Assessment of the Role for Rho Family GTPases in NADPH Oxidase Activation
Assessment of the Role for Rho Family GTPases in NADPH Oxidase Activation
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DOI：
10.1007/978-1-61779-442-1_14
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发表时间：
2012-01-01
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影响因子：
--
通讯作者：
Sumimoto, Hideki
中科院分区：
文献类型：
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作者：
Miyano, Kei;Sumimoto, Hideki
Rac是Rho家族小GTP酶的成员，在动物中Nox家族NADPH氧化酶的活化中起关键作用，Nox家族NADPH氧化酶是致力于产生活性氧物质如超氧化物的酶。吞噬细胞氧化酶Nox 2在吞噬过程中对杀微生物活性至关重要，其以完全依赖于Rac的方式被激活。GTP结合形式的Rac直接与氧化酶激活剂p67（phox）结合，后者又与Nox 2相互作用，导致超氧化物的产生。Rac也参与非吞噬性氧化酶Nox 1的激活;在这种情况下，GTP结合的Rac通过与Noxal相互作用而发挥作用，Noxal是Nox 1激活所需的一种p67（phox）相关蛋白。另一方面，在p67（phox）或Noxal存在下，Bar促进Nox 3产生超氧化物，这是内耳形成耳锥的原因，耳锥是感知平衡和重力所需的微小矿化结构。Rac的所有三种哺乳动物同源物（Rac 1、Rac 2和Rac 3），但不是Cdc 42或RhoA，都能够充当Nox 1 -3的激活剂。在这里，我们描述了用于测定Rae与p67（phox）和Noxal结合的方法，以及用于在无细胞和全细胞系统中重建Rac依赖的Nox活性的方法。
Rac, a member of the Rho family small GTPases, plays a crucial role in activation of Nox family NADPH oxidases in animals, enzymes dedicated to production of reactive oxygen species such as superoxide. The phagocyte oxidase Nox2, crucial for microbicidal activity during phagocytosis, is activated in a manner completely dependent on Rac. Rac in the GTP-bound form directly binds to the oxidase activator p67(phox), which in turn interacts with Nox2, leading to superoxide production. Rac also participates in activation of the nonphagocytic oxidase Nox1; in this case, GTP-bound Rac functions by interacting with Noxal, a p67(phox)-related protein that is required for Nox1 activation. On the other hand, in the presence of either p67(phox) or Noxal, Bar facilitates superoxide production by Nox3, which is responsible in the inner ear for formation of otoconia, tiny mineralized structures that are required for sensing balance and gravity. All the three mammalian homologs of Rac (Rac1, Rac2, and Rac3), but not Cdc42 or RhoA, are capable of serving as an activator of Nox1-3. Here, we describe methods for the assay of Rae binding to p67(phox) and Noxal and for the reconstitution of Rac-dependent Nox activity in cell-free and whole-cell systems.