, but an integral membrane in microsomes. Moreover, peroxisomes isolated from cultured skin fibroblasts from X-ALD patients with a mutation as well as a deletion in ALDP showed a normal amount of VLCAS. The consequence of VLCAS being localized to the luminal side of peroxisomes suggests that ALDP may be involved in stabilizing VLCAS activity, possibly through protein–protein interactions, and that loss or alterations in these interactions may account for the observed loss of peroxisomal VLCAS activity in X-ALD.
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ABSTRACT: The dysfunctioning of peroxisomes as observed in certain inherited disorders leads to defects in neuronal migration, myelin formation, lens development and bone ossification. Because of limited knowledge on how abnormal peroxisomal metabolism causes such a variety of abnormalities, mutant mice with defined peroxisomal defects are now in the spotlight. Although the mutant mice have the same biochemical defects as patients, the mutant phenotype of these mice is often rather different from the human disorders for which no effective therapy is currently available.Section Editor:Johan Auwerx—Institut de Genetique et Biologie Moleculaire et Cellulaire (IGBMC); and Institut Clinique de la Souris, StrasbourgThe characterization of rare disorders of peroxisomal biogenesis in humans has been instrumental in furthering our understanding of intracellular metabolism. These disorders have been extensively characterized by cell-based approaches, but in vitro studies have fallen short of the in vivo studies on mouse models. Only the careful study of such models has allowed us to grasp the full spectrum of abnormalities induced by dysfunctioning peroxisomes. The authors of this review are among the leading geneticists and biochemists studying peroxisomal disorders. They are well placed to summarize how these mouse models have changed our understanding of peroxisome function and metabolism.Drug Discovery Today Disease Models 12/2004;
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ABSTRACT: Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of β-oxidation enzyme activities as well as a set of auxiliary activities to provide the β-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of β-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p–Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in β-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in β-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.FEMS microbiology reviews 04/2003; 27(1):35-64. · 13.81 Impact Factor