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The host defense mechanism by protein complexes assembling on the p65/L-plastin scaffold.

通过在 p65/L-plastin 支架上组装蛋白质复合物的宿主防御机制。

基本信息

批准号：
17590394
负责人：
SHINNOMIYA Hiroto
金额：
$ 2.3万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590394/
关键词：
p65 L-plastin cytoskeleton macrophage host defense cell adhesion phosphorylation protein complex p65-scaffold 65 分子集合接着分子

项目摘要

A 65-kDa protein was identified as a phosphorylated protein in bacterial LPS-stimulated macrophages in our previous study and has turned out to be a murine homologue of human L-plastin, a protein originally identified as a transformation-induced phosphoprotein in cancer cells. The p65/L-plastin is a member of the plastin family characterized by a series of Ca^<2+>, calmodulin, and b-actin binding domains. The L-isoform is exclusively expressed in leukocytes, suggesting it plays a vital role in cellular functions unique to leukocytes. In the present study, we have investigated the dynamics of p65/L-plastin when macrophage adhesion via integrins was remarkably increased by stimulation with LPS. An inhibitor of an LPS-inducible serine kinase phosphorylating p65/L-plastin inhibited both p65/L-plastin rearrangement and cellular adhesion. In this p65/L-plastin-involved adhesion, the protein was clearly observed to locate densely in podosome-like structures close to the substratum. The increased adhesion and the rearrangement of p65/L-plastin were also characteristic for b_2-integrin-positive cells accumulated in the spleen of mice infected with Salmonella organisms. in vitro kinase assay using peptide substrates revealed that the kinase activity phosphorylating the p65/L-plastin-peptides was augmented both in peritoneal macrophages stimulated in vitro with LPS and in CD11b-positive cells infiltrated in the Salmonella-infected spleen. Furthermore, generation of hydrogen peroxide, a bactericidal substance, was much higher in LPS-stimulated macrophages where p65/L-plastin was translocated than in resting macrophages. These findings suggest that p65/L-plastin and b-actin are closely coupled dynamically in macrophages in response to bacterial stimulation to organize adhesion structures containing integrins, and that this may also function as cytoskeletal scaffolds that effectively organize the anti-bacterial activities of macrophages.

在我们以前的研究中，一种65 kDa的蛋白被鉴定为细菌LPS刺激的巨噬细胞中的磷酸化蛋白，并且已经证明是人L-plastin的鼠同源物，L-plastin是一种最初被鉴定为癌细胞中转化诱导的磷蛋白的蛋白质。p65/L-plastin是plastin家族的成员，其特征在于一系列Ca^2+、钙调蛋白和b-肌动蛋白结合结构域。L-亚型仅在白细胞中表达，表明其在白细胞特有的细胞功能中起着至关重要的作用。在本研究中，我们研究了p65/L-plastin的动力学时，巨噬细胞粘附通过整合素显着增加与LPS刺激。LPS诱导的丝氨酸激酶磷酸化p65/L-plastin的抑制剂抑制p65/L-plastin重排和细胞粘附。在这种p65/L-plastin参与的粘附中，清楚地观察到蛋白质密集地位于接近基质的豆荚状结构中。此外，b_2整合素阳性细胞在沙门氏菌感染小鼠脾脏中的聚集也表现为粘附增加和p65/L-plastin重排。使用肽底物的体外激酶测定显示，在用LPS体外刺激的腹腔巨噬细胞和在沙门氏菌感染的脾中浸润的CD 11b阳性细胞中，磷酸化p65/L-纤溶酶蛋白肽的激酶活性均增强。此外，产生过氧化氢，杀菌物质，是高得多的LPS刺激的巨噬细胞中的p65/L-plastin易位比在静息巨噬细胞。这些发现表明，p65/L-plastin和b-肌动蛋白在巨噬细胞中响应于细菌刺激而动态地紧密偶联，以组织含有整合素的粘附结构，并且这也可以作为有效地组织巨噬细胞的抗菌活性的细胞骨架支架。