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: 8552417
• 负责人: michele k evans
• 金额: $ 79.43万
• 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/8552417
• 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; Alleles; Amino Acid Substitution; Apoptosis; Apoptotic; Bacteria; Basal cell carcinoma; Base Excision Repairs; Binding; Binding Proteins; Biochemical; Brain; Breast Cancer Cell; Calcium; Calpain; Calpain I; Cancer Patient; Cancer cell line; Cell Extracts; Cells; Chronic; Clinical; Code; Crude Extracts; Cysteine; DNA; DNA Binding; DNA Damage; DNA Repair Enzymes; DNA biosynthesis; DNA glycosylase; Data; Defect; Embryo; Environmental Carcinogens; Enzymes; Ethnic Origin; Excision; Exposure to; Fibroblasts; Genes; Genetic Polymorphism; Genome Stability; Genomics; Guanine; Hela Cells; Histones; Homeostasis; Human; Human Cell Line; Hypersensitivity; In Vitro; Incidence; Incubated; Individual; Ionizing radiation; Ischemia; Knock-out; Knockout Mice; Lead; Lesion; Life Style; Location; Lyase; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of prostate; Measures; Metabolism; Mitochondria; Molecular; Molecular Conformation; Mus; Mutagenesis; Mutation; N-terminal; Nuclear; Nuclear Extract; Nuclear Proteins; Nucleosides; Outcome; Oxidative Stress; Pathway interactions; Patients; Peptide Hydrolases; Physiological; Play; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Population; Positioning Attribute; Predisposition; Process; Protein Binding; Protein Isoforms; Proteins; Reactive Oxygen Species; Renal carcinoma; Reperfusion Therapy; Reporting; Risk; Role; Serine; Single Strand Break Repair; Site; Surgical incisions; Temperature; Time; Tissues; Tumor Suppression; UV Radiation Exposure; Variant; Vitamin K 3; XRCC1 gene; base; brain tissue; cancer cell; carcinogenesis; cell type; comparative; follow-up; functional outcomes; high risk; in vivo; inhibitor/antagonist; killings; leukemia; malignant mouth neoplasm; mutant; novel; oxidative DNA damage; oxidative damage; prevent; protein expression; protein protein interaction; repaired; research study; response; sample fixation; sensor; stressor; transversion mutation

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 report a novel interaction between OGG1 and Poly(ADP-ribose) polymerase (PARP-1). We found that OGG1 binds directly to PARP-1 through the N-terminal region of OGG1, and this interaction is enhanced by oxidative stress. OGG1 appears to stimulate the poly(ADP-ribosyl)ation activity of PARP-1 both in vitro and in vivo, whereas, decreased poly(ADP-ribose) levels were observed in OGG1-/- cells compared to wild-type cells in response to DNA damage. Importantly, PARP-1 inhibits OGG1. Though the OGG1 polymorphic variant proteins R229Q and S326C bind to PARP-1, there was a reduction in poly(ADP-ribosyl)ation when the S326C protein was incubated with PARP-1. Furthermore, OGG1 -/- cells were more sensitive to PARP inhibitors alone or in combination with a DNA-damaging agent. These findings indicate that OGG1 binding to PARP-1 plays a functional role in the repair of oxidative DNA damage.We are pursing experiments focused on the type of binding and the functional outcomes of this binding to OGG1. Recently, two polymorphisms of OGG1, A53T and A288V, have been identified in brain tissues of AD patients, but little is known about how these polymorphisms may contribute to AD. Using purified proteins, we characterized the A53T and A288V polymorphic variants and detected a 55-75% reduction in the catalytic activity for both proteins. Additionally, the A53T polymorphism leads to significantly decreased substrate binding. We observed that both variants have decreased binding to known OGG1 binding partners PARP-1 and XRCC1. In vivo, we found OGG1-/- mouse embryo fibroblast cells expressing A53T and A288V OGG1 significantly more sensitive to DNA damage and had significantly decreased survival. Our results provide both biochemical and cellular evidence that A53T and A288V polymorphic proteins have deficiencies in catalytic and protein binding activities that could explain the increase in oxidative damage to DNA found in AD brains. These data suggest that OGG1 and these polymorphisms may have a role in AD susceptibility.

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。