Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest.
Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest.
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DOI：
10.1186/1743-422x-11-108
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发表时间：
2014-06-09
期刊：
影响因子：
4.8
通讯作者：
Landau NR
中科院分区：
文献类型：
--
作者：
Hakata Y;Miyazawa M;Landau NR
HIV-1 Vpr介导的G2细胞周期阻滞依赖于Vpr与E3泛素连接酶的相互作用，该连接酶含有损伤特异性DNA结合蛋白1 （DDB1）、Cullin 4A （Cul4A）、DDB1和cul4相关因子1 （DCAF1）和Rbx1。Vpr被认为与E3泛素连接酶复合体中的DCAF1直接相关，尽管复合体中蛋白质的确切相互作用模式尚未完全确定。SIVagm的Vpr诱导同源非洲绿猴（AGM）细胞的G2阻滞，但对人类细胞没有作用。SIVagm Vpr发挥其物种特异性功能的分子机制尚不清楚。E3泛素连接酶复合体中蛋白质的物理相互作用通过免疫共沉淀和western blotting进行评估。此外，通过共聚焦显微镜研究了蛋白质在细胞中的共定位。采用碘化丙啶染色和流式细胞术分析细胞周期。通过检测DNA损伤反应标志物H2AX的磷酸化水平来评估Vpr引起的DNA损伤反应。我们发现RNAi敲除DCAF1可以阻止DDB1与HIV-1 Vpr的共免疫沉淀，而DDB1敲除不影响Vpr与DCAF1的结合。具有L64P或R90K突变的HIV-1 Vpr突变体保持了与DCAF1结合的能力，但似乎没有与DDB1形成复合物。SIVagm Vpr与AGM DCAF1和DDB1相关，而在人细胞中，它与人DCAF1结合，但几乎不与人DDB1结合，导致H2AX的活化降低。Vpr突变体与DCAF1结合，但仅与DDB1结合较差，这表明DCAF1是必需的，但Vpr与DCAF1的简单结合不足以使Vpr与含DDB1的E3连接酶复合物结合。Vpr可能与E3连接酶复合体中的DCAF1和DDB1相互作用。或者，Vpr和DCAF1的相互作用可能诱导DCAF1或Vpr的构象变化，从而促进与DDB1的相互作用。SIVagm Vpr与DDB1而非DCAF1结合的能力可以解释SIVagm Vpr介导的G2阻滞的物种特异性。
HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown. Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response. We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX. The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the interaction with DDB1. The ability of SIVagm Vpr to associate with DDB1, but not DCAF1, can explain the species-specificity of SIVagm Vpr-mediated G2 arrest.