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）是细胞代谢的副产物，通过暴露于紫外线和电离辐射以及环境致癌物而产生。活性氧产生的主要碱基损伤是7,8-二氢-8-氧鸟嘌呤（8-oxoG）。与正常的鸟嘌呤不同，8-oxoG在DNA复制过程中有与腺嘌呤错配的倾向，导致G:C到T:A的翻转突变。氧化修饰的碱基，如8-oxoG，主要通过碱基切除修复途径（BER）进行修复，其第一步是由特定的DNA糖基化酶识别和切除受损的碱基。从DNA中去除8-oxoG的主要哺乳动物酶是8-oxo鸟嘌呤-DNA糖基化酶（OGG1）。OGG1是一种双功能酶，既具有8-oxoG切割活性，又具有较弱的碱性位点ap裂解酶链切割活性。在OGG1切除8-oxoG后，产生的基本位点在一系列步骤中被几种酶进一步加工以完成修复。