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。 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。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-oxoG1 切除活性。我们表征了 R229Q 多态性的酶活性，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。 我们还发现人 8-氧鸟嘌呤-DNA 糖基化酶 1 (OGG1) 是 Ca2+ 依赖性蛋白酶 Calpain I 的特定靶点。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/再灌注和已知会产生其他细胞应激源的组织中导致 8-氧鸟嘌呤修复活性降低和 8-氧鸟嘌呤水平升高。 扰动细胞内钙稳态，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。 BER 途径期间发生多种蛋白质-蛋白质相互作用，以协调该途径的高度复杂的过程。 我们正在使用公正的生化方法来确定 OGG1 的功能性结合伴侣。 利用这种方法，我们初步确定了PARP-1与OGG1特异性相互作用。 PARP-1 是 DNA 断裂的分子传感器，它通过物理结合或通过伙伴蛋白（包括各种核蛋白、组蛋白、单链断裂修复蛋白 (SSBR)、BER 蛋白和 PARP-1 本身）的聚 (ADP-核糖基) 化，在修复这些断裂中发挥关键作用。此外，PARP-1 会因 DNA 损伤而被激活，使用敲除细胞和 PARP-1 抑制剂的研究表明，PARP-1 对于维持基因组完整性很重要。我们报告了 OGG1 和聚（ADP-核糖）聚合酶（PARP-1）之间的一种新的相互作用。 我们发现OGG1通过OGG1的N末端区域直接与PARP-1结合，并且这种相互作用通过氧化应激而增强。 OGG1 似乎在体外和体内刺激 PARP-1 的聚（ADP-核糖）化活性，而与野生型细胞相比，在 OGG1-/- 细胞中观察到响应 DNA 损伤的聚（ADP-核糖）水平降低。重要的是，PARP-1 抑制 OGG1。尽管 OGG1 多态性变体蛋白 R229Q 和 S326C 与 PARP-1 结合，但当 S326C 蛋白与 PARP-1 一起孵育时，聚（ADP-核糖基）化作用减少。此外，OGG1 -/- 细胞对单独的 PARP 抑制剂或与 DNA 损伤剂组合的 PARP 抑制剂更敏感。这些发现表明，OGG1 与 PARP-1 的结合在修复氧化性 DNA 损伤中发挥着功能性作用。我们正在开展实验，重点关注结合类型以及与 OGG1 结合的功能结果。 最近，在 AD 患者的脑组织中发现了 OGG1 的两种多态性，A53T 和 A288V，但人们对这些多态性如何促进 AD 知之甚少。使用纯化的蛋白质，我们对 A53T 和 A288V 多态性变体进行了表征，并检测到这两种蛋白质的催化活性降低了 55-75%。此外，A53T 多态性导致底物结合显着降低。我们观察到这两种变体与已知的 OGG1 结合伙伴 PARP-1 和 XRCC1 的结合均减少。在体内，我们发现表达 A53T 和 A288V OGG1 的 OGG1-/- 小鼠胚胎成纤维细胞对 DNA 损伤显着更敏感，并且存活率显着降低。我们的结果提供了生化和细胞证据，表明 A53T 和 A288V 多态性蛋白在催化和蛋白结合活性方面存在缺陷，这可以解释 AD 大脑中 DNA 氧化损伤的增加。这些数据表明 OGG1 和这些多态性可能在 AD 易感性中发挥作用。