Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders
Physical and functional interaction between Dorfin and Valosin-containing protein that are colocalized in ubiquitylated inclusions in neurodegenerative disorders
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DOI：
10.1074/jbc.m406683200
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发表时间：
2004-12-03
影响因子：
4.8
通讯作者：
Sobue, G
中科院分区：
文献类型：
--
作者：
Ishigaki, S;Hishikawa, N;Sobue, G
Dorfin是一种RING-IBR型泛素连接酶（E3），可泛素化家族性肌萎缩侧索硬化症（ALS）的致病基因超氧化物歧化酶1（SOD 1）。Dorfin位于各种神经退行性疾病（如ALS和帕金森病（PD））中的泛素化包涵体（UBI）中。在这里，我们报告，Valosin-containing蛋白（VCP）直接结合Dorfin和VCP ATP酶活性深刻地有助于Dorfin的E3活性。使用质谱的高通量分析将VCP鉴定为Dorfin相关蛋白的候选者。甘油梯度离心分析表明，内源性Dorfin由400 - 600-kDa的复合物组成，并与内源性VCP共免疫沉淀。体外实验表明，Dorfin通过其C-末端区域与VCP直接相互作用。这两种蛋白质共定位于HEK 293细胞的侵袭体和ALS和PD受影响神经元的UBI中。VCPK 524 A是VCP的一种显性负性形式，它降低Dorfin对突变型超氧化物歧化酶1的E3活性，而对Parkin的autoubiquitylation没有影响。我们的研究结果表明，VCPs通过直接相互作用在功能上调节Dorfin，它们的功能相互作用可能与神经退行性疾病（如ALS或PD）中UBI形成的过程有关。
Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions ( UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400 - 600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCPK524A, a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.