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Cu/Zn Superoxide Dismutase (SOD1), is a ubiquitous antioxidant enzyme with several emerging roles outside of its canonical function. SOD1 is also emerging in central roles in cancer and neurodegenerative pathologies. Little is known about SOD1 regulation, particularly at a post-translational level. Post-translational modifications (PTMs) play an important role in enabling proteins to rapidly respond to their environment. Therefore, identifying specific PTMs involved in protein regulation represents a powerful opportunity to interfere with any associated pathologies. This work employs proteomics to identify mechanisms of post-translation regulation on cell survival signaling proteins. We focused on SOD1, which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Interestingly, we also found that K122 acyl mutants were sufficient to prevent mitochondrial accumulation of the G93A SOD1 clinical mutant. We observed increased autophagic activity in G93A expressing cells compared to WT or G93A/K122-acyl mimic double mutants, and found that this double mutant was just as prone to aggregate as G93A SOD1—suggesting that SOD1 aggregation is more toxic when in the mitochondria. We observed increased protein turnover rates in cells expressing SOD1 G93A, in support of increased autophagy. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a new interplay between SOD1 acylation, metabolic regulation, SOD1 aggregate toxicity, and SOD1-mediated cell survival.

Autophagy is a regulated and evolutionarily conserved catabolic process that serves to degrade superfluous or damaged organelles and recycle their biochemical components for use in energy production and other biosynthetic reactions. It is a crucial cellular response to various stresses including oxidative stress and starvation. Sirtuins are NAD+ dependent deacetylases that link transcriptional regulation to cellular energy homeostasis, DNA damage, ROS response, cell cycle control, apoptosis and autophagy. The role of autophagy in cancer is complex as autophagy exerts both cell protective and damaging functions depending on the circumstances. The purpose of this study is to investigate the role of individual Sirtuin family members in the regulation of gene expression of the known autophagy markers LC3 and Beclin 1 under DNA damage and oxidative stress. To investigate the regulation of autophagy, we followed the gene expression of autophagy genes LC3 and Beclin 1 under diverse stress conditions in human osteosarcoma cells (U2OS). The protein and mRNA levels of LC3-II and the formation of autophagosomes were increased in etoposide and rotenone treated cells. While LC3-II and LC3-1 protein expressions were increased in Sirt1 overexpressing cells, no significant change was observed in Beclin 1 protein level. However, inhibition of Sirt1 by siRNA did not affect the cellular levels of the autophagy markers suggesting the potential involvement of other Sirtuin family members in the regulation of autophagy. Elevated LC3 mRNA was observed in cells overexpressing any of the Sirtuins family members; however etoposide treatment selectively inhibited Sirt1 and Sirt2 dependent upregulation of LC3 mRNA. Induction of LC3-Luc reporter activity was observed in Sirt5 transfected cells, which was further increased by etoposide treatment. Sirt5 carrying mutation in its catalytic domain is not able to induce autophagy and Sirt5 mediating autophagy is under the control of NF-KB transcription factor. These results support the notion that Sirtuins are important regulators of autophagy and the function of each member is to differentially regulate DNA damage and oxidative stress responses.

Dietary supplementation of nicotinamide adenine dinucleotide (NAD+) precursors has been suggested as a treatment for non-alcoholic fatty liver disease and obesity. In the liver, NAD+ is primarily generated by nicotinamide phosphoribosyltransferase (NAMPT), and hepatic levels of NAMPT and NAD+ have been reported to be dependent on age and body composition. The aim of the present study was to identify time course-dependent changes in hepatic NAD content and NAD+ salvage capacity in mice challenged with a high-fat diet (HFD). We fed 7-week-old C57BL/6JBomTac male mice either regular chow or a 60% HFD for 6, 12, 24, and 48 weeks, and we evaluated time course-dependent changes in whole body metabolism, liver steatosis, and abundance of hepatic NAD-associated metabolites and enzymes. Mice fed a 60% HFD rapidly accumulated fat and hepatic triglycerides with associated changes in respiratory exchange ratio (RER) and a disruption of the circadian feeding pattern. The HFD did not alter hepatic NAD+ levels, but caused a decrease in NADP+ and NADPH levels. Decreased NADP+ content was not accompanied by alterations in NAD kinase (NADK) abundance in HFD-fed mice, but NADK levels increased with age regardless of diet. NAMPT protein abundance did not change with age or diet. HFD consumption caused a severe decrease in protein lysine malonylation after six weeks, which persisted throughout the experiment. This decrease was not associated with changes in SIRT5 abundance. In conclusion, hepatic NAD+ salvage capacity is resistant to long-term HFD feeding, and hepatic lipid accumulation does not compromise the hepatic NAD+ pool in HFD-challenged C57BL/6JBomTac male mice.