School of Medicine, Institute of Biochemistry, Justus-Liebig-University, Giessen, Germany.
Diabetes. 2010 Mar;59(3):670-8. doi: 10.2337/db08-1565. Epub 2009 Dec 15.
The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo.
MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2(Akita) mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice.
In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2(Akita), STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-beta2 subunit was increased.
Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.
泛素-蛋白酶体系统是细胞内异常蛋白质的主要降解机制。研究表明,糖尿病损伤细胞内的活性二羰基甲基乙二醛(MGO)水平升高,导致蛋白质修饰及其功能改变。本研究旨在体外和体内研究 MGO 衍生的晚期糖基化终产物(AGE)形成对蛋白酶体活性的影响。
通过质谱分析、免疫沉淀和 Western blot 分析 MGO 衍生的 AGE 对蛋白酶体亚基的修饰。使用蛋白酶体特异性荧光底物分析蛋白酶体活性。实验模型包括牛视网膜内皮细胞、糖尿病 Ins2(Akita)小鼠、糖氧还蛋白 1(GLO1)敲低小鼠和链脲佐菌素(STZ)注射糖尿病小鼠。
体外与 MGO 孵育会导致几种 20S 蛋白酶体亚基蛋白形成加合物。在培养的内皮细胞中,催化 20S 蛋白酶体亚基的表达水平没有改变,但蛋白酶体糜蛋白酶样活性显著降低。相比之下,调节 19S 蛋白酶体蛋白水平降低。在糖尿病 Ins2(Akita)、STZ 糖尿病和非糖尿病及糖尿病 G101 敲低小鼠中,糜蛋白酶样活性也降低,20S-β2 亚基的 MGO 修饰增加。
高血糖诱导的 MGO 形成共价修饰 20S 蛋白酶体,降低糖尿病肾脏中的活性,并减少多聚泛素受体 19S-S5a。结果表明高血糖与细胞功能障碍之间存在新的联系。