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，暴露在紫外线、电离辐射和环境致癌物中。ROS产生的一个主要碱基损伤是7，8-二氢-8-氧鸟嘌呤(8-oxoG)。与正常的鸟嘌呤不同，8-oxoG在DNA复制过程中容易与腺嘌呤错误配对，导致G：C到T：A颠倒突变的固定。氧化修饰的碱基，如8-oxoG，主要通过碱基切除修复途径(BER)修复，第一步是通过特定的DNA糖基酶识别和切除受损的碱基。哺乳动物中去除DNA中8-oxoG的主要酶是8-oxoguanine-DNA糖基酶(OGG1)。OGG1是一种双功能酶，在碱性部位既有8-oxoG切割活性，又有微弱的解链切割活性。在8-oxoG被OGG1切除后，所产生的基本位点被几种酶按顺序进一步处理以完成修复。 对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-氧鸟嘌呤修复活性。人们普遍认为，随着时间的推移，DNA氧化损伤的积累会导致癌症。在人类肺癌和肾癌中，OGG1染色体位点的频繁丢失，以及肺癌患者中OGG1活性显著低于对照组，表明OGG1在肿瘤抑制中发挥了作用。在8-oxoG修复缺陷的基因敲除小鼠中，晚发性肺癌的增加，肺癌患者肺组织中8-oxoG水平的升高，以及在几个人类癌细胞系中8-oxoG修复能力的降低，表明癌症与8-oxoG修复能力可能有关。OGG1编码序列的改变导致氨基酸替换，从而影响功能、丰度或细胞内位置，预计会影响基因组8-oxoG水平，从而影响基因组的稳定性和致癌作用。 已有多个OGG1基因多态被报道，并与多种癌症呈正相关。OGG1 C末端326位丝氨酸取代半胱氨酸是一种常见的多态现象。在人群中测得的S326C OGG1等位基因频率从0.13到0.62不等，且在种族之间存在显著差异。相关研究已经证实，S326C OGG1等位基因纯合的个体增加了肺癌、前列腺癌和口咽癌的发病率。先前的一项研究发现，纯化的多态S326C OGG1的催化效率(kcat/Km)降低，而另一项研究发现S326C基因型与体内8-氧鸟嘌呤修复能力降低有关。我们对纯化的S326C的糖基酶和解酶活性以及DNA损伤结合亲和力进行了表征，发现与野生型酶相比，OGG1的多态存在新的功能缺陷和明显的二聚体DNA结合构象。我们的结果证实了S326C对与C配对的8-oxoG的修复活性降低，进一步表明当病变与T或G相反时，S326C OGG1尤其缺乏8-oxoguanine切除活性。APE1刺激野生型OGG1导致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步骤。我们发现，与野生型酶不同，S326C OGG1不受APE1的显著刺激，因此在S326C OGG1修复8-氧鸟嘌呤的过程中，BER的协调可能会受到干扰。我们观察到在人类细胞中表达的S326C OGG1的修复活性和二聚体构象降低，因此S326C OGG1突变体的活性和二聚体化学计量比的改变可能与体内相关。