FBXO25-associated nuclear domains: A novel subnuclear structure
FBXO25-associated nuclear domains: A novel subnuclear structure
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DOI：
10.1091/mbc.e07-08-0815
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发表时间：
2008-05-01
影响因子：
3.3
通讯作者：
Gomes, Marcelo D.
中科院分区：
文献类型：
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作者：
Manfiolli, Adriana O.;Maragno, Ana Leticia G. C.;Gomes, Marcelo D.
Skp1、Cul1、Rbx1和FBXO25蛋白形成功能性泛素连接酶复合物。本研究采用免疫化学和生化方法研究了FBXO25的细胞分布及其与某些核蛋白的共定位。用亲和纯化的抗体对FBXO25序列2-62残基的重组片段进行检测。免疫印迹分析显示，FBXO25蛋白在除横纹肌外的所有小鼠组织中均有表达。共聚焦分析显示，内源性FBXO25部分集中在一个新的点状核结构域，不同于破碎体和其他已被充分表征的结构。这些核室含有高浓度的泛素偶联物和至少两种泛素-蛋白酶体系统的其他组分：20S蛋白酶体和Skp1。我们建议将这些区室命名为fbxo25相关核结构域。有趣的是，放线菌素D或热休克处理对转录的抑制极大地影响了含fbxo25结构的核组织，表明它们是受细胞转录活性影响的动态区室。此外，我们提供证据表明，FBXO25依赖性泛素连接酶活性可以阻止重组含聚谷氨酰胺的亨廷顿蛋白在人胚胎肾293细胞的细胞核中聚集，这表明该蛋白可能是核FBXO25介导的泛素化的靶标。
Skp1, Cul1, Rbx1, and the FBXO25 protein form a functional ubiquitin ligase complex. Here, we investigate the cellular distribution of FBXO25 and its colocalization with some nuclear proteins by using immunochemical and biochemical approaches. FBXO25 was monitored with affinity-purified antibodies raised against the recombinant fragment spanning residues 2-62 of the FBXO25 sequence. FBXO25 protein was expressed in all mouse tissues tested except striated muscle, as indicated by immunoblot analysis. Confocal analysis revealed that the endogenous FBXO25 was partially concentrated in a novel dot-like nuclear domain that is distinct from clastosomes and other well-characterized structures. These nuclear compartments contain a high concentration of ubiquitin conjugates and at least two other components of the ubiquitin-proteasome system: 20S proteasome and Skp1. We propose to name these compartments FBXO25-associated nuclear domains. Interestingly, inhibition of transcription by actinomycin D or heat-shock treatment drastically affected the nuclear organization of FBXO25-containing structures, indicating that they are dynamic compartments influenced by the transcriptional activity of the cell. Also, we present evidences that an FBXO25-dependent ubiquitin ligase activity prevents aggregation of recombinant polyglutamine-containing huntingtin protein in the nucleus of human embryonic kidney 293 cells, suggesting that this protein can be a target for the nuclear FBXO25 mediated ubiquitination.