An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus
An N-terminal segment of the active component of the bacterial genotoxin cytolethal distending toxin B (CDTB) directs CDTB into the nucleus
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DOI：
10.1074/jbc.m305062200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Sugai, M
中科院分区：
文献类型：
--
作者：
Nishikubo, S;Ohara, M;Sugai, M
细胞致死性膨胀毒素（CDT）是由伴放线放线杆菌产生的一种致病因子。它是G（2）/M转换的细胞周期特异性抑制剂。CDTB是CDT全毒素的亚基之一，在进入靶细胞后参与遗传毒性作用，由此染色体损伤诱导检查点磷酸化级联反应。显微注射到细胞质中的CDTB显示定位于细胞核中并诱导染色质塌陷。为了研究参与CDTB核转运的分子机制，我们使用了CDTB-绿色荧光蛋白（GFP）融合蛋白的瞬时表达和显微注射。显微注射后，His标记的CDTB-GFP在3-4 h内进入细胞核。瘦霉素B没有增加融合蛋白的进入速度，表明融合蛋白的相对缓慢的进入不是由于蛋白质的CRM 1依赖性核输出。CDTB-GFP的核定位是温度依赖性的。体外转运试验表明，CDTB的核定位是由主动转运介导的。使用一系列截短的CDTB-GFP融合蛋白的瞬时表达的测定揭示，残基48-124构成参与CDTB的核转运的最小区域。用SV 40 T核定位信号对CDTB的核转运区域进行结构域交换实验表明，CDTB由两个结构域组成，一个是核转运的N-末端结构域，一个是C-末端活性结构域。我们的研究结果强烈表明，核定位的CDTB是所需的全毒素诱导细胞扩张和细胞周期阻滞。这是第一次证明具有独特的核转运结构域的细菌毒素通过主动转运转移到动物细胞核。
Cytolethal distending toxin (CDT), produced by Actinobacillus actinomycetemcomitans, is a putative virulence factor in the pathogenesis of periodontal diseases. It is a cell cycle specific inhibitor at the G(2)/M transition. CDTB, one of the subunits of the CDT holotoxin, is implicated in a genotoxic role after entering the target cells, whereby chromosomal damage induces checkpoint phosphorylation cascades. CDTB microinjected into the cytoplasm was shown to localize in the nucleus and induce chromatin collapse. To investigate the molecular mechanism involved in nuclear transport of CDTB, we used transient expression and microinjection of a CDTB-green fluorescent protein (GFP) fusion protein. After microinjection, His-tagged CDTB-GFP entered the nucleus in 3-4 h. Leptomycin B did not increase the speed of entry of the fusion protein, suggesting that the relatively slow entry of the fusion protein is not due to the CRM1-dependent nuclear export of the protein. Nuclear localization of the CDTB-GFP was temperature-dependent. An in vitro transport assay demonstrated that the nuclear localization of CDTB is mediated by active transport. An assay using transient expression of a series of truncated CDTB-GFP fusion proteins revealed that residues 48-124 constitute the minimum region involved in nuclear transport of CDTB. A domain swapping experiment of the region involved in nuclear transport of CDTB with an SV40 T nuclear localization signal indicated that CDTB is composed of two domains, an N-terminal domain for nuclear transport and a C-terminal active domain. Our results strongly suggest that nuclear localization of CDTB is required for the holotoxin to induce cytodistension and cell cycle block. This is the first demonstration that a bacterial toxin possessing a unique domain for nuclear transport is transferred to the animal cell nucleus by active transport.