O'Brien Marisa, Chalker Julia, Slade Liam, Gardiner Danielle, Mailloux Ryan J
Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
Department of Biochemistry, Memorial University of Newfoundland, 230 Elizabeth Ave, St. John's, Newfoundland, Canada A1B 3X9.
Free Radic Biol Med. 2017 May;106:302-314. doi: 10.1016/j.freeradbiomed.2017.02.046. Epub 2017 Feb 27.
Pyruvate dehydrogenase (Pdh) is a vital source of reactive oxygen species (ROS) in several different tissues. Pdh has also been suggested to serve as a mitochondrial redox sensor. Here, we report that O/ HO emission from pyruvate dehydrogenase (Pdh) is altered by S-glutathionylation. Glutathione disulfide (GSSG) amplified O/ HO production by purified Pdh during reverse electron transfer (RET) from NADH. Thiol oxidoreductase glutaredoxin-2 (Grx2) reversed these effects confirming that Pdh is a target for S-glutathionylation. S-glutathionylation had the opposite effect during forward electron transfer (FET) from pyruvate to NAD lowering O/ HO production. Immunoblotting for protein glutathione mixed disulfides (PSSG) following diamide treatment confirmed that purified Pdh can be S-glutathionylated. Similar observations were made with mouse liver mitochondria. S-glutathionylation catalysts diamide and disulfiram significantly reduced pyruvate or 2-oxoglutarate driven O/ HO production in liver mitochondria, results that were confirmed using various Pdh, 2-oxoglutarate dehydrogenase (Ogdh), and respiratory chain inhibitors. Immunoprecipitation of Pdh and Ogdh confirmed that either protein can be S-glutathionylated by diamide and disulfiram. Collectively, our results demonstrate that the S -glutathionylation of Pdh alters the amount of ROS formed by the enzyme complex. We also confirmed that Ogdh is controlled in a similar manner. Taken together, our results indicate that the redox sensing and ROS forming properties of Pdh and Ogdh are linked to S-glutathionylation.
丙酮酸脱氢酶（Pdh）是几种不同组织中活性氧（ROS）的重要来源。Pdh也被认为可作为线粒体氧化还原传感器。在此，我们报告丙酮酸脱氢酶（Pdh）的O₂/ H₂O₂释放会因S-谷胱甘肽化而改变。在从NADH进行逆向电子传递（RET）过程中，谷胱甘肽二硫化物（GSSG）增强了纯化的Pdh产生O₂/ H₂O₂的能力。硫醇氧化还原酶谷氧还蛋白-2（Grx2）逆转了这些效应，证实Pdh是S-谷胱甘肽化的靶点。在从丙酮酸到NAD的正向电子传递（FET）过程中，S-谷胱甘肽化具有相反的作用，降低了O₂/ H₂O₂的产生。用二酰胺处理后对蛋白质谷胱甘肽混合二硫化物（PSSG）进行免疫印迹证实纯化的Pdh可以被S-谷胱甘肽化。对小鼠肝脏线粒体也有类似的观察结果。S-谷胱甘肽化催化剂二酰胺和双硫仑显著降低了肝脏线粒体中丙酮酸或2-氧代戊二酸驱动的O₂/ H₂O₂产生，使用各种Pdh、2-氧代戊二酸脱氢酶（Ogdh）和呼吸链抑制剂证实了这些结果。对Pdh和Ogdh进行免疫沉淀证实这两种蛋白质都可以被二酰胺和双硫仑S-谷胱甘肽化。总体而言，我们的结果表明Pdh的S-谷胱甘肽化改变了酶复合物形成的ROS量。我们还证实Ogdh也以类似方式受到调控。综上所述，我们的结果表明Pdh和Ogdh的氧化还原传感及ROS形成特性与S-谷胱甘肽化有关。
