Parkin与细胞结构相关蛋白相互作用：分子机制与功能

发布时间：2018-05-22 20:10

本文选题：Parkin + 乳腺癌　；参考：《南开大学》2010年博士论文

【摘要】：Parkin基因(也被称为PARK2)位于6号染色体的25.2-27区的脆性位点区域,是一个编码泛素连接酶的基因,能够促进蛋白底物的泛素化,使之通过蛋白酶体进行降解。多年的研究证明Parkin基因的突变是导致帕金森症发生的主要原因之一。Parkin在多种肿瘤中的表达显著降低或者不表达,并且有报道表明Parkin表达的降低主要是由于它的启动子的异常甲基化所引起的。这些实验结果表明Parkin可能是一个潜在的肿瘤抑制因子。看来,Parkin在帕金森症以及癌症发生过程中都起重要作用,其分子机制有待进一步研究。最近的报道表明,Parkin是一个微管结合蛋白,并且Parkin与微管的结合不受Parkin泛素连接酶活性丧失的点突变的影响。微管与Parkin的这种共定位关系有可能使Parkin锚定于细胞质中从而调节Parkin的泛素连接酶活性。考虑到微管在细胞内的多种重要作用以及微管作为肿瘤化疗靶点的研究,我们推断Parkin与微管的结合可能具有重要的生理意义,有可能会影响紫杉醇等以微管为靶点的抗肿瘤药物的敏感性。在我们的研究中发现Parkin能够结合在微管的外部,增强了微管与紫杉醇之间的结合,从而增强了由紫杉醇所诱导的微管的组装以及微管的稳定性。进一步研究的实验结果表明Parkin能够增加由紫杉醇所诱导的细胞多核化以及细胞凋亡,使得乳腺癌细胞对紫杉醇更加敏感。更为重要的是,在临床上采用紫杉醇联合用药化疗的病人组织样本中,Parkin的表达与肿瘤患者的病理反应成正相关。并且在原代培养的乳腺癌细胞中,Parkin的表达水平与紫杉醇的敏感性也是正相关的。这些研究表明Parkin可以促进乳腺癌对紫杉醇药物的敏感性,因此Parkin的表达水平可以做为一个诊断的标志,帮助预测乳腺癌患者是否适合含有紫杉醇的方案进行化疗。除此之外,我们的研究还表明,Parkin可以作为化疗药物开发的靶点,用以提高紫杉醇的敏感性。 Parkin基因的突变是导致帕金森症发生的主要原因之一,50%以上的早发性青少年帕金森症中都检测到了Parkin的突变。Parkin的突变导致帕金森症的原因可能是由于Parkin的突变致使泛素连接酶活性丧失,使得底物蛋白不能够被泛素化通过蛋白酶体进行降解,从而在细胞内过度累积,对细胞产生了极大的毒性。近年来的研究证明帕金森症患者中线粒体的形态,动态性以及功能都会发生异常。线粒体是一个高度动态性的器官,处于不断的断裂与融合的过程中,提供生物体所需要的大部分能量。线粒体动态性的失调与神经元细胞的存活以及多种神经性疾病的发生相关。大量的实验证明Parkin在调节线粒体动态性上起到了重要的作用,它能够与线粒体动态性相关蛋白相互作用来调节线粒体的断裂或者融合,然而调节的具体机制尚未明确,并且也还没有发现有一个线粒体相关的蛋白会在帕金森症患者的脑部发生累积。在我们的研究中,发现Parkin能够与线粒体断裂相关蛋白Drp1相互作用,通过泛素48位赖氨酸形成的多聚泛素化链介导Drp1的泛素化,使其通过蛋白酶体进行降解。沉默细胞内的Parkin能够使Drp1的表达水平明显的升高,从而导致线粒体的片断化。除此之外,用可以诱导帕金森症模型的神经毒素处理细胞时,能够使Parkin的表达水平明显的降低,而Drp1的表达水平显著增高。当沉默细胞内Drp1的表达水平时,能够抑制由神经毒素所诱导的线粒体的片断化以及细胞凋亡,并且能够有效的抑制帕金森症小鼠脑部黑质密质层部位多巴胺神经元的丢失。更为重要的是,通过免疫组化和生化分析帕金森症病人的临床组织样本,我们发现Parkin与Drp1的表达水平是负相关的,Drp1的表达水平明显的增加,而Parkin的表达水平显著降低。这些证据表明Drp1是一个典型的Parkin的底物,Parkin表达水平的降低以及突变能够导致Drp1蛋白的累积,从而使线粒体发生片断化,功能异常。我们的研究揭示了帕金森症中Parkin基因的突变导致线粒体形态功能异常的重要机制。
[Abstract]:The Parkin gene (also known as PARK2), a fragile site in the 25.2-27 region of chromosome 6, is a gene encoding a ubiquitin ligase that promotes ubiquitination of protein substrates and degradate them through proteasomes. Years of studies have shown that the mutation of the Parkin gene is one of the main causes of the occurrence of Parkinson's disease,.Parkin The expression of a variety of tumors is significantly reduced or unexpressed, and there have been reports that the decrease in Parkin expression is mainly due to the abnormal methylation of its promoter. These results suggest that Parkin may be a potential tumor suppressor. It appears that Parkin plays an important role in the process of Parkinson's and cancer. Its molecular mechanism remains to be further studied.
Recent reports suggest that Parkin is a microtubule binding protein, and the combination of Parkin with microtubules is not affected by the point mutation of the loss of Parkin ubiquitin ligase activity. This colocalization relationship between microtubules and Parkin may cause Parkin to be anchored in the cytoplasm to regulate the ubiquitin ligase activity of Parkin. A variety of important roles and microtubules as a target for cancer chemotherapy, we infer that the combination of Parkin and microtubules may have important physiological significance and may affect the sensitivity of paclitaxel and other antitumor drugs targeting microtubules. In our study, we found that Parkin can be combined with the external microtubules to enhance microtubules and purple. The combination of taxol enhanced the assembly of microtubules and the stability of microtubules induced by taxol. Further studies showed that Parkin could increase cell nucleation and cell apoptosis induced by taxol, making breast cancer cells more sensitive to taxol. The expression of Parkin was positively