Li Zhen, Liu Rongrong, Liu Yingying, Zhao Mengmeng, Luan Jun, Wang Yan, Shang Wenjing, Song Xicheng, Sun Yan, Han Fengchan
Key Laboratory for Genetic Hearing Disorders in Shandong, Department of Biochemistry and Molecular Biology, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China; Department of Otorhinolaryngology Head and Neck Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, No. 20 East, Yuhuangding Road, Yantai, 264000, Shandong, PR China; Shandong Provincial Clinical Research Center for Otorhinolaryngologic Diseases, No. 20 East, Yuhuangding Road, Yantai, 264000, Shandong, PR China.
Key Laboratory for Genetic Hearing Disorders in Shandong, Department of Biochemistry and Molecular Biology, Binzhou Medical University, 346 Guanhai Road, Yantai, 264003, Shandong, PR China; Department of Otorhinolaryngology Head and Neck Surgery, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, No. 20 East, Yuhuangding Road, Yantai, 264000, Shandong, PR China.
Biochem Biophys Res Commun. 2022 Jul 5;612:134-140. doi: 10.1016/j.bbrc.2022.04.104. Epub 2022 Apr 26.
A/J mouse is a typical animal model of age-related deafness. Previous studies have shown that the mice suffer from progressive hearing loss and degeneration of cochlear cells, and a variation of H55 N in citrate synthase (CS) causes about 40% the hearing loss. CS is a key enzyme in the tricarboxylic acid cycle, which is transported from cytoplasm to mitochondria after synthesis, sorted by the mitochondrial targeting sequence (MTS). To explore the mechanism of CS (H55 N) variation in affecting its function, HEI-OC1 cells were infected with lentivirus particles to express CS-Flag or CS(H55 N)-Flag. The results showed that H55 N variation in CS, as purified by co-immunoprecipitation, decreased the enzyme activity by about 50%. Confocal microscope co-localization indicated that the CS (H55 N) variation led to a decrement in its mitochondrial content. Western blot also showed the amount of CS(H55 N)-Flag was more than that of CS(WT)-Flag in the cytosol. The results suggest H55 N variation in CS lead to decrement of its enzyme activity and targeting transport to mitochondria. We therefore conclude that decrement in CS activity and mitochondrial delivery contributes to the degeneration of cochlear cells and thus the hearing loss in A/J mice.
A/J小鼠是典型的年龄相关性耳聋动物模型。先前的研究表明，这些小鼠患有进行性听力丧失和耳蜗细胞退化，柠檬酸合酶（CS）中H55N的变异导致约40%的听力丧失。CS是三羧酸循环中的关键酶，合成后从细胞质转运到线粒体，由线粒体靶向序列（MTS）进行分选。为了探究CS（H55N）变异影响其功能的机制，用慢病毒颗粒感染HEI-OC1细胞以表达CS-Flag或CS（H55N）-Flag。结果表明，通过免疫共沉淀纯化的CS中的H55N变异使酶活性降低了约50%。共聚焦显微镜共定位表明，CS（H55N）变异导致其线粒体含量减少。蛋白质印迹法还显示，胞质溶胶中CS（H55N）-Flag的量比CS（WT）-Flag的量多。结果表明，CS中的H55N变异导致其酶活性降低以及向线粒体的靶向转运减少。因此，我们得出结论，CS活性降低和线粒体递送减少导致耳蜗细胞退化，从而导致A/J小鼠听力丧失。
