Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria
Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria
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DOI：
10.1093/emboj/20.10.2404
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发表时间：
2001-05-15
期刊：
影响因子：
11.4
通讯作者：
Ziegler, M
中科院分区：
文献类型：
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作者：
Herrero-Yraola, A;Bakhit, SMA;Ziegler, M
线粒体adp核糖基化导致两种类似于26和53 kDa的蛋白质的修饰，这些蛋白质的性质以及它们修饰的生理后果仍然未知。在这里，55kda蛋白谷氨酸脱氢酶（GDH）被确定为线粒体中酶促半胱氨酸特异性adp核糖基化的特异性受体。该修饰蛋白从线粒体制备中分离得到，通过n端测序和色氨酸消化的质谱分析鉴定为GDH。用[C-14]腺嘌呤对人肝癌细胞进行体内培养，证实了这种修饰的发生。纯化的GDH在半胱氨酸残基中被adp核糖基化，线粒体活性将adp核糖从NAD（+）转移到受体位点。GDH的adp核糖基化导致其催化活性受到实质性抑制。结合adp核糖和GDH亚基之间的化学计量表明，每个催化活性同六聚体的一个亚基的修饰导致酶失活。分离的adp核糖基化的GDH被Mg2+依赖的线粒体adp核糖半胱氨酸水解酶重新激活，GDH是一种高度调节的酶，是第一个鉴定出的活性可能被adp核糖基化调节的线粒体蛋白。
Mitochondrial ADP-ribosylation leads to modification of two proteins of similar to 26 and 53 kDa, The nature of these proteins and, hence, the physiological consequences of their modification have remained unknown. Here, a 55 kDa protein, glutamate dehydrogenase (GDH), was established as a specific acceptor for enzymatic, cysteine-specific ADP-ribosylation in mitochondria. The modified protein was isolated from the mitochondrial preparation and identified as GDH by N-terminal sequencing and mass spectrometric analyses of tryptic digests. Incubation of human hepatoma cells with [C-14]adenine demonstrated the occurrence of the modification in vivo. Purified GDH was ADP-ribosylated in a cysteine residue in the presence of the mitochondrial activity that transferred the ADP-ribose from NAD(+) onto the acceptor site. ADP-ribosylation of GDH led to substantial inhibition of its catalytic activity. The stoichiometry between incorporated ADP-ribose and GDH subunits suggests that modification of one subunit per catalytically active homohexamer causes the inactivation of the enzyme. Isolated, ADP-ribosylated GDH was reactivated by an Mg2+-dependent mitochondrial ADP-ribosylcysteine hydrolase, GDH, a highly regulated enzyme, is the first mitochondrial protein identified whose activity may be modulated by ADP-ribosylation.