Side-by-side comparison of recombinant human glutathione peroxidases identifies overlapping substrate specificities for soluble hydroperoxides.
Side-by-side comparison of recombinant human glutathione peroxidases identifies overlapping substrate specificities for soluble hydroperoxides.
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重组人谷胱甘肽过氧化物酶的并列比较确定了可溶性过氧化氢的重叠底物特异性。
DOI：
10.1016/j.redox.2022.102593
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发表时间：
2023-02
期刊：
影响因子：
11.4
通讯作者：
Kipp, Anna P.
中科院分区：
文献类型：
--
作者：
Schwarz, Maria;Loeser, Alina;Cheng, Qing;Wichmann-Costaganna, Mareike;Schaedel, Patrick;Werz, Oliver;Arner, Elias S. J.;Kipp, Anna P.
八种人类谷胱甘肽过氧化物酶(GPX)中有五种是硒蛋白，代表的蛋白质含有硒作为氨基酸硒半胱氨酸的一部分。GPX对于减少谷胱甘肽消耗方式中的过氧化氢，从而调节细胞氧化还原动态平衡是重要的。GPX1、GPX2和Gpx4是细胞内三种主要的GPXs，但它们的表达模式不同，其中GPX1和Gpx4普遍表达，而GPX2主要在上皮细胞表达。已有研究表明，GPX1和GPX2可以减少可溶性的过氧化氢，而Gpx4则可以减少复杂的脂质过氧化，从而保护细胞免受脂质过氧化和铁下垂的伤害。但这些数据中的大多数来自缺乏其中一种异构体的细胞，因此，补偿或其他细胞效应可能会影响结论。到目前为止，将分离的重组人硒蛋白谷胱甘肽过氧化物酶用于纯酶分析还没有被用来同时研究它们的底物特异性。使用在大肠杆菌中生产的重组GPX1、GPX2和GPX4，我们在这里以17种不同的过氧化物酶(均为50μM)为底物，通过消耗NADPH的谷胱甘肽还原酶偶联试验来评估它们的GPx活性。Gpx4显然是唯一能够还原磷脂酰胆碱氢过氧化氢的异构体。相比之下，三种异构体均可还原小分子可溶性过氧化氢，如过氧化氢、过氧化异丙苯和叔丁基过氧化氢，但与GPX2和Gpx4相比，GPX1的还原效率约高10倍。此外，三种异构体都能还原几种脂肪酸衍生的过氧化氢，而且GPX1的活性也是最高的。有趣的是，脂肪酸衍生的过氧化氢的立体异构体明显影响了GPX酶的活性。总体而言，Gpx4具有明显的底物特异性，但当比较GPX1和GPX2时就不是这样了。显然，就还原可溶性和脂肪酸衍生的过氧化氢的周转率而言，GPX1是三种GPX中最有效的异构体。分离的重组人谷胱甘肽过氧化物酶被并排比较。Gpx4是唯一能还原磷脂酰胆碱氢过氧化氢的异构体。GPX1、2和4可还原小分子可溶性和脂肪酸衍生的过氧化氢。尚未发现GPX1和GPX2具有底物特异性。就底物周转率而言，GPX1是三种GPX中最有效的异构体。
Five out of eight human glutathione peroxidases (GPXs) are selenoproteins, representing proteins that contain selenium as part of the amino acid selenocysteine. The GPXs are important for reducing hydroperoxides in a glutathione-consuming manner and thus regulate cellular redox homeostasis. GPX1, GPX2, and GPX4 represent the three main cytosolic GPXs, but they differ in their expression patterns with GPX1 and GPX4 being expressed ubiquitously, whereas GPX2 is mainly expressed in epithelial cells. GPX1 and GPX2 have been described to reduce soluble hydroperoxides, while GPX4 reduces complex lipid hydroperoxides, thus protecting cells from lipid peroxidation and ferroptosis. But most of these data are derived from cells that are devoid of one of the isoforms and thus, compensation or other cellular effects might affect the conclusions. So far, the use of isolated recombinant human selenoprotein glutathione peroxidases in pure enzyme assays has not been employed to study their substrate specificities side by side. Using recombinant GPX1, GPX2, and GPX4 produced in E. coli we here assessed their GPX activities by a NADPH-consuming glutathione reductase-coupled assay with 17 different peroxides (all at 50 μM) as substrates. GPX4 was clearly the only isoform able to reduce phosphatidylcholine hydroperoxide. In contrast, small soluble hydroperoxides such as H2O2, cumene hydroperoxide, and tert-butyl hydroperoxide were reduced by all three isoforms, but with approximately 10-fold higher efficiency for GPX1 in comparison to GPX2 and GPX4. Also, several fatty acid-derived hydroperoxides were reduced by all three isoforms and again GPX1 had the highest activity. Interestingly, the stereoisomerism of the fatty acid-derived hydroperoxides clearly affected the activity of the GPX enzymes. Overall, distinct substrate specificity is obvious for GPX4, but not so when comparing GPX1 and GPX2. Clearly GPX1 was the most potent isoform of the three GPXs in terms of turnover in reduction of soluble and fatty-acid derived hydroperoxides. Isolated recombinant human glutathione peroxidases are compared side by side. GPX4 is the only isoform able to reduce phosphatidylcholine hydroperoxide. Small soluble and fatty acid-derived hydroperoxides are reduced by GPX1, 2 and 4. No substrate specificity has been identified for GPX1 and GPX2. GPX1 was the most potent isoform of the three GPXs in terms of substrate turnover.
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DOI：
10.1111/j.1432-1033.1983.tb07687.x
发表时间：
1983-01-01
期刊：
EUROPEAN JOURNAL OF BIOCHEMISTRY
影响因子：
--
作者：
GROSSMANN, A;WENDEL, A
通讯作者：
WENDEL, A
DOI：
10.1016/bs.mie.2021.10.004
发表时间：
2022-01-01
期刊：
SELENOPROTEIN STRUCTURE AND FUNCTION
影响因子：
--
作者：
Cheng, Qing;Arner, Elias S. J.
通讯作者：
Arner, Elias S. J.
影响因子：
21.3
作者：
Friedmann Angeli JP;Schneider M;Proneth B;Tyurina YY;Tyurin VA;Hammond VJ;Herbach N;Aichler M;Walch A;Eggenhofer E;Basavarajappa D;Rådmark O;Kobayashi S;Seibt T;Beck H;Neff F;Esposito I;Wanke R;Förster H;Yefremova O;Heinrichmeyer M;Bornkamm GW;Geissler EK;Thomas SB;Stockwell BR;O'Donnell VB;Kagan VE;Schick JA;Conrad M
通讯作者：
Conrad M
影响因子：
4.8
作者：
de Haan, JB;Bladier, C;Kola, I
通讯作者：
Kola, I
影响因子：
1.9
作者：
FUNK, MO;ISAAC, R;PORTER, NA
通讯作者：
PORTER, NA