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: 7963983
• 负责人: michele k evans
• 金额: $ 62.95万
• 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/7963983
• 关键词: 7,8-dihydro-8-oxoguanine; 8-Oxo-2' -Deoxyguanosine; 8-Oxoguanine DNA Glycosylase; 8-hydroxy-2' -deoxyguanosine; 8-hydroxyguanosine; Address; Adenine; Affect; Affinity; Age; Aging; Alleles; Alzheimer' s Disease; Amino Acid Sequence; Amino Acid Substitution; Apoptosis; Apoptotic; Bacteria; Basal cell carcinoma; Base Excision Repairs; Binding; Binding Proteins; Calcium; Calpain; Calpain I; Cancer Patient; Cancer cell line; Cell Extracts; Cells; Chronic; Cisplatin; Clinical; Co-Immunoprecipitations; Code; Crude Extracts; Cysteine; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA glycosylase; Data; Defect; Digestion; Disease; Disease Progression; Environmental Carcinogens; Enzymes; Ethnic Origin; Excision; Exposure to; Follow-Up Studies; Gene Frequency; Genes; Genetic Polymorphism; Genome Stability; Genomics; Genotype; Glutamic Acid; Guanine; Hela Cells; Homeostasis; Human; Human Cell Line; Hydrogen Peroxide; Hypersensitivity; In Vitro; Incidence; Individual; Ionizing radiation; Ischemia; Knockout Mice; Lead; Lesion; Life Style; Link; Location; Lung; Lung Neoplasms; Lyase; Malignant Neoplasms; Malignant neoplasm of lung; Measures; Mediating; Metabolism; Mind; Mitochondria; Molecular Conformation; Mutagenesis; Mutation; Neurodegenerative Disorders; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Nuclear Extract; Nucleosides; Outcome; Oxidative Stress; Pathway interactions; Peptide Hydrolases; Peptide Sequence Determination; Physiological; Play; Population; Positioning Attribute; Predisposition; Process; Proline; Prostate; Protein Isoforms; Proteins; Proteolysis; Reactive Oxygen Species; Regulation; Renal carcinoma; Reperfusion Injury; Reperfusion Therapy; Reporting; Resistance; Risk; Role; Serine; Site; Somatic Mutation; Structure of parenchyma of lung; Surgical incisions; Temperature; Threonine; Time; Tissues; Tumor Suppression; Variant; Vitamin K 3; base; calpain inhibitor; calpeptin; cancer cell; carcinogenesis; cell killing; comparative; high risk; human APEX1 protein; in vivo; inhibitor/antagonist; killings; leukemia; malignant breast neoplasm; malignant mouth neoplasm; mutant; novel; novel marker; oxidative DNA damage; prevent; protein expression; repaired; research study; sample fixation; stoichiometry; stressor; thermostability; transversion mutation; ultraviolet

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. We characterized the enzymatic activity of R229Q and determined the effect of R229Q expression on KG-1 survival following exposure to DNA damaging agents. Our results show 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. Initially reported as a unique somatic mutation in KG-1 cells, we report that the R229Q allele is a documented polymorphism in human populations. With the significant incidence of the allele in the population, our observations of OGG1 structural destabilization and increased cell killing following induction of oxidative DNA damage resulting from the R229Q polymorphism suggest that the variant may be a potential marker for cancer susceptibility. These results suggest that decreased 8-oxoguanine repair in KG-1 is due to thermolability of R229Q OGG1 and that the enzyme variant increases cellular susceptibility to killing resulting from oxidative DNA damage. The R229Q OGG1 variant is a validated polymorphism prevalent in world populations and not an isolated mutation in KG-1 cells, thus the R229Q OGG1 allele may be a novel marker for cancer susceptibility. With that in mind, we have begun looking a age-associated diseases and deficient repair activity for 8-hydroxy-2-deoxyguanine (8-oxoguanine),since it has been observed in affected tissues in neurodegenerative diseases of aging, such as Alzheimers 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 Ca2+-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. 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.

