Actin is ADP‐ribosylated by the Salmonella enterica virulence‐associated protein SpvB
Actin is ADP‐ribosylated by the Salmonella enterica virulence‐associated protein SpvB
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DOI：
10.1046/j.1365-2958.2001.02258.x
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发表时间：
2001-02
影响因子：
3.6
通讯作者：
Dilek Tezcan‐Merdol;T. Nyman;U. Lindberg;F. Haag;F. Koch-Nolte;M. Rhen
中科院分区：
文献类型：
--
作者：
Dilek Tezcan‐Merdol;T. Nyman;U. Lindberg;F. Haag;F. Koch-Nolte;M. Rhen
沙门氏菌毒力相关蛋白SpvB最近被证明含有羧基末端单（ADP-核糖基）转移酶结构域。我们在此证明，SpvB的催化结构域以及含有全长SpvB的细菌提取物修饰了来自巨噬细胞样J774-A.1和上皮MDCK细胞的43 kDa蛋白，如从[32 P]-烟酰胺腺嘌呤二核苷酸（NAD）到43 kDa蛋白的标记转移所示。当通过二维凝胶电泳分析时，相同的蛋白质在感染S.肠球菌血清变体都柏林菌株SH 9325感染，而用同基因spvB突变菌株感染不导致修饰。使用SH 9325感染的细胞进行的免疫沉淀和免疫印迹实验将修饰的蛋白质鉴定为肌动蛋白。SpvB的分离的催化结构域介导32 P从[32 P]-NAD转移到体外各种来源的肌动蛋白，而分离的真核对照蛋白或细菌蛋白未被修饰。在体外肌动蛋白聚合试验中，分离的催化SpvB结构域阻止G肌动蛋白转化为F肌动蛋白。显微镜检查MDCK细胞感染SH 9325显示形态学变化和损失的丝状肌动蛋白的内容，而细胞感染的spvB突变体几乎不受影响。我们得出结论，肌动蛋白是SpvB介导的修饰的靶点，最有可能是ADP核糖基化，并且G肌动蛋白的修饰干扰肌动蛋白聚合。
The Salmonella enterica virulence‐associated protein SpvB was recently shown to contain a carboxy‐terminal mono(ADP‐ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full‐length SpvB modifies a 43 kDa protein from macrophage‐like J774‐A.1 and epithelial MDCK cells as shown by label transfer from [32P]‐nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two‐dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325‐infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]‐NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB‐mediated modification, most probably ADP‐ribosylation, and that the modification of G actin interferes with actin polymerization.