Yang Hanlin, Gao Shikai, Lu Guangyang, He Junhui, Dong Jinru, Zhang Xinyi, Liu Luya, Zhong Kai, Zha Guangming, Han Liqiang, Guo Shuang, Li Heping, Wang Yueying
Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China; Key Laboratory of Veterinary Biotechnology of Henan Province, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China; Ministry of Education Key Laboratory for Animal Pathogens and Biosafety, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China.
Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China; Key Laboratory of Veterinary Biotechnology of Henan Province, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, Henan, China.
Cell Signal. 2025 Mar;127:111570. doi: 10.1016/j.cellsig.2024.111570. Epub 2024 Dec 16.
Sirtuin 5 (SIRT5) in mitochondria possesses a strong capacity for lysine desuccinylation, involving in various biological processes. Our previous research demonstrated that NH regulated autophagy dependent on SIRT5 in bovine mammary epithelial cells (bMECs). Interestingly, we discovered that SIRT5 reduced the content of NH and glutamate by inhibiting GLS activity in bMECs, the ratio of ADP/ATP also declined. In this study, we identified that SIRT5 interacted with endogenous GLS and GDH through Co-IP assay, but had no effect on endogenous GLS and GDH expression. SIRT5 made the succinylation levels of GLS and GDH significantly declined and resulted in the reduction of GLS and GDH activity. Next, the content of ammonia and glutamate, as well as the related autophagy markers were measured, we found that SIRT5 affected the glutamine metabolism, which attenuated ammonia release in MAC-T cells, accompanying with cellular autophagy decline, reducing the formation of autophagosome. Deletion of SIRT5 gene in MAC-T cells by means of CRISPR-cas9, we found the content of NH and glutamate increased, as well as autophagy promoted, which could be alleviated by SIRT5 overexpression. SIRT5 KO also resulted in increase of succinylation of GLS and GDH, as well as autophagy response in bMECs. Furthermore, SIRT5 promoted the maintenance of mitochondria homeostasis. Mechanistically, SIRT5 reduced ammonia release by modulating the succinylation levels and enzymatic activities of GLS and GDH in mitochondria and promoted the maintenance of mitochondria homeostasis, as well as further attenuated ammonia-stimulated autophagy in bovine mammary epithelial cells.
线粒体中的沉默调节蛋白5（SIRT5）具有很强的赖氨酸去琥珀酰化能力，参与多种生物学过程。我们之前的研究表明，NH在牛乳腺上皮细胞（bMECs）中依赖SIRT5调节自噬。有趣的是，我们发现SIRT5通过抑制bMECs中的GLS活性降低了NH和谷氨酸的含量，ADP/ATP的比率也下降了。在本研究中，我们通过免疫共沉淀实验确定SIRT5与内源性GLS和GDH相互作用，但对内源性GLS和GDH的表达没有影响。SIRT5使GLS和GDH的琥珀酰化水平显著下降，并导致GLS和GDH活性降低。接下来，我们测量了氨和谷氨酸的含量以及相关的自噬标志物，发现SIRT5影响谷氨酰胺代谢，减弱了MAC-T细胞中的氨释放，同时细胞自噬下降，减少了自噬体的形成。通过CRISPR-cas9删除MAC-T细胞中的SIRT5基因，我们发现NH和谷氨酸的含量增加，自噬增强，而SIRT5过表达可以缓解这种情况。SIRT5基因敲除还导致bMECs中GLS和GDH的琥珀酰化增加以及自噬反应增强。此外，SIRT5促进了线粒体稳态的维持。机制上，SIRT5通过调节线粒体中GLS和GDH的琥珀酰化水平和酶活性来减少氨释放，促进线粒体稳态的维持，并进一步减弱牛乳腺上皮细胞中氨刺激的自噬。
