ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in walker a motif
ATPase activity of p97/valosin-containing protein is regulated by oxidative modification of the evolutionally conserved cysteine 522 residue in walker a motif
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DOI：
10.1074/jbc.m509700200
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发表时间：
2005-12-16
影响因子：
4.8
通讯作者：
Kakizuka, A
中科院分区：
文献类型：
--
作者：
Noguchi, M;Takata, T;Kakizuka, A
Valosin-containing protein（p97/VCP）在肿瘤、神经退行性变等多种生理和病理过程中发挥着重要作用。我们以前表明，VCP（K524 A），ATP酶活性阴性的VCP突变体，诱导空泡化，积累的泛素化蛋白，和细胞死亡，表型通常观察到的神经退行性疾病。然而，其ATP酶活性的任何调节机制尚未阐明。在这里，我们表明，氧化应激很容易失活VCP ATP酶活性。利用液相色谱/串联质谱，我们发现至少有三个半胱氨酸残基被氧化应激修饰。其中，第522位半胱氨酸（Cys-522）被确定为负责VCP氧化失活的位点。VCP（C522 T），一个单一的氨基酸取代突变体从半胱氨酸到苏氨酸，赋予几乎完全的抗氧化失活。在氧化应激反应中，VCP加强了与Np 14和Ufd 1的相互作用，这两者在内质网相关蛋白降解中是必不可少的。Cys-522位于第二个ATP结合基序中，在多细胞生物体中高度保守，但在单细胞生物体中不高度保守。Cdc 48 p（酵母VCP）在相应的氨基酸中含有苏氨酸，在体外表现出抗氧化失活的能力。此外，酵母突变体（Δ cdc 48 + cdc 48 [T532 C]）显示对氧化剂诱导的生长抑制和细胞死亡敏感。这些结果清楚地表明，VCP ATP酶活性是由Cys-522残基的氧化修饰调节的。这种调节机制可能在多细胞生物体中氧化应激向内质网应激反应的转化以及各种神经退行性疾病的病理过程中发挥关键作用。
Valosin-containing protein (p97/VCP) has been proposed as playing crucial roles in a variety of physiological and pathological processes such as cancer and neurodegeneration. We previously showed that VCP(K524A), an ATPase activity-negative VCP mutant, induced vacuolization, accumulation of ubiquitinated proteins, and cell death, phenotypes commonly observed in neurodegenerative disorders. However, any regulatory mechanism of its ATPase activity has not yet been clarified. Here, we show that oxidative stress readily inactivates VCP ATPase activity. With liquid chromatography/tandem mass spectrometry, we found that at least three cysteine residues were modified by oxidative stress. Of them, the 522nd cysteine (Cys-522) was identified as the site responsible for the oxidative inactivation of VCP. VCP(C522T), a single-amino acid substitution mutant from cysteine to threonine, conferred almost complete resistance to the oxidative inactivation. In response to oxidative stress, VCP strengthened the interaction with Npl4 and Ufd1, both of which are essential in endoplasmic reticulum-associated protein degradation. Cys-522 is located in the second ATP binding motif and is highly conserved in multicellular but not unicellular organisms. Cdc48p ( yeast VCP) has threonine in the corresponding amino acid, and it showed resistance to the oxidative inactivation in vitro. Furthermore, a yeast mutant (Delta cdc48 + cdc48[T532C]) was shown to be susceptible to oxidants-induced growth inhibition and cell death. These results clearly demonstrate that VCP ATPase activity is regulated by the oxidative modification of the Cys-522 residue. This regulatory mechanism may play a key role in the conversion of oxidative stress to endoplasmic reticulum stress response in multicellular organisms and also in the pathological process of various neurodegenerative disorders.