Islinger Markus, Lüers Georg H, Li Ka Wan, Loos Maarten, Völkl Alfred
Department of Anatomy and Cell Biology, Ruprecht-Karl University, 69120 Heidelberg, Germany.
J Biol Chem. 2007 Aug 10;282(32):23055-69. doi: 10.1074/jbc.M610910200. Epub 2007 May 22.
Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal beta-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-of-flight/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 alpha-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 fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptoralpha-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).
已知贝特类药物可诱导过氧化物酶体增殖及过氧化物酶体β-氧化酶的表达。为分析贝特类药物诱导的复杂代谢网络变化,我们比较了对照大鼠和苯扎贝特处理大鼠肝脏过氧化物酶体的蛋白质组。对高度纯化的过氧化物酶体进行亚分级分离,然后用iTRAQ试剂标记胰蛋白酶肽段后,通过基质辅助激光解吸电离飞行时间/飞行时间质谱分析由此获得的基质、外周和整合膜亚分级中的蛋白质。借助这种定量技术,我们能够鉴定出134种个体蛋白质,涵盖了大部分已知的过氧化物酶体蛋白质组。通过cDNA克隆验证了10个预测的新开放阅读框,其中7个可通过免疫细胞化学定位到过氧化物酶体。此外,定量质谱证实了苯扎贝特处理后大多数已知的过氧化物酶体增殖物激活受体α调节的过氧化物酶体蛋白的诱导作用,证明了iTRAQ程序在大规模实验中的适用性。然而,并非所有蛋白质的反应程度相似,而是呈现出贝特类药物特异性的诱导模式,显示出过氧化物酶体增殖物激活受体α介导的对特定配体反应的变异性。根据我们的数据,大鼠肝脏过氧化物酶体显然并非专门用于隔离极长链脂肪酸(C22-C26),而是优先代谢长链脂肪酸(C16-18)。