Hill Jeff W, Hu Jennifer J, Evans Michele K
Division of Cancer Prevention and Control, Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL 33136-1002, United States.
DNA Repair (Amst). 2008 Apr 2;7(4):648-54. doi: 10.1016/j.dnarep.2008.01.003. Epub 2008 Feb 21.
Deficient repair activity for 8-hydroxy-2'-deoxyguanine (8-oxoguanine), a premutagenic oxidative DNA damage, has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimer's disease, and in ischemia/reperfusion injury, type 2 diabetes mellitus, and cancer. These conditions have in common the accumulation of oxidative DNA damage, which is believed to play a role in disease progression, and loss of intracellular calcium regulation. These observations suggest that oxidative DNA damage repair capacity may be influenced by fluctuations in cellular calcium. We have identified human 8-oxoguanine-DNA glycosylase 1 (OGG1), the major 8-oxoguanine repair activity, as a specific target of the Ca(2+)-dependent protease Calpain I. Protein sequencing of a truncated partially calpain-digested OGG1 revealed that calpain recognizes OGG1 for degradation at a putative PEST (proline, glutamic acid, serine, threonine) sequence in the C-terminus of the enzyme. Co-immunoprecipitation experiments showed that OGG1 and Calpain I are associated in human cells. Exposure of HeLa cells to hydrogen peroxide or cisplatin resulted in the degradation of OGG1. Pretreatment of cells with the calpain inhibitor calpeptin resulted in inhibition of OGG1 proteolysis and suggests that OGG1 is a target for calpain-mediated degradation in vivo during oxidative stress- and cisplatin-induced apoptosis. Polymorphic OGG1 S326C was comparatively resistant to calpain digestion in vitro, yet was also degraded by a calpain-dependent pathway in vivo following DNA damaging agent exposure. The degradation of OGG1 by calpain may contribute to decreased 8-oxoguanine repair activity and elevated levels of 8-oxoguanine reported in tissues undergoing chronic oxidative stress, ischemia/reperfusion, and other cellular stressors known to produce perturbations of intracellular calcium homeostasis which activate calpain.
在诸如阿尔茨海默病等衰老相关神经退行性疾病的受累组织中,以及在缺血/再灌注损伤、2型糖尿病和癌症中,均观察到针对8-羟基-2'-脱氧鸟嘌呤(8-氧代鸟嘌呤,一种致突变性氧化DNA损伤)的修复活性不足。这些病症的共同之处在于氧化DNA损伤的积累,据信其在疾病进展中起作用,以及细胞内钙调节的丧失。这些观察结果表明,氧化DNA损伤修复能力可能受细胞钙波动的影响。我们已确定人类8-氧代鸟嘌呤-DNA糖基化酶1(OGG1),即主要的8-氧代鸟嘌呤修复活性酶,是钙依赖性蛋白酶钙蛋白酶I的特定作用靶点。对经部分钙蛋白酶消化的截短OGG1进行蛋白质测序显示,钙蛋白酶识别OGG1并在该酶C末端的一个假定的PEST(脯氨酸、谷氨酸、丝氨酸、苏氨酸)序列处将其降解。免疫共沉淀实验表明,OGG1和钙蛋白酶I在人类细胞中相互关联。将HeLa细胞暴露于过氧化氢或顺铂会导致OGG1降解。用钙蛋白酶抑制剂钙肽素对细胞进行预处理可抑制OGG1的蛋白水解,这表明在氧化应激和顺铂诱导的细胞凋亡过程中,OGG1在体内是钙蛋白酶介导降解的靶点。多态性OGG1 S326C在体外对钙蛋白酶消化具有相对抗性,但在暴露于DNA损伤剂后,在体内也通过钙蛋白酶依赖性途径被降解。钙蛋白酶对OGG1的降解可能导致在经历慢性氧化应激、缺血/再灌注以及其他已知会导致细胞内钙稳态紊乱从而激活钙蛋白酶的细胞应激源的组织中,8-氧代鸟嘌呤修复活性降低以及8-氧代鸟嘌呤水平升高。