Nox1-dependent reactive oxygen generation is regulated by Rac1
Nox1-dependent reactive oxygen generation is regulated by Rac1
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DOI：
10.1074/jbc.m512751200
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发表时间：
2006-06-30
影响因子：
4.8
通讯作者：
Lambeth, J. David
中科院分区：
文献类型：
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作者：
Cheng, Guangjie;Diebold, Becky A.;Lambeth, J. David
Rac1在多种细胞类型中都与活性氧（ROS）的产生有关，但其产生ROS的酶学来源尚未得到证实。目前的研究表明，吞噬细胞NADPH氧化酶成分gp91（phox）的同源物Nox1可被Rac1激活。当Nox1与其调节亚基NOXO1和NOXA1共同表达时，可观察到大量ROS产生。在此，组成型激活的Rac1（G12V）共同表达（而非野生型Rac1）会显著进一步刺激其活性。使用小干扰RNA降低Rac1表达可减少依赖于Nox1的ROS。CDC42（G12V）未能增加活性，且针对CDC42的小干扰RNA未能降低活性，这表明了对Rac的特异性。干扰Rac1结合的NOXA1的TPR结构域突变体在支持依赖于Nox1的ROS产生方面无效。免疫沉淀实验表明存在一种包含Rac1（G12V）、NOXO1、NOXA1和Nox1的复合物。CDC42（G12V）在这种复合物中不能替代Rac1（G12V）。Nox1与Rac1（G12V）形成一种复合物，该复合物不依赖于NOXA1和NOXO1，这与Rac1（G12V）与Nox1的直接结合一致。Nox1和NOXO1增强了Rac1（G12V）与NOXA1的相互作用，表明存在协同结合。提出了一个模型，比较了Nox1与gp91（phox）（Nox2）的调节亚基的激活情况，其中Rac1激活是急性激活依赖于Nox1的ROS产生的主要触发因素。
Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild- type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1( G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.