Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes - Formation of S-(2-succinyl)cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress
Inactivation of glyceraldehyde-3-phosphate dehydrogenase by fumarate in diabetes - Formation of S-(2-succinyl)cysteine, a novel chemical modification of protein and possible biomarker of mitochondrial stress
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DOI：
10.2337/db07-0838
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发表时间：
2008-01-01
期刊：
影响因子：
7.7
通讯作者：
Baynes, John W.
中科院分区：
文献类型：
--
作者：
Blatnik, Matthew;Frizzell, Norma;Baynes, John W.
反式-S-（2-琥珀酰基）半胱氨酸（2SC）是通过克雷布斯循环中间体富马酸盐与蛋白质中的半胱氨酸残基的迈克尔加成反应形成的。我们研究了富马酸盐在化学修饰和抑制巯基酶，甘油醛-3-磷酸脱氢酶（GAPDH），在体外和组织中的糖尿病rates.RESEARCH设计和方法GAPDH的作用与富马酸盐在PBS中孵育，以评估富马酸盐对酶活性的影响，在体外。通过高效液相色谱-四极杆/飞行时间质谱法分析胰蛋白酶肽确定2SC形成的位点。分别采用液相色谱/串联质谱法和气相色谱/质谱法测定对照组和链脲佐菌素诱导的糖尿病大鼠腓肠肌中的2SC和富马酸盐。用免疫沉淀法从肌肉中分离GAPDH，用质谱分析确定GAPDH的修饰位点，结果在体外和体内均发现2SC，其活性位点Cys-149和亲核Cys-244几乎相等。体外富马酸盐对GAPDH的灭活与2SC的形成相关。在糖尿病大鼠中，与对照大鼠相比，富马酸盐和2SC浓度增加约5倍，同时GAPDH比活性降低约25%。在糖尿病与对照组动物中，GAPDH的2SC分数修饰显著增加，与糖尿病动物肌肉中GAPDH的比活性降低一致。2SC可能是糖尿病线粒体应激的一个有用的生物标志物。富马酸盐对GAPDH和其他酶和蛋白质的修饰可能导致糖尿病并发症发生的代谢变化。
OBJECTIVE-S-(2-succinyl)cysteine (2SC) is formed by a Michael addition reaction of the Krebs cycle intermediate, fumarate, with cysteine residues in protein. We investigated the role of fumarate in chemical modification and inhibition of the sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in vitro and in tissues of diabetic rats.RESEARCH DESIGN AND METHODS-GAPDH was incubated with fumarate in PBS to assess effects of fumarate on enzyme activity, in vitro. Sites of 2SC formation were determined by analysis of tryptic peptides by high-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry. 2SC and fumarate in gastrocnemius muscle of control and streptozotocin-induced diabetic rats were measured by liquid chromatography/tandem mass spectrometry and by gas chromatography/ mass spectrometry, respectively. GAPDH was isolated from muscle by immunoprecipitation, and sites of modification of GAPDH were determined by mass spectrometry analysis.RESULTS-2SC was found, both in vitro and in vivo, about equally at active-site Cys-149 and nucleophilic Cys-244. Inactivation of GAPDH by fumarate in vitro correlated with formation of 2SC. In diabetic compared with control rats, fumarate, and 2SC concentration increased approximately fivefold, accompanied by an similar to 25% decrease in GAPDH specific activity. The fractional modification of GAPDH by 2SC was significantly increased in diabetic versus control animals, consistent with the decreased specific activity of GAPDH in muscle of diabetic animals.CONCLUSIONS-Fumarate contributes to inactivation of GAPDH in diabetes. 2SC may be a useful biomarker of mitochondrial stress in diabetes. Modification of GAPDH and other enzymes and proteins by fumarate may contribute to the metabolic changes underlying the development of diabetes complications.