Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity
Pirin regulates pyruvate catabolism by interacting with the pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity
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DOI：
10.1128/jb.00710-06
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Lai, Hsin-Chih
中科院分区：
文献类型：
--
作者：
Soo, Po-Chi;Horng, Yu-Tze;Lai, Hsin-Chih
蛋白质pirin参与多种生物过程，从原核微生物、真菌和植物到哺乳动物都是保守的。它在哺乳动物中作为转录辅因子或凋亡相关蛋白，并参与植物种子萌发和幼苗发育。在原核生物中，虽然吡林是应力诱导的蓝细菌，并可能作为槲皮素酶在大肠杆菌，吡林直向同源物的功能仍然大多未被确定。我们发现，粘质沙雷氏菌pirin（pirin（Sm））基因编码的pirin蛋白质的直系同源物。蛋白质下拉和细菌双杂交试验，然后由十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和电喷雾电离-串联质谱分析表明，丙酮酸脱氢酶（PDH）E1亚基作为一个组件与pirin（Sm），基因相互作用。功能分析表明，在S. marcescens CH-1。色葡萄marcescens CH-1 pirin（Sm）基因通过插入-缺失同源重组进行突变。相应地，在S. marcescens CH-1 pirin（Sm）突变体。与此同时，细胞的NADH/NAD（+）的比例增加的pirin（Sm）突变体，表明增加的三羧酸（TCA）循环活性。我们的研究结果表明，pirin（Sm）基因通过与PDH E1亚基相互作用，抑制PDH酶复合物的活性，在S. marcescens CH-1，并且他们表明pirin（Sm）是参与决定丙酮酸代谢朝向TCA循环或发酵途径的方向的重要蛋白质。
The protein pirin, which is involved in a variety of biological processes, is conserved from prokaryotic microorganisms, fungi, and plants to mammals. It acts as a transcriptional cofactor or an apoptosis-related protein in mammals and is involved in seed germination and seedling development in plants. In prokaryotes, while pirin is stress induced in cyanobacteria and may act as a quercetinase in Escherichia coli, the functions of pirin orthologs remain mostly uncharacterized. We show that the Serratia marcescens pirin (pirin(Sm)) gene encodes an ortholog of pirin protein. Protein pull-down and bacterial two-hybrid assays followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization-tandem mass spectrometry analyses showed the pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with the pirin(Sm), gene. Functional analyses showed that both PDH El subunit activity and PDH enzyme complex activity are inhibited by the pirin(Sm) gene in S. marcescens CH-1. The S. marcescens CH-1 pirin(Sm) gene was subsequently mutated by insertion-deletion homologous recombination. Accordingly, the PDH Ell and PDH enzyme complex activities and cellular ATP concentration increased up to 250%, 140%, and 220%, respectively, in the S. marcescens CH-1 pirin(Sm) mutant. Concomitantly, the cellular NADH/NAD(+) ratio increased in the pirin(Sm) mutant, indicating increased tricarboxylic acid (TCA) cycle activity. Our results show that the pirin(Sm) gene plays a regulatory role in the process of pyruvate catabolism to acetyl coenzyme A through interaction with the PDH El subunit and inhibiting PDH enzyme complex activity in S. marcescens CH-1, and they suggest that pirin(Sm) is an important protein involved in determining the direction of pyruvate metabolism towards either the TCA cycle or the fermentation pathways.