Meng Feilong, Cang Xiaohui, Peng Yanyan, Li Ronghua, Zhang Zhengyue, Li Fushan, Fan Qingqing, Guan Anna S, Fischel-Ghosian Nathan, Zhao Xiaoli, Guan Min-Xin
From the Division of Medical Genetics and Genomics, Zhejiang Children's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, China.
the Institute of Genetics and.
J Biol Chem. 2017 Feb 17;292(7):2881-2892. doi: 10.1074/jbc.M116.749374. Epub 2017 Jan 3.
Nuclear modifier gene(s) was proposed to modulate the phenotypic expression of mitochondrial DNA mutation(s). Our previous investigations revealed that a nuclear modifier allele (A10S) in TRMU (methylaminomethyl-2-thiouridylate-methyltransferase) related to tRNA modification interacts with 12S rRNA 1555A→G mutation to cause deafness. The A10S mutation resided at a highly conserved residue of the N-terminal sequence. It was hypothesized that the A10S mutation altered the structure and function of TRMU, thereby causing mitochondrial dysfunction. Using molecular dynamics simulations, we showed that the A10S mutation introduced the Ser dynamic electrostatic interaction with the Lys residue of helix 4 within the catalytic domain of TRMU. The Western blotting analysis displayed the reduced levels of TRMU in mutant cells carrying the A10S mutation. The thermal shift assay revealed the value of mutant TRMU protein, lower than that of the wild-type counterpart. The A10S mutation caused marked decreases in 2-thiouridine modification of U34 of tRNA, tRNA and tRNA However, the A10S mutation mildly increased the aminoacylated efficiency of tRNAs. The altered 2-thiouridine modification worsened the impairment of mitochondrial translation associated with the m.1555A→G mutation. The defective translation resulted in the reduced activities of mitochondrial respiration chains. The respiratory deficiency caused the reduction of mitochondrial ATP production and elevated the production of reactive oxidative species. As a result, mutated TRMU worsened mitochondrial dysfunctions associated with m.1555A→G mutation, exceeding the threshold for expressing a deafness phenotype. Our findings provided new insights into the pathophysiology of maternally inherited deafness that was manifested by interaction between mtDNA mutation and nuclear modifier gene.
核修饰基因被认为可调节线粒体DNA突变的表型表达。我们之前的研究表明，与tRNA修饰相关的TRMU（甲基氨基甲基-2-硫尿苷酸甲基转移酶）中的一个核修饰等位基因（A10S）与12S rRNA 1555A→G突变相互作用导致耳聋。A10S突变位于N端序列的一个高度保守残基处。据推测，A10S突变改变了TRMU的结构和功能，从而导致线粒体功能障碍。通过分子动力学模拟，我们发现A10S突变在TRMU催化结构域内引入了丝氨酸与螺旋4中赖氨酸残基的动态静电相互作用。蛋白质免疫印迹分析显示，携带A10S突变的突变细胞中TRMU水平降低。热稳定性分析表明，突变型TRMU蛋白的熔点低于野生型对应蛋白。A10S突变导致tRNA、tRNA和tRNA的U34的2-硫尿苷修饰显著降低。然而，A10S突变轻微提高了tRNA的氨酰化效率。2-硫尿苷修饰的改变加剧了与m.1555A→G突变相关的线粒体翻译损伤。翻译缺陷导致线粒体呼吸链活性降低。呼吸功能缺陷导致线粒体ATP生成减少，活性氧化物质生成增加。结果，突变的TRMU加剧了与m.1555A→G突变相关的线粒体功能障碍，超过了表达耳聋表型的阈值。我们的研究结果为母系遗传性耳聋的病理生理学提供了新的见解，这种耳聋表现为线粒体DNA突变与核修饰基因之间的相互作用。
