The role of the Cockayne syndrome proetin

Cockayne综合征(CS)是一种以神经变性、恶病质和加速衰老为特征的常染色体隐性遗传病。80%的病例是由CS互补B组(CSB)基因突变引起的，已知的CSB基因涉及DNA修复和转录。在CS细胞中，核和线粒体DNA氧化损伤的修复存在缺陷，这可能是该病的主要潜在原因。以前我们发现CSB缺陷细胞在氧化应激后积累氧化碱基8-羟基鸟嘌呤和8-羟基腺嘌呤，这与观察到CSB和8-oxoG修复的主要DNA糖基酶OGG1在体内处于一个复合体中是一致的。我们还发现CSB蛋白与Nei样DNA糖基酶NEIL1物理上相互作用，NEIL1也参与氧化碱基的修复。这种相互作用显著地刺激了NEIL1的催化活性，包括糖基酶和解酶。观察到CSB缺陷小鼠在脑组织中积累了显著较高水平的几种氧化DNA碱基，包括法比腺嘌呤和法比鸟嘌呤，这支持了CSB蛋白在体内清除氧化损伤中的作用。此前，我们证明了CSB蛋白也与PARP1蛋白相互作用，PARP1蛋白参与单链断裂修复的早期步骤，这两种蛋白在细胞对氧化应激的反应中相互作用。CSB是PARP-1核糖化的底物，这两种蛋白很可能在碱基切除过程中共同发挥作用。我们的结果表明，CSB蛋白在DNA氧化损伤的修复中起着重要作用，未修复损伤的积累，特别是在靶组织，如脑，可能与以严重的早发性神经变性为特征的CS病理有关。为了进一步探讨CSB在线粒体中的作用，我们评估了氧化应激后CSB的线粒体定位。我们发现，甲萘二酮治疗后CSB对线粒体的定位增加，这会导致一种形式的氧化应激。此外，我们发现与野生型细胞相比，CSB缺陷细胞中8-氧鸟嘌呤、尿嘧啶和5-羟基尿嘧啶的切割活性降低。这种缺陷与线粒体内膜相关的BER活性丧失有关。这些依赖于CSB的改变具有功能后果，因为我们在CSB缺陷细胞中观察到mtDNA突变增加。综上所述，结果表明CSB通过帮助招募、稳定和/或保留修复复合体中与内膜相关的修复复合体中的BER蛋白，在线粒体BER中发挥直接作用。 CSB不仅在线粒体DNA修复中发挥作用，我们还在研究CSB在线粒体中的作用，以调节线粒体的质量，从而调节线粒体的生物能量学。携带CSB突变的小鼠的临床特征包括听力损失、小胶质细胞激活和恶病质，与人类CS的灾难性疾病表型相比轻微。我们最近的研究揭示了CSB小鼠模型的新的复杂特征，包括代谢率升高和自噬改变。在CSB缺陷的细胞中，线粒体含量增加，而自噬下调，这可能是由于p62的招募和线粒体泛素化的缺陷所致。CSB缺陷细胞表现为自由基生成增加和受损线粒体的积累。因此，用自噬刺激剂氯化锂或雷帕霉素处理可以逆转CSB缺陷细胞的生物能量表型。我们的数据表明，CSB作为线粒体DNA损伤传感器，诱导线粒体在应激反应中的自噬，自噬的药物调节剂是这种加速衰老表型的潜在治疗选择。在未来的研究中，我们计划调查CSB小鼠是否表现出遗传决定的代谢缺陷。 &#8195；

Cockayne syndrome (CS) is a devastating autosomal recessive disease characterized by neurodegeneration, cachexia, and accelerated aging. 80% of the cases are caused by mutations in the CS complementation group B (CSB) gene known to be involved in DNA repair and transcription. In CS cells, there are deficiencies in the repair of oxidative DNA damage in the nuclear and mitochondrial DNA, and this may be a major underlying cause of the disease. Previously we found that CSB-deficient cells accumulate oxidized bases, 8-hydroxyguanine and 8-hydroxyadenine, after oxidative stress, consistent with the observation that CSB and oxoguanine DNA glycosylase (OGG1), the major DNA glycosylase for 8-oxoG repair, are in a complex in vivo. We also found that the CSB protein physically interacts with the Nei-like DNA glycosylase, NEIL1, which is also involved in the repair of oxidized bases. This interaction significantly stimulates NEIL1 catalytic activities, both the glycosylase and the AP-lyase. The observation that CSB-deficient mice accumulate significantly higher levels of several oxidized DNA bases in brain tissue, including fapyadenine and fapyguanine, supports a role for the CSB protein in the removal of oxidized lesions in vivo. Previously we demonstrated that the CSB protein also interacts with PARP1, a protein involved in the early steps of single-strand break repair, and that these two proteins cooperate in the cellular responses to oxidative stress. CSB is a substrate for PARP-1 ribosylation and it is likely that these two proteins function together in the process of base excision. Our results indicate that the CSB protein plays an important role in the repair of oxidative DNA damage and that accumulation of unrepaired lesions, particular in target tissues, like the brain, may be relevant to the CS pathology, which is characterized by severe early onset neurodegeneration. To further explore the role of CSB in mitochondria, we evaluated the mitochondrial localization of CSB following oxidative stress. We found increased CSB localization to mitochondria following menadione treatment, which causes a form of oxidative stress. Additionally, we found reduced 8-oxo-guanine, uracil, and 5-hydroxy-uracil incision activities in CSB-deficient cells compared to wild-type cells. This deficiency correlated with a loss of mitochondrial inner membrane associated BER activities. These CSB-dependent changes had a functional consequence because we observed elevated mtDNA mutations in CSB deficient cells. Together the results suggest that CSB plays a direct role in mitochondrial BER by helping to recruit, stabilize, and/or retain BER proteins in repair complexes that in mitochondria are associated with the inner membrane. Not only does CSB play a role in mtDNA repair, we are also pursuing the proposal that CSB functions in mitochondria to modulate mitochondrial quality and thereby mitochondrial bioenergetics. The clinical features of mice carrying a mutation in CSB involve hearing loss, microglial activation and cachexia, and are mild compared to the catastrophic disease phenotype of CS in human patients. Our recent studies reveal novel complex features of the CSB mouse model, including elevated metabolic rate and altered autophagy. Mitochondrial content is increased in CSB-deficient cells, whereas autophagy is down-regulated, presumably as a result of defects in the recruitment of P62 and mitochondrial ubiquitination. CSB-deficient cells show increased free radical production and an accumulation of damaged mitochondria. Accordingly, treatment with the autophagic stimulators lithium chloride or rapamycin reverses the bioenergetic phenotype of CSB-deficient cells. Our data imply that CSB acts as an mtDNA damage sensor, inducing mitochondrial autophagy in response to stress, and that pharmacological modulators of autophagy are potential treatment options for this accelerated aging phenotype. In future studies, we plan to investigate whether the CSB mouse displayed a genetically-determined metabolic defect.