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

1)在RAW 264.7细胞中，PMA协同增强干扰素-γ诱导的诱导型一氧化氮合酶活性，但不能单独提高诱导型一氧化氮合酶活性。在RAW 264.7细胞中，PMA可能与干扰素-γ作为共信号调节诱导型一氧化氮合酶的诱导，因为PMA的协同作用是通过IRF-1介导的。PMA和干扰素-γ联合应用可增加iNOSmRNA量，但不影响iNOSmRNA量，提示PMA对干扰素-γ诱导的iNOSmRNA量的协同作用可能依赖于转录速率的提高。2)经干扰素-γ和PMA处理的RAW 264.7细胞裂解产物经SDS/PAGE后用抗iNOS抗体免疫印迹。免疫印迹显示，与单独的干扰素-γ或PMA相比，在130kDa的条带密度上，诱导型一氧化氮合酶的蛋白水平显著增加。免疫印迹观察到诱导型一氧化氮合酶的高分子复合体(&gt；130 kDa)的拖尾，特别是在蛋白酶体抑制剂处理后，尤其是在蛋白酶体抑制剂处理后，如乳糖素、MG132和N-乙酰-L-亮氨酰-L-亮氨酰-去甲亮氨酸处理后。3)蛋白酶体抑制剂处理导致诱导型一氧化氮合酶的积累，相反，降钙素和类胰蛋白酶抑制剂不会导致诱导型一氧化氮合酶的积累，溶酶体蛋白酶抑制剂胡萝卜素和胃蛋白酶-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是泛素化的，它的降解需要iNOS的泛素化。4)部分纯化的iNOS和结合酶在^&lt；125&gt；i-GST-泛素和ATP的存在下孵育。用抗iNOS抗体进行免疫沉淀后，用十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法和放射自显影观察~GT；130 KDa的I-偶联。

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.