Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions
Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions
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DOI：
10.1042/bj20090854
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发表时间：
2009-10-15
影响因子：
4.1
通讯作者：
Lee, Kong-Joo
中科院分区：
文献类型：
--
作者：
Hwang, Na Rae;Yim, Seung-Hee;Lee, Kong-Joo
了解ROS（活性氧）的细胞靶点及其调节是理解ROS介导的信号传导的必要前提。GAPDH（甘油醛-3-磷酸脱氢酶）是已知的氧化应激中的主要靶蛋白，并且在其活性位点处变得巯基化。然而，氧化GAPDH（非活性形式）的分子和功能变化尚未得到表征。为了研究氧化应激下GAPDH的修饰，我们通过二维凝胶电泳分离氧化产物，并使用nanoLC-ESI-q-TOF MS/MS（纳米柱液相色谱耦合电喷雾电离四极杆飞行时间串联MS）对其进行鉴定。细胞内GAPDH经氧化应激后在双向凝胶电泳上分离出多个酸性斑点，经鉴定为活性位点Cys（152）上的半胱氨酸二硫化物和半胱氨酸。我们鉴定了氧化失活GAPDH的相互作用蛋白为p54 nrb（54 kDa核RNA结合蛋白）和PSF（聚嘧啶片段结合蛋白相关剪接因子），已知这两种蛋白均以异二聚体形式存在并与RNA和DNA结合。在GAPDH活性位点突变体C152 S的表达后，氧化GAPDH和p54 nrb之间的相互作用被消除。p54 nrb的C-末端以依赖于过氧化氢剂量的方式与胞质溶胶中的GAPDH结合。GAPDH-p54 nrb复合物通过p54 nrb-PSF结合增强内在拓扑异构酶I活化。这些结果表明，GAPDH发挥糖酵解以外的其他功能，并且氧化修饰的GAPDH通过改变其相互作用蛋白质来调节其细胞功能，即通过与p54 nrb-PSF复合物相互作用来进行RNA剪接。
Knowledge of the cellular targets of ROS (reactive oxygen species) and their regulation is an essential prerequisite for understanding ROS-mediated signalling. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is known as a major target protein in oxidative stresses and becomes thiolated in its active site. However, the molecular and functional changes of oxidized GAPDH, the inactive form, have not yet been characterized. To examine the modifications of GAPDH under oxidative stress, we separated the oxidation products by two-dimensional gel electrophoresis and identified them using nanoLC-ESI-q-TOF MS/MS (nano column liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem MS). Intracellular GAPDH subjected to oxidative stress separated into multiple acidic spots on two-dimensional gel electrophoresis and were identified as cysteine disulfide and cysteic acids on Cys(152) in the active site. We identified the interacting proteins of oxidized inactive GAPDH as p54nrb (54 kDa nuclear RNA-binding protein) and PSF (polypyrimidine tract-binding protein-associated splicing factor), both of which are known to exist as heterodimers and bind to RNA and DNA. Interaction between oxidized GAPDH and p54nrb was abolished upon expression of the GAPDH active site mutant C152S. The C-terminal of p54nrb binds to GAPDH in the cytosol in a manner dependent on the dose of hydrogen peroxide. The GAPDH-p54nrb complex enhances the intrinsic topoisomerase I activation by p54nrb-PSF binding. These results suggest that GAPDH exerts other functions beyond glycolysis, and that oxidatively modified GAPDH regulates its cellular functions by changing its interacting proteins, i.e. the RNA splicing by interacting with the p54nrb-PSF complex.