Involvement of Vesicle-Associated Membrane Protein 7 in Human Gastric Epithelial Cell Vacuolation Induced by Helicobacter pylori-Produced VacA
Involvement of Vesicle-Associated Membrane Protein 7 in Human Gastric Epithelial Cell Vacuolation Induced by Helicobacter pylori-Produced VacA
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DOI：
10.1128/iai.01573-07
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发表时间：
2008-03
影响因子：
3.1
通讯作者：
H. Mashima;J. Suzuki;T. Hirayama;Yukako Yoshikumi;H. Ohno;H. Ohnishi;H. Yasuda;T. Fujita;M. Omata
中科院分区：
文献类型：
--
作者：
H. Mashima;J. Suzuki;T. Hirayama;Yukako Yoshikumi;H. Ohno;H. Ohnishi;H. Yasuda;T. Fujita;M. Omata
幽门螺杆菌产生的细胞毒素VacA诱导细胞内空泡形成。VacA诱导的空泡被认为代表细胞内运输的病理状态。内体的融合机制需要在Q-SNARE和R-SNARE之间形成紧密复合物。我们最近报道，syntaxin 7，Q-SNARE蛋白的家族成员，参与VacA诱导的空泡形成。为了进一步阐明其分子机制，我们确定了囊泡相关膜蛋白7（VAMP 7）作为syntaxin 7的伴侣参与其中。免疫细胞化学显示内源性VAMP 7定位于VacA诱导的空泡中。北方印迹研究表明，VacA中毒增加VAMP 7 mRNA的时间依赖性的方式。VAMP 7与突触融合蛋白7共免疫沉淀，并且分别与突触融合蛋白7和VAMP 7结合的内源性VAMP 7和突触融合蛋白7的量响应于VacA而增加。使用小干扰RNA下调VAMP 7抑制了VacA诱导的空泡化，并且瞬时转染VAMP 7的N端结构域显性阴性突变体VAMP 7也抑制了空泡化。因此，我们得出结论，R-SNARE VAMP 7作为Q-SNARE syntaxin 7的伴侣在VacA诱导的空泡化中起着重要作用。
ABSTRACT Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The VacA-induced vacuole is assumed to represent the pathological status of intracellular trafficking. The fusion mechanism of the endosomes requires the formation of a tight complex between the Q-SNAREs and the R-SNAREs. We recently reported that syntaxin 7, a family member of the Q-SNARE protein, is involved in VacA-induced vacuole formation. In order to further elucidate the molecular mechanism, we identified the participation of vesicle-associated membrane protein 7 (VAMP7) as a partner of syntaxin 7. Immunocytochemistry revealed endogenous VAMP7 to be localized to the vacuoles induced by VacA. A Northern blotting study demonstrated that VacA intoxication increased VAMP7 mRNA in a time-dependent manner. VAMP7 was coimmunoprecipitated with syntaxin 7, and the amounts of endogenous VAMP7 and syntaxin 7 bound to syntaxin 7 and VAMP7, respectively, increased in response to VacA. The down-regulation of VAMP7 using small interfering RNA inhibited VacA-induced vacuolation, and the transient transfection of dominant-negative mutant VAMP7, the N-terminal domain of VAMP7, also inhibited the vacuolation. We therefore conclude that R-SNARE VAMP7 plays an important role in VacA-induced vacuolation as a partner of Q-SNARE syntaxin 7.