Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes
Real-time monitoring of basal H2O2 levels with peroxiredoxin-based probes
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DOI：
10.1038/nchembio.2067
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发表时间：
2016-06-01
影响因子：
14.8
通讯作者：
Dick, Tobias P.
中科院分区：
文献类型：
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作者：
Morgan, Bruce;Van Laer, Koen;Dick, Tobias P.
基于过氧化氢敏感蛋白OxyR和Orp1的基因编码的探针大大提高了在刺激或应激细胞中检测升高的过氧化氢水平的能力。然而，这些蛋白质不够敏感，不足以监测代谢的过氧化氢基线水平。以酵母菌为探针开发平台，我们开发了两种基于过氧化还蛋白的过氧化氢探针：roGFP2-Tsa2 Delta C-R和roGFP2-Tsa2 Delta C-P Delta C-R。这些探针类似于在正常的非压力条件下被氧化的50%，并且同样对过氧化氢的增加和减少做出反应。因此，它们可以完全动态、实时地测量活细胞中基础过氧化氢水平，并具有亚细胞分辨率。我们证明这些探针的表达不会改变内源性过氧化氢的动态平衡。RoGFP2-TSA2 Delta C-R探针显示了基础过氧化氢水平和分氧压之间的实时相互作用。此外，它还揭示了胞浆和线粒体基质之间过氧化氢运输的不对称性，以及基质过氧化氢水平与细胞生长速度之间的强烈相关性。
Genetically encoded probes based on the H2O2-sensing proteins OxyR and Orp1 have greatly increased the ability to detect elevated H2O2 levels in stimulated or stressed cells. However, these proteins are not sensitive enough to monitor metabolic H2O2 baseline levels. Using yeast as a platform for probe development, we developed two peroxiredoxinbased H2O2 probes, roGFP2-Tsa2 Delta C-R and roGFP2-Tsa2 Delta C-P Delta C-R, that afford such sensitivity. These probes are similar to 50% oxidized under 'normal' unstressed conditions and are equally responsive to increases and decreases in H2O2. Hence, they permit fully dynamic, real-time measurement of basal H2O2 levels, with subcellular resolution, in living cells. We demonstrate that expression of these probes does not alter endogenous H2O2 homeostasis. The roGFP2-Tsa2 Delta C-R probe revealed real-time interplay between basal H2O2 levels and partial oxygen pressure. Furthermore, it exposed asymmetry in H2O2 trafficking between the cytosol and mitochondrial matrix and a strong correlation between matrix H2O2 levels and cellular growth rate.