Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner
Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner
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DOI：
10.1074/jbc.m310340200
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发表时间：
2004-01-23
影响因子：
4.8
通讯作者：
Horiguchi, Y
中科院分区：
文献类型：
--
作者：
Matsuzawa, T;Fukui, A;Horiguchi, Y
百日咳博德特氏菌皮肤坏死毒素 (DNT) 可激活细胞内 Rho GTP 酶，是由 N 端受体结合结构域和 C 端酶结构域组成的单链多肽。我们发现 DNT 在 Arg(44) 的 C 端被弗林蛋白酶（一种哺乳动物内切蛋白酶）切割，生成几乎对应于受体结合结构域的 N 端片段和包含酶促结构域的 C 端剩余部分 (DeltaB)。即使在裂解后，这两个片段仍保持关联并形成切口形式。对弗林蛋白酶不敏感的 DNT 突变体没有细胞效应，而带切口的毒素比完整形式更有效，表明弗林蛋白酶的切口是作用的先决条件。 DeltaB，但不是带切口的毒素，与人工脂质体相关，并在由于缺乏表面受体而对 DNT 产生抗性的细胞中激活 Rho。这些结果意味着从结合结构域解离的DeltaB完全具有穿过脂质双层膜进入细胞质的能力。发现 DeltaB 的易位能力可归因于包含氨基酸 45 - 166 的 N 端区域，包括推定的跨膜结构域。使用干扰囊泡运输的各种试剂进行的药理学分析表明，易位既不需要内体酸化，也不需要逆行囊泡转运到更深的细胞器，尽管 DNT 似乎是通过动力依赖性内吞作用内化的。我们得出结论，DNT 与其受体结合并内化到发生蛋白水解加工的内体中。处理后从结合结构域中释放的 DeltaB 开始将酶结构域转移到细胞质中。
Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (DeltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that DeltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of DeltaB was found to be attributable to the N-terminal region encompassing amino acids 45 - 166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.