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Molecular mechanism of intracellular trafficking of TLR4

TLR4细胞内运输的分子机制

基本信息

批准号：
17591034
负责人：
OTA Yasuo
金额：
$ 2.24万
依托单位：
The University of Tokyo
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-17591034/
关键词：
innate immunity TLR LPS TLR4 cellular localization LL motif 細胞内輸送トラフィック

项目摘要

Essential Components of the innate immune system are TLRs, which recognize microbial products termed pathogen-associated molecular patterns (PAMPs). PAMP recognition leads to activation of the innate immune system, which in turn activates adaptive immunity. However, it remains to be determined that the mechanisms by which TLR4 were transported to the cell surface and activated. In this study, we tried to elucidate some important motifs which determined cellular localization of TLR4.Focused on LL motif, we made various truncation mutations of TLR4. These TLR4 truncation mutations and MD2 were transfected into and expressed on HEK293T cells, and LPS-induced activities of NF-κB were compared by a luciferase assay. LPS-induced NF-κB activities were observed in cells transfected with 1-826 TLR4 mutant as well as wild type TLR4. However, they were not observed in cells transfected with 1-815 and shorter TLR4 truncation mutants. In addition, localization of 1-826 TLR4 mutant was similar to that of wild type TLR4. However, cell surface expression was not observed in truncation mutants of 1-815 and shorter TLR4. These results indicated that there were some motifs which determined localization of TLR4 around position of 815. Next, we made a series of single truncation mutants of TLR4 at the positions of 813, 815, 816, and 817. We found that cell surface expression of a TLR4 mutant (L815A) was not observed. LPS-induced NF-κB activities were not observed in cells transfected with a TLR4 mutant (L815A) in the absence of CD14. While, they were reinstated in the presence of CD14. Thus, we identified an important region which determines cellular localization of TLR4.

先天免疫系统的基本组成部分是TLR，其识别称为病原体相关分子模式（PAMP）的微生物产物。PAMP识别导致先天免疫系统的激活，这反过来激活了适应性免疫。然而，TLR 4被转运到细胞表面并被激活的机制仍有待确定。本研究试图阐明决定TLR 4细胞定位的一些重要模体，并以LL模体为重点，对TLR 4进行了各种截短突变。将这些TLR 4截短突变体和MD 2转染并在HEK 293 T细胞上表达，并通过荧光素酶测定比较LPS诱导的NF-κB活性。在转染1-826 TLR 4突变体和野生型TLR 4的细胞中观察到LPS诱导的NF-κB活性。然而，在用1-815和更短的TLR 4截短突变体转染的细胞中没有观察到它们。此外，1-826 TLR 4突变体的定位与野生型TLR 4相似。然而，在1-815和较短TLR 4的截短突变体中未观察到细胞表面表达。这些结果表明，在815位附近存在决定TLR 4定位的模体。接下来，我们在813、815、816和817位置制备了一系列TLR 4的单截短突变体。我们发现没有观察到TLR 4突变体（L 815 A）的细胞表面表达。LPS诱导的NF-κB活性在转染TLR 4突变体（L 815 A）的细胞中在不存在CD 14的情况下未观察到。同时，它们在CD 14存在下恢复。因此，我们确定了决定TLR 4细胞定位的重要区域。