SIRT5 regulation of ammonia-induced autophagy and mitophagy

Abstract

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.

A Tale of Two Glutaminases: Homologous Enzymes with Distinct Roles in Tumorigenesis

“…Sebastian et al showed that SirT6 represses MYC, and, by extension, the expression of GLS [112]. A more direct interaction was demonstrated by Poletta and colleagues, who used immunoprecipitation assays to show that GLS binds directly to SirT5 [113]. They additionally showed that GLS was succinylated in the absence of SirT5, and suggested that the de succinylation activity of SirT5 acted to regulate GLS activity.…”

Sirtuin 5: a review of structure, known inhibitors and clues for developing new inhibitors

“…SIRT1-3 have been well studied for decades (Bell and Guarente, 2011;Donmez and Outeiro, 2013;Mellini et al, 2015), whereas SIRT4-7 have received relatively little attention until recently. SIRT5 has become an active topic of research towards its biological and physiological functions (Du et al, 2011;Nishida et al, 2015;Polletta et al, 2015;Tan et al, 2014). Several studies have demonstrated that SIRT5 actually has relatively weak deacetylase activity comparing with other sirtuins.…”

“…In contrast, SIRT5 has robust desuccinylation, demalonylation and deglutarylation activities in vitro and in vivo, ∼1,000-fold higher catalytic efficiency than deacetylation activity . Many studies also revealed that SIRT5 plays crucial roles in the regulation of ammonia detoxification (Nakagawa et al, 2009;Polletta , 2015), fatty acid oxidation (Park et al, 2013;Zhang et al, 2015), cellular respiration (Li et al, 2015a;Park et al, 2013), ketone body formation (Rardin et al, 2013), and reactive oxygen species (ROS) management , and disregulation or uncontrolled activation of SIRT5 can cause several human diseases, for instance cancer, Alzheimer's disease, and Parkinson's disease (Kumar and Lombard, 2015;Lai et al, 2013;Liu et al, 2015;Lu et al, 2014;Parihar et al, 2015).…”

SHMT2 Desuccinylation by SIRT5 Drives Cancer Cell Proliferation

“…It targets carbamoyl phosphate synthetase 1 (CPS1), urate oxidase (UOX), and glutaminase (GLS) to regulate the balance of the urea cycle and mitophagy (22)(23)(24)(25). It controls fatty acid oxidation through desuccinylating 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), enoyl-CoA hydratase a-subunit (ECHA), and demalonylating malonyl-CoA decarboxylase (26)(27)(28).…”