related to the pathological response of the tumor patients in the tissue samples of the patients treated with taxol, and the expression level of Parkin was also positively related to the sensitivity of taxol in the primary cultured breast cancer cells. These studies suggest that Parkin can promote the sensitivity of breast cancer to paclitaxel drugs, so Par The expression level of kin can be used as a diagnostic marker to help predict whether patients with breast cancer are suitable for chemotherapy with paclitaxel. Our study also suggests that Parkin can be used as a target for the development of chemotherapeutic drugs to improve the sensitivity of taxol.
Mutation of the Parkin gene is one of the main causes of the occurrence of Parkinson's disease. More than 50% of early adolescent Parkinson's disease has detected a mutation of the Parkin mutation.Parkin, which may cause the loss of the ubiquitin ligase activity due to the mutation of the Parkin, so that the substrate protein can not be generalized by ubiquitination. The proteasome is degraded and overaccumulated in the cell, producing great toxicity to the cells. In recent years, studies have shown that the morphology, dynamics and function of mitochondria in the patients with Parkinson's disease are abnormal. Mitochondria are a highly dynamic organ that provides organisms in the process of breaking and fusion. Most of the energy needed. The imbalance of mitochondrial dynamics is associated with the survival of neurons and the occurrence of a variety of neuropathic diseases. A large number of experiments have shown that Parkin plays an important role in regulating mitochondrial dynamics, and it can interact with mitochondrial dynamic related proteins to regulate mitochondrial disruption or fusion. However, the specific mechanism of regulation is not yet clear, and no mitochondrial associated protein has been found to accumulate in the brain of patients with Parkinson's disease.
In our study, we found that Parkin can interact with mitochondrial fracture related protein Drp1 and mediate the ubiquitination of Drp1 through a polyubiquitin 48 - bit lysine - based polyubiquitin chain that degradates it through proteasome. The silence of Parkin in cells can cause a flat rise in the expression of Drp1, leading to the fragmentation of the mitochondria. In addition, when the neurotoxin, which can induce Parkinson's disease model, can reduce the expression level of Parkin obviously, and the expression level of Drp1 is significantly higher. When the expression level of Drp1 in the silent cell, it can inhibit the fragmentation of the mitochondria and apoptosis induced by the neurotoxin, and can be used. The loss of dopamine neurons in the substantia nigra of the brain of Parkinson's mice was inhibited. More importantly, we found that the expression level of Parkin was negatively correlated with the expression level of Drp1 by immunohistochemical and biochemical analysis of the clinical tissue samples of the patients with Parkinson's disease. The expression level of Drp1 was significantly increased, while the expression level of Parkin was significant. These evidences suggest that Drp1 is a typical substrate for Parkin, the decrease of Parkin expression level and the mutation can lead to the accumulation of Drp1 protein, resulting in the fragmentation and dysfunction of mitochondria. Our study revealed the important mechanism of the abnormal mitochondrial morphologic function in Parkinson's disease.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329;Q51

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