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) 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。缺乏 8-oxoG 修复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 修复能力可能存在关联。 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-氧代鸟嘌呤切除活性。APE1刺激野生型OGG1导致8-oxoG切除率增加，并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。 我们表征了 R229Q 的酶活性，并确定了暴露于 DNA 损伤剂后 R229Q 表达对 KG-1 存活的影响。 我们的结果表明，R229Q OGG1 具有高度热不稳定性，并且在体外和体内生理温度下会迅速失活。 核和线粒体 R229Q OGG1 的表达使 KG-1 细胞在暴露于甲萘醌和 8-oxodG 后通过凋亡途径敏感地被杀伤，因此 R229Q 在 ROS 和氧化核苷暴露后促进细胞凋亡。 最初报道为 KG-1 细胞中的独特体细胞突变，我们报道 R229Q 等位基因是人类群体中已记录的多态性。 由于该等位基因在人群中的显着发生率，我们对 OGG1 结构不稳定和 R229Q 多态性引起的氧化 DNA 损伤诱导后细胞杀伤增加的观察表明，该变异可能是癌症易感性的潜在标志。 这些结果表明，KG-1 中 8-氧代鸟嘌呤修复的减少是由于 R229Q OGG1 的热稳定性造成的，并且酶变体增加了细胞对氧化 DNA 损伤造成的杀伤的敏感性。 R229Q OGG1 变异是世界人群中普遍存在的经过验证的多态性，而不是 KG-1 细胞中的孤立突变，因此 R229Q OGG1 等位基因可能是癌症易感性的新标记。考虑到这一点，我们开始寻找与年龄相关的疾病和 8-羟基-2-脱氧鸟嘌呤 (8-oxoguanine) 的修复活性缺陷，因为它已在衰老神经退行性疾病（如阿尔茨海默病）以及缺血/再灌注损伤、2 型糖尿病和癌症的受影响组织中观察到。 这些病症的共同点是氧化 DNA 损伤的积累（据信这在疾病进展中发挥着一定作用）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到细胞钙波动的影响。 我们已经鉴定出人 8-氧鸟嘌呤-DNA 糖基酶 1 (OGG1)（主要的 8-氧鸟嘌呤修复活性）作为 Ca2+ 依赖性蛋白酶 Calpain I 的特定靶标。对截短的部分钙蛋白酶消化的 OGG1 进行蛋白质测序表明，钙蛋白酶可识别 OGG1，并在假定的 PEST（脯氨酸、谷氨酸、丝氨酸、 苏氨酸）位于酶 C 末端的序列。 免疫共沉淀实验表明 OGG1 和 Calpain I 在人类细胞中相关。 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预处理细胞会抑制 OGG1 蛋白水解，表明 OGG1 是氧化应激和顺铂诱导的细胞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抵抗力，但在暴露于 DNA 损伤剂后，体内也会被钙蛋白酶依赖性途径降解。 钙蛋白酶对 OGG1 的降解可能会导致 8-氧鸟嘌呤修复活性降低，并在经历慢性氧化应激、缺血/再灌注和其他已知会产生细胞内钙稳态扰动的组织中升高 8-氧鸟嘌呤水平，从而激活钙蛋白酶。 今年，我们开始解决以下问题：对于识别和处理氧化诱导的 DNA 损伤可能至关重要的其他蛋白质是否与 OGG1 多态性存在不同的相互作用。 我们继续在基线上检查野生型 OGG1 与 DNA 损伤传感蛋白的结合。 这使我们能够更直接地了解 OGG1 及其多态性变体在可能更容易受到氧化应激影响的个体细胞中处理和修复氧化 DNA 损伤中的可能作用。