de Cubas Laura, Pak Valeriy V, Belousov Vsevolod V, Ayté José, Hidalgo Elena
Oxidative Stress and Cell Cycle Group, Universitat Pompeu Fabra, C/ Dr. Aiguader 88, 08003 Barcelona, Spain.
Laboratory of Experimental Oncology, Pirogov Russian National Research Medical University, 117997 Moscow, Russia.
Antioxidants (Basel). 2021 May 6;10(5):731. doi: 10.3390/antiox10050731.
Fluorescent protein-based reporters used to measure intracellular HO were developed to overcome the limitations of small permeable dyes. The two major families of genetically encoded redox reporters are the reduction-oxidation sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins and HyPer and derivatives. We have used the most sensitive probes of each family, roGFP2-Tpx1.C169S and HyPer7, to monitor steady-state and fluctuating levels of peroxides in fission yeast. While both are able to monitor the nanomolar fluctuations of intracellular HO, the former is two-five times more sensitive than HyPer7, and roGFP2-Tpx1.C169S is partially oxidized in the cytosol of wild-type cells while HyPer7 is fully reduced. We have successfully expressed HyPer7 in the mitochondrial matrix, and it is ~40% oxidized, suggesting higher steady-state levels of peroxides, in the low micromolar range, than in the cytosol. Cytosolic HyPer7 can detect negligible HO in the cytosol from mitochondrial origin unless the main HO scavenger, the cytosolic peroxiredoxin Tpx1, is absent, while mitochondrial HyPer7 is oxidized to the same extent in wild-type and cells. We conclude that there is a bidirectional flux of HO across the matrix and the cytosol, but Tpx1 rapidly and efficiently scavenges mitochondrial-generated peroxides and stops their steady-state cytosolic levels rising.
为克服小分子可渗透染料的局限性，人们开发了基于荧光蛋白的报告基因来测量细胞内的过氧化氢（HO）。基因编码的氧化还原报告基因的两个主要家族是与过氧化物酶和HyPer及其衍生物融合的对还原 - 氧化敏感的绿色荧光蛋白（roGFP）家族的蛋白质。我们使用了每个家族中最敏感的探针，即roGFP2 - Tpx1.C169S和HyPer7，来监测裂殖酵母中过氧化物的稳态水平和波动水平。虽然两者都能够监测细胞内HO的纳摩尔级波动，但前者比HyPer7敏感两到五倍，并且roGFP2 - Tpx1.C169S在野生型细胞的细胞质中部分氧化，而HyPer7完全还原。我们已成功在线粒体基质中表达HyPer7，其氧化程度约为40% , 这表明在低微摩尔范围内，过氧化物的稳态水平高于细胞质中的水平。除非主要的HO清除剂——细胞质过氧化物酶Tpx1不存在，否则细胞质中的HyPer7几乎检测不到来自线粒体的细胞质中的HO，而线粒体中的HyPer7在野生型细胞和[此处原文缺失细胞类型相关内容]细胞中被氧化到相同程度。我们得出结论，HO在线粒体基质和细胞质之间存在双向流动，但Tpx1能快速有效地清除线粒体产生的过氧化物，并阻止其在细胞质中的稳态水平升高。
