SIRT5 regulation of ammonia-induced autophagy and mitophagy

In liver the mitochondrial sirtuin, SIRT5, controls ammonia detoxification by regulating CPS1, the first enzyme of the urea cycle. However, while SIRT5 is ubiquitously expressed, urea cycle and CPS1 are only present in the liver and, to a minor extent, in the kidney. To address the possibility that SIRT5 is involved in ammonia production also in nonliver cells, clones of human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12, overexpressing or silenced for SIRT5 were produced. Our results show that ammonia production increased in SIRT5-silenced and decreased in SIRT5-overexpressing cells. We also obtained the same ammonia increase when using a new specific inhibitor of SIRT5 called MC3482. SIRT5 regulates ammonia production by controlling glutamine metabolism. In fact, in the mitochondria, glutamine is transformed in glutamate by the enzyme glutaminase, a reaction producing ammonia. We found that SIRT5 and glutaminase coimmunoprecipitated and that SIRT5 inhibition resulted in an increased succinylation of glutaminase. We next determined that autophagy and mitophagy were increased by ammonia by measuring autophagic proteolysis of long-lived proteins, increase of autophagy markers MAP1LC3B, GABARAP, and GABARAPL2, mitophagy markers BNIP3 and the PINK1-PARK2 system as well as mitochondrial morphology and dynamics. We observed that autophagy and mitophagy increased in SIRT5-silenced cells and in WT cells treated with MC3482 and decreased in SIRT5-overexpressing cells. Moreover, glutaminase inhibition or glutamine withdrawal completely prevented autophagy. In conclusion we propose that the role of SIRT5 in nonliver cells is to regulate ammonia production and ammonia-induced autophagy by regulating glutamine metabolism.

在肝脏中，线粒体沉默信息调节因子5（SIRT5）通过调控尿素循环（urea cycle）的首个酶氨甲酰磷酸合成酶1（CPS1），实现氨解毒过程。然而，尽管SIRT5呈广泛表达模式，但尿素循环与CPS1仅在肝脏中存在，在肾脏中仅少量表达。为探究SIRT5在非肝脏细胞中同样参与氨生成的可能性，我们构建了过表达或沉默SIRT5的人乳腺癌细胞系MDA-MB-231与小鼠成肌细胞C2C12的克隆株。研究结果显示，SIRT5沉默的细胞中氨生成量升高，而SIRT5过表达的细胞中氨生成量降低。采用新型特异性SIRT5抑制剂MC3482处理后，同样观测到氨生成量升高。SIRT5通过调控谷氨酰胺代谢调节氨生成。实际上，在线粒体中，谷氨酰胺经谷氨酰胺酶（glutaminase）催化转化为谷氨酸，该反应伴随氨的产生。我们发现SIRT5与谷氨酰胺酶存在免疫共沉淀相互作用，且抑制SIRT5会导致谷氨酰胺酶的琥珀酰化（succinylation）水平升高。我们进一步通过检测长寿命蛋白的自噬性降解、自噬标志物MAP1LC3B、GABARAP及GABARAPL2的表达上调、线粒体自噬（mitophagy）标志物BNIP3与PINK1-PARK2系统的激活，以及线粒体形态与动力学变化，证实氨可诱导自噬与线粒体自噬。我们观察到，在SIRT5沉默的细胞与经MC3482处理的野生型细胞中，自噬与线粒体自噬水平升高，而在SIRT5过表达的细胞中则显著降低。此外，抑制谷氨酰胺酶或撤除谷氨酰胺，可完全阻断上述自噬现象。综上，我们提出：在非肝脏细胞中，SIRT5通过调控谷氨酰胺代谢，进而调节氨生成及氨诱导的自噬过程。