Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany.
Functional Proteomics, SFB 815 Core Unit, Goethe-University, Frankfurt, Germany.
Redox Biol. 2019 Feb;21:101125. doi: 10.1016/j.redox.2019.101125. Epub 2019 Jan 29.
NADPH oxidase (Nox) -derived reactive oxygen species have been implicated in redox signaling via cysteine oxidation in target proteins. Although the importance of oxidation of target proteins is well known, the specificity of such events is often debated. Only a limited number of Nox-oxidized proteins have been identified thus far; especially little is known concerning redox-targets of the constitutively active NADPH oxidase Nox4. In this study, HEK293 cells with tetracycline-inducible Nox4 overexpression (HEK-tet-Nox4), as well as podocytes of WT and Nox4-/- mice, were utilized to identify Nox4-dependent redox-modified proteins.
TGFβ1 induced an elevation in Nox4 expression in podocytes from WT but not Nox4-/- mice. Using BIAM based redox switch assay in combination with mass spectrometry and western blot analysis, 142 proteins were identified as differentially oxidized in podocytes from wild type vs. Nox4-/- mice and 131 proteins were differentially oxidized in HEK-tet-Nox4 cells upon Nox4 overexpression. A predominant overlap was found for peroxiredoxins and thioredoxins, as expected. More interestingly, the GRB2-associated-binding protein 1 (Gab1) was identified as being differentially oxidized in both approaches. Further analysis using mass spectrometry-coupled BIAM switch assay and site directed mutagenesis, revealed Cys374 and Cys405 as the major Nox4 targeted oxidation sites in Gab1.
INNOVATION & CONCLUSION: BIAM switch assay coupled to mass spectrometry is a powerful and versatile tool to identify differentially oxidized proteins in a global untargeted way. Nox4, as a source of hydrogen peroxide, changes the redox-state of numerous proteins. Of those, we identified Gab1 as a novel redox target of Nox4.
NADPH 氧化酶（Nox）衍生的活性氧通过靶蛋白半胱氨酸氧化参与氧化还原信号转导。尽管靶蛋白氧化的重要性是众所周知的，但此类事件的特异性经常存在争议。迄今为止，仅鉴定出有限数量的 Nox 氧化蛋白；特别是关于组成型活性 NADPH 氧化酶 Nox4 的氧化还原靶标知之甚少。在这项研究中，使用四环素诱导的 Nox4 过表达（HEK-tet-Nox4）的 HEK293 细胞以及 WT 和 Nox4-/-小鼠的足细胞，鉴定 Nox4 依赖性氧化还原修饰蛋白。
TGFβ1 诱导 WT 但不是 Nox4-/-小鼠的足细胞中 Nox4 表达升高。使用 BIAM 基于氧化还原开关测定法结合质谱和 Western blot 分析，在 WT 与 Nox4-/-小鼠的足细胞中，有 142 种蛋白被鉴定为差异氧化，在 Nox4 过表达的 HEK-tet-Nox4 细胞中有 131 种蛋白被鉴定为差异氧化。预期会发现过氧化物酶和硫氧还蛋白的主要重叠。更有趣的是，GRB2 相关结合蛋白 1（Gab1）被鉴定为这两种方法中差异氧化的蛋白。使用质谱偶联 BIAM 开关测定法和定点突变进一步分析，揭示 Cys374 和 Cys405 是 Gab1 中 Nox4 靶向氧化的主要位点。
BIAM 开关测定法与质谱联用是一种强大而通用的工具，可用于以非靶向方式鉴定差异氧化蛋白。Nox4 作为过氧化氢的来源，改变了许多蛋白质的氧化还原状态。在这些蛋白质中，我们确定 Gab1 是 Nox4 的一种新型氧化还原靶标。
