权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

ユビキチン様蛋白質が関与する細胞内蛋白質の修飾反応

涉及泛素样蛋白的细胞内蛋白修饰反应

基本信息

批准号：
12670117
负责人：
TANIGAWA Yoshinori
金额：
$ 2.37万
依托单位：
Shimane Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670117/
关键词：
RAW 264.7 cell iNOS ubiquitin proteasome ユビキチン免疫抑制

项目摘要

1) In RAW 264.7 cells, PMA synergistically increased IFN-γ-induced iNOS activity, but PMA alone failed to increase iNOS activity. PMA might modulate iNOS induction as a cosignal with IFN-γ in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1. PMA together with IFN-γ increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-γ-induced iNOS mRNA production may be depend on the elevation of the transcription rate.2) The lysates from RAW 264.7 cells treated with IFN-γ and PMA were subjected to SDS/PAGE followed by immunoblotting with an anti-iNOS antibody. In addition to markedly augmented iNOS protein level in the density of 130 KDa band compared with the IFN-γ or PMA alone, the tailing to higher molecular complexes (>130 KDa) of iNOS were observed by immunoblots, particularly after the treatment of proteasomal inhibitors, such as lactacyctin, MG132 and N-acetyl-L-leucinyl-L-leucinyl-norleucinal.3) Treatment with the proteasomal inhibitors resulted in the accumulation of iNOS, in contrast, calpastatin inhibitor and trypsn-like protease inhibitor did not lead to the accumulation of iNOS, and similar results were obtained using the lysosomal protease inhibitors lupeptin and pepstatin-A. These results suggested that the accumulated iNOS after proteasomal inhibition is ubiquitinated and the ubiquitination of iNOS is required for its degradation.4) Partially purified iNOS and conjugase was incubated in the presence of ^<125>I-GST-ubiqutin and ATP. After the immunoprecipitation using anti-iNOS antibody, samples were subjected to SDS/PAGE ^<125>I-conjugation of >130 KDa was observed by autoradiography.

1)在RAW 264.7细胞中，PMA可协同提高IFN-γ诱导的iNOS活性，但单独PMA不能提高iNOS活性。在RAW 264.7细胞中，PMA可能作为IFN-γ的协同信号调节iNOS的诱导，因为PMA的协同作用是通过IRF-1介导的。PMA联合IFN-γ增加了iNOS mRNA，但不影响iNOS mRNA的降解，提示PMA对IFN-γ诱导的iNOS mRNA产生的协同作用可能依赖于转录率的升高。2)经IFN-γ和PMA处理的RAW 264.7细胞裂解物进行SDS/PAGE检测，然后用抗inos抗体进行免疫印迹。除了与IFN-γ或单独的PMA相比，在130 KDa密度处iNOS蛋白水平显著增加外，免疫印迹还观察到iNOS向更高分子复合物（bbb130 KDa）的转移，特别是在蛋白酶体抑制剂（如乳酸素、MG132和n -乙酰基-l -亮氨酸-l -亮氨酸-去亮氨酸）的处理后。3)用蛋白酶体抑制剂治疗导致iNOS的积累，而calpastatin抑制剂和胰蛋白酶样蛋白酶抑制剂不导致iNOS的积累，溶酶体蛋白酶抑制剂lupeptin和pepstatin-A也得到类似的结果。这些结果表明，蛋白酶体抑制后积累的iNOS被泛素化，而iNOS的泛素化是其降解所必需的。4)部分纯化的iNOS和偶联酶在^<125> i - gst泛素和ATP存在下孵育。用抗inos抗体免疫沉淀后，对样品进行SDS/PAGE ^<125> i-射线自显影观察>偶联130 KDa。