SIRT5-mediated GLS and GDH desuccinylation attenuates the autophagy of bovine mammary epithelial cells induced by ammonia

Abstract

Sirtuin 5 (SIRT5) in mitochondria possesses a strong capacity for lysine desuccinylation, involving in various biological processes. Our previous research demonstrated that NH₃ regulated autophagy dependent on SIRT5 in bovine mammary epithelial cells (bMECs). Interestingly, we discovered that SIRT5 reduced the content of NH₃ 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 NH₃ 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.