Proteolytic disregulation of the S326C mutant OGG1 DNA r

S326C 突变体 OGG1 DNA r 的蛋白水解失调

• 批准号: 7327033
• 负责人: michele k evans
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7327033
• 关键词: Proteolytic; disregulation; S326C; mutant; OGG1

Reactive oxygen species (ROS) are produced as a by-product of cellular metabolism and through exposure to ultraviolet and ionizing radiation and environmental carcinogens. A major base damage produced by ROS is 7,8-dihydro-8-oxoguanine (8-oxoG). Unlike normal guanine, 8-oxoG has the propensity to mispair with adenine during DNA replication, resulting in the fixation of G:C to T:A transversion mutations. Oxidatively modified bases, such as 8-oxoG, are repaired primarily by the base excision repair pathway (BER), the first steps of which are the recognition and excision of the damaged base by a specific DNA glycosylase. The major mammalian enzyme for removing 8-oxoG from DNA is 8-oxoguanine-DNA glycosylase (OGG1). OGG1 is a bifunctional enzyme, having both 8-oxoG excision activity and a weak AP-lyase strand incision activity at abasic sites. Following excision of 8-oxoG by OGG1, the resultant abasic site is further processed in sequential steps by several enzymes to complete repair. Studies of OGG1 knockout mice and immunodepletion experiments suggest that OGG1 is the major mammalian 8-oxoguanine repair activity in non-transcribed DNA. It is widely accepted that accumulation of oxidative DNA damage over time can lead to cancer. A role for OGG1 in tumor suppression is suggested by the frequent loss of the OGG1 chromosomal locus in human lung and renal cancers and by significantly lower OGG1 activity among lung cancer patients compared to controls. Increased late-onset lung tumors in knockout mice deficient in repair of 8-oxoG, elevated 8-oxoG levels in lung tissue of lung cancer patients and decreased repair of 8-oxoG demonstrated in several human cancer cells lines suggest that cancer and 8-oxoG repair capacity may be linked. Changes in the OGG1 coding sequence that result in amino acid substitutions that affect function, abundance, or intracellular location could be anticipated to impact genomic 8-oxoG levels, and thereby influence genomic stability and carcinogenesis. Several OGG1 polymorphisms have been reported and positively correlate with a variety of cancers. A frequently occurring polymorphism results in the substitution of serine for cysteine at position 326 in the C-terminus of OGG1. The allele frequency of S326C OGG1 measured in human populations ranges from 0.13 to as high as 0.62 and varies significantly with ethnicity . Association studies have identified that individuals homozygous for the S326C OGG1 allele have increased incidence of lung, prostate, and orolaryngeal cancers. A previous study found decreased catalytic efficiency (kcat/Km) of purified polymorphic S326C OGG1 , while another study implicated the S326C genotype with decreased 8-oxoguanine repair capacity in vivo. We characterized the glycosylase and AP-lyase activities and DNA damage binding affinity of purified S326C and found novel functional defects in the polymorphic OGG1 and a distinct dimeric DNA binding conformation compared to the wild-type enzyme. Our results confirm that S326C has decreased repair activity towards 8-oxoG paired with C and further show that S326C OGG1 is particularly deficient in 8-oxoguanine excision activity when the lesion is opposite T or G. The stimulation of wild-type OGG1 by APE1 results in increased rates of 8-oxoG excision, and is believed to be an important step in the regulation and coordination of base excision repair in vivo. We show that S326C OGG1 is not significantly stimulated by APE1, unlike the wild-type enzyme, thereby the coordination of BER may be perturbed during repair of 8-oxoguanine by S326C OGG1. We observed decreased repair activity and dimeric conformation of S326C OGG1 expressed in human cells, thus the altered activity and dimeric stoichiometry of the S326C OGG1 variant may be relevant in vivo.

活性氧 (ROS) 是细胞代谢的副产品，是通过暴露于紫外线、电离辐射和环境致癌物质而产生的。 ROS 产生的主要碱基损伤是 7,8-二氢-8-氧代鸟嘌呤 (8-oxoG)。与正常鸟嘌呤不同，8-oxoG 在 DNA 复制过程中容易与腺嘌呤错配，导致 G:C 固定为 T:A 颠换突变。氧化修饰的碱基，例如 8-oxoG，主要通过碱基切除修复途径 (BER) 进行修复，该途径的第一步是特定 DNA 糖基酶识别和切除受损碱基。从 DNA 中去除 8-oxoG 的主要哺乳动物酶是 8-氧代鸟嘌呤-DNA 糖基化酶 (OGG1)。 OGG1 是一种双功能酶，具有 8-oxoG 切除活性和脱碱基位点弱 AP 裂解酶链切割活性。 OGG1 切除 8-oxoG 后，所得脱碱基位点将通过多种酶按顺序步骤进一步加工以完成修复。 OGG1基因敲除小鼠的研究和免疫耗竭实验表明，OGG1是哺乳动物非转录DNA中主要的8-氧代鸟嘌呤修复活性。人们普遍认为，随着时间的推移，氧化 DNA 损伤的积累会导致癌症。人类肺癌和肾癌中 OGG1 染色体位点的频繁丢失以及肺癌患者与对照组相比 OGG1 活性显着降低表明了 OGG1 在肿瘤抑制中的作用。 8-oxoG 修复缺陷的敲除小鼠中晚发性肺肿瘤增加、肺癌患者肺组织中 8-oxoG 水平升高以及几种人类癌细胞系中 8-oxoG 修复减少表明癌症和 8-oxoG修复能力可能是相关的。 OGG1 编码序列的变化会导致影响功能、丰度或细胞内位置的氨基酸取代，预计会影响基因组 8-oxoG 水平，从而影响基因组稳定性和致癌作用。 多种 OGG1 多态性已被报道，并且与多种癌症呈正相关。经常发生的多态性导致 OGG1 C 末端 326 位的丝氨酸取代半胱氨酸。在人群中测得的 S326C OGG1 等位基因频率范围为 0.13 至 0.62，并且随种族的不同而存在显着差异。关联研究发现，S326C OGG1 等位基因纯合的个体患肺癌、前列腺癌和口咽癌的发病率增加。之前的一项研究发现纯化的多态性 S326C OGG1 的催化效率（kcat/Km）降低，而另一项研究则表明 S326C 基因型体内 8-氧鸟嘌呤修复能力降低。我们表征了纯化的 S326C 的糖基酶和 AP 裂解酶活性以及 DNA 损伤结合亲和力，并发现与野生型酶相比，多态性 OGG1 存在新的功能缺陷以及独特的二聚体 DNA 结合构象。我们的结果证实，S326C 对与 C 配对的 8-oxoG 的修复活性降低，并进一步表明，当病变与 T 或 G 相对时，S326C OGG1 特别缺乏 8-oxoG1 切除活性。 APE1 结果对野生型 OGG1 的刺激8-oxoG 切除率增加，被认为是体内碱基切除修复调节和协调的重要一步。我们发现，与野生型酶不同，S326C OGG1 不会受到 APE1 的显着刺激，因此在 S326C OGG1 修复 8-氧代鸟嘌呤期间，BER 的协调可能会受到干扰。我们观察到人类细胞中表达的 S326C OGG1 的修复活性和二聚体构象降低，因此 S326C OGG1 变体的活性和二聚体化学计量的改变可能与体内相关。