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。在肺癌患者肺组织中肺部修复缺乏8-oxog修复，8-oxog水平升高的敲除小鼠中的晚期肺部肿瘤增加，并且在几个人类癌细胞系列中证明的8-氧气的修复降低表明癌症和8-oxog修复能力可能会联系起来。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。在人类种群中测得的S326C OGG1的等位基因频率范围从0.13到0.62，并且随着种族的差异很大。协会研究已经确定，S326C OGG1等位基因纯合子的个体增加了肺，前列腺和口服癌的发病率。先前的一项研究发现，纯化多态性S326C OGG1的催化效率（KCAT/km），而另一项研究则暗示了S326C基因型，体内8-氧气的修复能力降低。 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。 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.我们表明，与野生型酶不同，APE1并未显着刺激S326C OGG1，因此在S326C OGG1修复8-氧气的8-氧气时，BER的配位可能会受到干扰。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的降低，因此S326C OGG1变体的活性和二聚体化学计量的变化可能在体内与之相关。 我们表征了R229Q的酶促活性，并确定了R229Q表达对DNA损伤剂后KG-1存活的影响。 我们的结果表明，R229Q OGG1是高度热的，并且在体外和体内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。 最初据报道是KG-1细胞中独特的体细胞突变，我们报告说R229Q等位基因是人类种群中有记录的多态性。 随着等位基因在人群中的显着发病率，我们对OGG1结构不稳定的观察结果和诱导氧化性DNA损伤的细胞杀伤增加，这表明R229Q多态性导致了氧化性DNA损伤，这表明该变体可能是癌症易感性的潜在标记。 这些结果表明，kg-1中的8-氧气修复降低是由于R229Q OGG1的可热性引起的，并且酶变体增加了细胞的易感性，导致氧化性DNA损伤导致杀伤。 R229Q OGG1变体是在世界人群中普遍存在的经过验证的多态性，而不是KG-1细胞中孤立的突变，因此R229Q OGG1等位基因可能是癌症易感性的新颖标志。考虑到这一点，我们已经开始寻找与年龄相关的疾病和对8-羟基-2-脱氧瓜氨酸（8-氧气）（8-氧气）的修复活动，因为在衰老的神经退行性疾病中已经观察到了它在阿尔茨海默氏病的神经退行性疾病中观察到，例如阿尔茨海默氏病疾病，以及在等级/癌症和癌症中，类型为2-糖尿病。 这些疾病具有氧化性DNA损伤的积累，据信在疾病进展中起作用，并且细胞内钙调节的丧失。 这些观察结果表明，氧化性DNA损伤修复能力可能受细胞钙波动的影响。 我们已经确定了人类8-氧气 - 糖苷-DNA糖基酶1（OGG1），主要的8-氧气修复活性，是Ca2+依赖性蛋白酶钙蛋白酶钙蛋白酶的特定靶标。酶的C末端序列。 共免疫沉淀实验表明，OGG1和CALPAIN I与人类细胞相关。 HeLa细胞暴露于过氧化氢或顺铂导致OGG1降解。 用钙蛋白酶抑制剂钙调蛋白对细胞进行预处理导致OGG1蛋白水解抑制，并表明OGG1是在氧化应激和顺铂诱导的凋亡过程中钙蛋白酶介导的体内降解的靶标。 多态性OGG1 S326C在体外对钙蛋白酶消化具有相对抗性，但在DNA损伤剂暴露后，体内的钙蛋白酶依赖性途径也会降解。 Calpain对OGG1的降解可能导致8-氧气的修复活性降低，并且在经历了慢性氧化应激，缺血/再灌注的组织中报道的8-氧气的水平升高，其他已知的细胞胁迫和其他细胞胁迫产生脑内稳态的细胞内钙化脑稳态的扰动。 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。