Mitochondrial protein targets of thiol-reactive electrophiles
Mitochondrial protein targets of thiol-reactive electrophiles
复制标题
DOI：
10.1021/tx700433m
复制
发表时间：
2008-04-01
影响因子：
4.1
通讯作者：
Liebler, Daniel C.
中科院分区：
文献类型：
--
作者：
Wong, Hansen L.;Liebler, Daniel C.
线粒体在细胞凋亡或程序性细胞死亡的调节中起关键作用。最近的研究表明，反应性亲电试剂诱导依赖于细胞凋亡。我们推测，反应性亲电试剂对特定线粒体蛋白的共价修饰是导致细胞凋亡启动的触发因素。在这项研究中，我们通过液相色谱-串联质谱法（LC-TAS-MS）鉴定了HEK 293细胞线粒体组分中模型生物素标记的亲电探针N-碘乙酰基-N-生物素基己二胺（IAB）和1-生物素酰胺基-4（4 '-[马来酰亚胺乙基环己烷]甲酰氨基）丁烷（BMCC）的蛋白质靶标。这些亲电体可重复地加合了总共1693个半胱氨酸残基，映射到809个蛋白质。蛋白质修饰是选择性的，在1255个半胱氨酰肽加合物中只有438个半胱氨酸位点（35%），809个鉴定的蛋白质靶点中有362个（45%）被两种亲电试剂加合。其中，大约三分之一的蛋白质数据库分析后被注释到线粒体。IAB引发细胞凋亡事件，包括细胞色素c释放、半胱天冬酶-3激活。和聚（ADP-核糖）聚合酶（PARP）裂解，而BMCC没有。在IAB和BMCC鉴定的靶点中，44个是骨化相关蛋白，并且两种探针对这些靶点的加合位点特异性不同。这两种亲电体之间的修饰位点的差异可能揭示其修饰触发细胞凋亡的烷基化位点。
Mitochondria serve a pivotal role in the regulation of apoptosis or programmed cell death. Recent studies have demonstrated that reactive electrophiles induce mitochondrion-dependent apoptosis. We hypothesize that covalent modification of specific mitochondrial proteins by reactive electrophiles serves as a trigger leading to the initiation of apoptosis. In this study, we identified protein targets of the model biotin-tagged electrophile probes N-iodoacetyl-N-biotinylhexylene-diamine (IAB) and 1-biotinamido-4(4'-[maleimidoethylcyclohexane]carboxamido)butane (BMCC) in HEK293 cell mitochondrial fractions by liquid chromatography-tandem mass spectrometry (LC-TAS-MS). These electrophiles reproducibly adducted a total of 1693 cysteine residues that mapped to 809 proteins. Protein modifications were selective in that only 438 cysteine sites in 1255 cysteinyl peptide adducts (35%) and 362 of the 809 identified protein targets (45%) were adducted by both electrophiles. Of these, approximately one-third were annotated to the mitochondria following protein database analysis. IAB initiated apoptotic events including cytochrome c release, caspase-3 activation., and poly(ADP-ribose)polymerase (PARP) cleavage, whereas BMCC did not. Of the identified targets of IAB and BMCC, 44 were apoptosis-related proteins, and adduction site specificity on these targets differed between the two probes. Differences in sites of modification between these two electrophiles may reveal alkylation sites whose modification triggers apoptosis.