CERKL突变导致视网膜变性的分子机制

发布时间：2018-01-22 13:52

本文关键词： CERKL 视网膜变性 TRX2 活性氧物质感光细胞凋亡斑马鱼　出处：《华中科技大学》2013年博士论文　论文类型：学位论文

【摘要】：CERKL是2004年被克隆的一个新基因,因与神经酰胺激酶CERK高度同源而被命名为神经酰胺类似蛋白(ceramide kinase-like protein, CERKL),该基因的突变会导致严重的视网膜退行性疾病,包括视网膜色素变性(retinitis pigmentosa, RP)和视锥视杆细胞营养不良(cone rod dystrophy, CRD),但CERKL在视网膜中的分子功能一直未取得重要进展,该基因突变引起视网膜变性的病理机制还不清楚。为阐释该基因的致病分子机制,本论文通过酵母双杂交、免疫共沉淀等实验手段,发现并确定了CERKL与线粒体硫氧还原蛋白TRX2存在相互作用。进一步分析这种相互作用的生理意义,发现CERKL能够调控TRX2的氧化还原状态。细胞中过表达CERKL可维持氧化压力下TRX2的还原态,从而保护细胞免受由氧化压力引起的细胞凋亡；用siRNA抑制CERKL表达,在氧胁迫条件下TRX2的还原态比例显著降低,细胞内活性氧(reactive oxygen species, ROS)积累明显,进而导致细胞凋亡。CERKL的两个致病错义突变p.R106S和p.C125W,显著破坏其与TRX2的相互作用,并失去氧化条件下保护细胞抗氧化的能力。用siRNA抑制细胞中TRX2的表达、或通过添加TRX2还原酶(TrxR2)抑制剂(auranofin)抑制TRX2还原态的转化,均使得CERKL保护细胞抗氧化损伤的能力大幅减弱或消失,提示TRX2在CERKL保护细胞抗氧化的过程中是必须的。进一步应用斑马鱼进行体内实验,结果发现在斑马鱼中CERKL主要表达于视网膜感光细胞的视锥细胞中。Morpholino抑制斑马鱼CERKL的表达,斑马鱼中出现明显的类似于视网膜变性症状,视网膜感光细胞层明显变短,感光细胞排列紊乱。TUNEL结果显示,视网膜的多个细胞层中均产生明显的细胞凋亡信号。在CERKL敲低的斑马鱼视网膜中,核酸、蛋白、脂类这些生物大分子都发生了明显的氧化损伤信号,感光细胞层的氧化损伤尤为明显。Redox Westernblot检测,发现CERKL缺陷鱼中氧化态的TRX2含量显著增加。以上斑马鱼in vivo的研究结果与我们在细胞实验中得到的结果相互验证,提示CERKL通过调控TRX2,保护视网膜细胞抵抗氧胁迫引起的细胞凋亡。本文首次从分子水平上阐述了CERKL突变引起视网膜变性的分子机制,尤为重要的是,论文揭示了一条机体内新的抗ROS引起的细胞凋亡途径。发现CERKL是一个新的调控因子,在细胞中它通过调控TRX2相关的抗ROS通路,保护细胞中的氧化还原平衡,从而起到在氧胁迫情况下的抗凋亡作用。由于代谢过程中ROS积累是视网膜变性以及其它神经退行性疾病的共同机制,本文的发现有可能揭示视网膜变性、黄斑变性等视网膜疾病以及其它重要的神经退行性疾病新的发病机制,并为这些重要的人类疾病的预防和治疗提供了新的靶点。
[Abstract]:CERKL is a new gene cloned in 2004. Because of its high homology with ceramide kinase CERK, it is named ceramide kinase-like protein (CERKL). Mutations in the gene can lead to severe retinal degenerative diseases, including retina pigmentosa initis pigmentosa. CERKL and cone rod dystrophysis, but the molecular function of CERKL in the retina has not made any significant progress. The pathological mechanism of retinal degeneration caused by this gene mutation is unclear. In order to elucidate the molecular mechanism of the gene, this paper uses yeast two-hybrid, immuno-coprecipitation and other experimental means. The interaction between CERKL and mitochondrial thioxy reducing protein (TRX2) was found and confirmed. The physiological significance of this interaction was further analyzed. It was found that CERKL could regulate the redox state of TRX2. Overexpression of CERKL in cells could maintain the reduced state of TRX2 under oxidative pressure. Thus protecting cells from apoptosis caused by oxidative pressure; When CERKL expression was inhibited by siRNA, the reduction rate of TRX2 decreased significantly under oxygen stress, and reactive oxygen species was induced by reactive oxygen species in cells. Ros) accumulation, which resulted in two missense mutations, P. R106S and p.C125W, of apoptosis. CERKL significantly destroyed the interaction between R106S and TRX2. SiRNA was used to inhibit the expression of TRX2 in the cells. Or by adding TRX2 reductase TrxR2) inhibitor to inhibit the transformation of TRX2 reductive state. The ability of CERKL to protect cells from antioxidant damage was significantly weakened or disappeared, suggesting that TRX2 is necessary in the process of CERKL protection. Zebrafish were further used for in vivo experiments. The results showed that CERKL was mainly expressed in cones of retinal photoreceptor cells in zebrafish. Morpholino inhibited the expression of CERKL in zebrafish. Zebrafish showed obvious symptoms similar to retinal degeneration, retinal photoreceptor cell layer became shorter, photoreceptor cells were disordered. Tunel results showed. In many layers of the retina, there are obvious cell apoptosis signals. In the retina of zebrafish with CERKL knockout, the biological macromolecules such as nucleic acid, protein and lipid have obvious oxidative damage signal. The oxidative damage of photoreceptor cell layer was especially obvious. Redox Westernblot was detected. It was found that the content of oxidized TRX2 in CERKL deficient fish increased significantly. The above results of zebrafish in vivo were verified by our results in cell experiments. The results suggest that CERKL protects retinal cells from apoptosis induced by oxygen stress by regulating TRX 2. In this paper, the molecular mechanism of retinal degeneration caused by CERKL mutation is discussed for the first time at the molecular level. A novel anti-apoptotic pathway induced by ROS was revealed in this paper. It was found that CERKL is a new regulatory factor in cells by regulating the TRX2 related anti-#en3# pathway. Protect the redox balance in the cells, thus play the role of anti-apoptosis under oxygen stress. The accumulation of ROS during metabolism is the common mechanism of retinal degeneration and other neurodegenerative diseases. The findings of this paper may reveal new pathogenesis of retinal diseases such as retinal degeneration, macular degeneration and other important neurodegenerative diseases. It also provides new targets for the prevention and treatment of these important human diseases.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1;Q75

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