Abstract
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.
Highlights
Immunohistochemical examinations demonstrated that VCP co-localized with abnormal protein aggregates or ubiquitin-positive inclusions observed in several human neurodegenerative disorders, such as nuclear inclusions in Huntington disease [9], Lewy and Marinesco bodies in Parkinson disease [18], intracellular inclusions in motor neuron disease and dementia [18], dystrophic neurites of the senile plaque in Alzheimer disease [18], etc
VCP was preincubated with GSH or diamide alone, no bands were detected with the anti-GSH antibody, and VCP was detected at a position corresponding to 97 kDa
When VCP was preincubated with diamide plus GSH, GSSG, or GSNO, VCP was detected with the anti-GSH antibody, and its migration positions shifted to the upper positions
Summary
Immunohistochemical examinations demonstrated that VCP co-localized with abnormal protein aggregates or ubiquitin-positive inclusions observed in several human neurodegenerative disorders, such as nuclear inclusions in Huntington disease [9], Lewy and Marinesco bodies in Parkinson disease [18], intracellular inclusions in motor neuron disease and dementia [18], dystrophic neurites of the senile plaque in Alzheimer disease [18], etc These results have led us to propose that VCP functions as a sensor for the accumulation of misfolded proteins in cells [2, 17]. VCP(K524A), an ATPase activity-negative mutant has been shown to cause ER stress, vacuole formation, and accumulation of ubiquitinated proteins in the membrane fraction followed by cell death [17] These phenotypes are very similar to those observed in the pathology of several human neurodegenerative disorders (see above); we have called VCP vacuole-creating protein [9, 18]. No target molecule of oxidative stress has as yet been identified in such pathological processes
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