Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic - Insights into the interaction of ebselen with mitochondria
Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic - Insights into the interaction of ebselen with mitochondria
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DOI：
10.1074/jbc.m501148200
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发表时间：
2005-06-24
影响因子：
4.8
通讯作者：
Murphy, MP
中科院分区：
文献类型：
--
作者：
Filipovska, A;Kelso, GF;Murphy, MP
线粒体过氧化产物的产生在病理学和氧化还原信号中都是一个关键事件。因此，它们在线粒体内的选择性降解引起了相当大的兴趣。在这里，我们探索了过氧化物酶模拟物ebselen与线粒体的相互作用。我们特别感兴趣的是ebselen是否被线粒体谷胱甘肽(GSH)和硫氧还蛋白激活，确定ebselen部分是否可以通过连接到亲脂阳离子来靶向线粒体，以及探索ebselen与线粒体蛋白质结合的性质。为了实现这些目标，我们合成了2-[4-(4-三苯基膦碘丁氧基)苯基]-1，2-苯并异硒唑)-3(2H)-酮碘化物(MitoPOX)，它含有一个与三苯基膦(TPP)阳离子共价连接的ebselen部分。TPP的固定正电荷促进了质谱分析，表明Ebselen部分被GSH还原为Selenol形式，随后与过氧化氢反应重新生成Ebselen部分。丝裂原过氧化物酶和依布硒是有效的抗氧化剂，可以降解磷脂过氧化氢，防止脂质过氧化，保护线粒体免受氧化损伤。这两种过氧化物酶模拟物都需要被线粒体GSH或硫氧还蛋白激活才能成为有效的抗氧化剂。令人惊讶的是，由于ebselen部分与蛋白质的共价结合，与TPP阳离子的结合只导致线粒体对ebselen的摄取略有增加。使用针对TPP部分的抗血清，我们显示了那些共价连接到ebselen部分的蛋白质。这一分析表明，线粒体中存在的大部分ebselen通过可逆的硒硫键与蛋白质硫醇结合。线粒体过氧化物酶和ebselen均可减少氧化应激诱导的细胞凋亡，提示它们可减轻线粒体氧化应激。这一探索使人们对线粒体内过氧化物酶模拟物的行为有了新的见解，并将其用于研究线粒体的氧化损伤。
Mitochondrial production of peroxides is a critical event in both pathology and redox signaling. Consequently their selective degradation within mitochondria is of considerable interest. Here we have explored the interaction of the peroxidase mimetic ebselen with mitochondria. We were particularly interested in whether ebselen was activated by mitochondrial glutathione (GSH) and thioredoxin, in determining whether an ebselen moiety could be targeted to mitochondria by conjugating it to a lipophilic cation, and in exploring the nature of ebselen binding to mitochondrial proteins. To achieve these goals we synthesized 2-[4-(4-triphenylphosphoniobutoxy) phenyl]-1,2-benzisoselenazol)-3(2H)-one iodide (MitoPeroxidase), which contains an ebselen moiety covalently linked to a triphenylphosphonium (TPP) cation. The fixed positive charge of TPP facilitated mass spectrometric analysis, which showed that the ebselen moiety was reduced by GSH to the selenol form and that subsequent reaction with a peroxide reformed the ebselen moiety. MitoPeroxidase and ebselen were effective antioxidants that degraded phospholipid hydroperoxides, prevented lipid peroxidation, and protected mitochondria from oxidative damage. Both peroxidase mimetics required activation by mitochondrial GSH or thioredoxin to be effective antioxidants. Surprisingly, conjugation to the TPP cation led to only a slight increase in the uptake of ebselen by mitochondria due to covalent binding of the ebselen moiety to proteins. Using antiserum against the TPP moiety we visualized those proteins covalently attached to the ebselen moiety. This analysis indicated that much of the ebselen present within mitochondria is bound to protein thiols through reversible selenenylsulfide bonds. Both MitoPeroxidase and ebselen decreased apoptosis induced by oxidative stress, suggesting that they can decrease mitochondrial oxidative stress. This exploration has led to new insights into the behavior of peroxidase mimetics within mitochondria and to their use in investigating mitochondrial oxidative damage.