Rat Liver Peroxisomes after Fibrate Treatment

Abstract

Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal ␤-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-offlight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor ␣-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibratespecific induction scheme showing the variability of peroxisome proliferator-activated receptor ␣-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22-C26) but rather metabolize preferentially long chain fatty acids (C16 -18).Peroxisomes, often described as multipurpose organelles, are involved in numerous biochemical pathways of lipid and peroxide metabolism. The existence of more than 15 peroxisomal inherited diseases like the Zellweger syndrome or X-linked adrenoleukodystrophy, which are often lethal, emphasizes the vital importance of these organelles (1-3). Cells are able to remodel the protein composition of their peroxisomes and to adjust the number of these organelles in response to physiological or environmental stimuli to cope with the metabolic needs. Consequently, peroxisomes are regarded as highly modular organelles, with different protein composition depending on the metabolic function or cell type. Fibrates, used as hypolipidemic drugs, induce both the proliferation of peroxisomes as well as the ␤-oxidation of fatty acids (4). These alterations of the metabolic functions of peroxisomes are accompanied by changes of proteins involved in the biogenesis of the organelles as well as in enzymes for the breakdown of fatty acids (5, 6). Several investigators in the past have used fibrate-induced peroxisome proliferation as a model to analyze selected aspects of peroxisome biogenesis or lipid metabolism, using techniques like Western blotting, enzyme assays, immunocytochemistry, Northern blotting, or in situ hybridization (6 -11). However, complex rearrangements of the entire peroxisomal proteome could not be analyzed with these techniques.In this respect, mass spectrometry represents a straightfo...

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“…S2E" type="url"/>). ACBD5 is another potential TA protein recently detected at peroxisomes (Islinger et al, 2007; Nazarko et al, 2014; Wiese et al, 2007). Overexpressed and endogenous ACBD5 showed peroxisomal localization in COS-7 cells (Fig.…”

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