Proteolytic disregulation of the S326C mutant OGG1 DNA repair enzyme

S326C 突变体 OGG1 DNA 修复酶的蛋白水解失调

• 批准号: 8148267
• 负责人: michele k evans
• 金额: $ 53.04万
• 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/8148267
• 关键词: DNA Repair Enzymes; OGG1 gene; mutant

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. 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. 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. We characterized the enzymatic activity of the R229Q polymorphism and determined the effect of R229Q expression on KG-1 survival following exposure to DNA damaging agents. Our results showed that R229Q OGG1 is highly thermolabile and rapidly inactivated at physiological temperatures both in vitro and in vivo. Expression of both nuclear and mitochondrial R229Q OGG1 sensitized KG-1 cells to killing via an apoptotic pathway following exposure to menadione and 8-oxodG, thus R229Q promotes apoptosis following ROS and oxidized nucleoside exposure. We have also identified human 8-oxoguanine-DNA glycosylase 1 (OGG1) as a specific target of the Ca2+-dependent protease Calpain I. 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. This year we have begun to address the question of whether other proteins that may be vital to recognition and processing of oxidatively induced DNA damage interact differently with polymorphic forms of OGG1. We have proceeded to examine at baseline the binding of wild type OGG1 to DNA damage sensing proteins. This has enabled us to understand more directly the possible role of OGG1 and its polymorphic variants in the processing and repair of oxidative DNA damage in cells from individuals who may be more vulnerable to the effects of oxidative stress. Multiple protein-protein interactions occur during the BER pathway in order to coordinate the highly intricate process of this pathway. We are using an unbiased biochemical approach in order to determine functional binding partners for OGG1. Using this approach, we preliminarily have determined that PARP-1 specifically interacts with OGG1. PARP-1 is a molecular sensor of DNA breaks and it plays a key role in repair of these breaks by either physically associating with or also by poly(ADP-ribosyl)ation of partner proteins including various nuclear proteins, histones, single-strand break repair proteins (SSBR), BER proteins and on PARP-1 itself. Furthermore, PARP-1 is activated in response to DNA damage and studies using knockout cells and PARP-1 inhibitors show that PARP-1 is important for maintaining genomic integrity. We are pursing experiments focused on the type of binding and the functional outcomes of this binding to OGG1.

活性氧（ROS）作为细胞代谢的副产品，通过暴露于紫外线和电离辐射和环境致癌物。 ROS产生的主要基本损害为7,8-二氢-8-氧甲烷（8-oxog）。与正常的鸟嘌呤不同，8-oxog在DNA复制过程中倾向于用腺嘌呤混乱，从而导致g：c：c至t：t：a transtression突变。氧化修饰的碱基（例如8-oxog）主要是通过碱基切除修复途径（BER）修复的，其第一步是通过特定的DNA糖基酶对受损碱的识别和切除。从DNA中去除8-氧的主要哺乳动物酶是8-氧气甘氨酸-DNA糖基酶（OGG1）。 OGG1是一种双功能酶，具有8-oxog切除活性，并且在abasic部位具有弱的AP酶链切口活性。通过OGG1切除8-oxog后，通过几种酶进行了顺序处理所得的无asic位点以完成修复。 OGG1基因敲除小鼠和免疫启动实验的研究表明，OGG1是未转录的DNA中主要的哺乳动物8-氧气修复活性。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。与对照组相比，肺癌和肾脏癌患者中OGG1染色体基因座的经常丧失在人类肺和肾脏癌中的频繁丧失，与对照组相比，OGG1染色体基因座的经常丧失明显降低了OGG1染色体位点，与OGG1染色体基因座的频繁丧失相比，与对照组相比，OGG1染色体基因座的频繁丧失明显降低，这表明了OGG1在肿瘤抑制中的作用。可以预期会影响功能，丰度或细胞内位置的氨基酸取代的OGG1编码序列的变化会影响基因组8-oxog水平，从而影响基因组稳定性和癌变。 已经报道了几种OGG1多态性，并与各种癌症呈正相关。经常发生的多态性导致丝氨酸在OGG1的C末端326处的半胱氨酸取代。我们表征了纯化的S326C的糖基化酶和AP-溶性活性以及DNA损伤结合亲和力，并在多态性OGG1中发现了新型的功能缺陷，并且与野生型酶相比，具有不同的二聚体DNA结合构象。我们的结果证实，S326C与C配对8-oxog的修复活性降低，并进一步表明，当病变相反T或G时，S326C OGG1在8-氧化氨基的切除活性中尤其缺乏。我们表征了R229Q聚构态的酶活性，并确定了R229Q表达的效果，以摄取R229Q的效果。 我们的结果表明，R229Q OGG1是高度热的，并且在体外和体内的生理温度下迅速灭活。 暴露于Menadione和8-OxoDG后，核和线粒体R229Q OGG1敏化kg-1细胞在通过凋亡途径中杀死，因此R229Q在ROS和氧化核苷暴露后促进凋亡。 我们还确定了人类8-氧气 - 糖苷-DNA糖基化酶1（OGG1）是Ca2+依赖性蛋白酶钙蛋白酶钙蛋白酶的特定靶标。CALPAINOGG1的降解可能会导致8-氧气素修复活性降低，并在8-氧气素修复活动中降低，并在8-氧气中升高，并在8-氧气中升高，并在8-氧气中升高，并导致其他氧化应激的氧化应激，氧化应激，依次的氧化氧化氧气，依次氧化氧气，质量为氧化应激的氧化应激率。激活钙蛋白酶的细胞内钙稳态的扰动。 今年，我们已经开始解决以下问题：其他对于氧化诱导的DNA损伤的识别和处理至关重要的蛋白质是否与OGG1的多态性形式不同。 我们已经在基线上检查了野生型OGG1与DNA损伤传感蛋白的结合。 这使我们能够更直接地理解OGG1及其多态性变体在处理和修复细胞中可能更容易受到氧化应激作用的人的氧化DNA损伤中的可能作用。为了协调该途径的高度复杂过程，多种蛋白质 - 蛋白质相互作用发生。 我们正在使用一种无​​偏的生化方法来确定OGG1的功能结合伙伴。 使用这种方法，我们初步确定PARP-1特异性与OGG1相互作用。 PARP-1是DNA断裂的分子传感器，它通过与伴侣蛋白（包括各种核蛋白质，单链蛋白，单链破裂修复蛋白（SSBR），BER蛋白和PARP-1本身）的聚（ADP-核糖基）物理缔合或通过物理缔合或通过物理相关性或通过聚（ADP-核糖基）的物理关联而在这些断裂中起关键作用。此外，响应DNA损伤而激活PARP-1，使用基因敲除细胞和PARP-1抑制剂进行研究表明，PARP-1对于维持基因组完整性很重要。我们正在追求重点是结合的类型和与OGG1结合的功能结果。