It is a well-established scientific observation that mammalian cells contain fidelity or watchdog proteins that maintain the correct function of cellular organelles. Over the past several years, the Sirtuin deacetylase family protein Sirt3 has emerged as a mitochondrial fidelity protein that directs energy generation and regulates reactive oxygen species (ROS) scavenging proteins. Loss of function or genetic mutation of these fidelity proteins has been shown to create a cellular environment that is permissive for the development of cellular damage associated with processes such as aging and carcinogenesis. Mitochondria are the primary organelles that direct oxidative metabolism for the production of ATP; however, this is also a significant source of ROS. Thus, it is reasonable to propose that mitochondria should contain proteins that would signal downstream target molecules and/or ROS scavenger enzymes to maintain mitochondrial and cellular homeostatic poise. It is also reasonable to hypothesize that the mitochondria contain fidelity proteins similar to those found in the nucleus and cytoplasm. We discuss a new role of Sirt3 in the direction of the primary superoxide scavenger protein, manganese superoxide dismutase (MnSOD), and how the acetylation or deacetylation of several specific lysines appears to direct MnSOD enzymatic dismutase activity. Aberrant downstream regulation of MnSOD by Sirt3 may be a potential source of cellular damage that accumulates with aging to create a tumor-permissive phenotype. Future studies can explore the role of MnSOD in age-related illness using this new mechanism of enzymatic regulation.

Aims: Adenosine triphosphate (ATP) synthase uses chemiosmotic energy across the inner mitochondrial membrane to convert adenosine diphosphate and orthophosphate into ATP, whereas genetic deletion of Sirt3 decreases mitochondrial ATP levels. Here, we investigate the mechanistic connection between SIRT3 and energy homeostasis. Results: By using both in vitro and in vivo experiments, we demonstrate that ATP synthase F ₁ proteins alpha, beta, gamma, and Oligomycin sensitivity-conferring protein (OSCP) contain SIRT3-specific reversible acetyl-lysines that are evolutionarily conserved and bind to SIRT3. OSCP was further investigated and lysine 139 is a nutrient-sensitive SIRT3-dependent deacetylation target. Site directed mutants demonstrate that OSCP ^K139 directs, at least in part, mitochondrial ATP production and mice lacking Sirt3 exhibit decreased ATP muscle levels, increased ATP synthase protein acetylation, and an exercise-induced stress-deficient phenotype. Innovation: This work connects the aging and nutrient response, via SIRT3 direction of the mitochondrial acetylome, to the regulation of mitochondrial energy homeostasis under nutrient-stress conditions by deacetylating ATP synthase proteins. Conclusion: Our data suggest that acetylome signaling contributes to mitochondrial energy homeostasis by SIRT3-mediated deacetylation of ATP synthase proteins. Antioxid. Redox Signal. 21, 551–564.

SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2–related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2^–/– mice had decreased iron levels, while this effect was reversed in Sirt2^–/– Nrf2^–/– double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.

Sirtuins connect energy generation and metabolic stress to the cellular acetylome. Currently, only the mitochondrial sirtuins (SIRT3-5) and SIRT1 have been shown to direct mitochondrial function, however SIRT2, the primary cytoplasmic sirtuin, is not yet reported to associate with mitochondria. This study revealed a novel physiological function of SIRT2: the regulation of mitochondrial function. First, the acetylation of several metabolic mitochondrial proteins was found to be altered in Sirt2-deficient mice, which was subsequently validated by immunoprecipitation experiments in which the acetylated mitochondrial proteins directly interacted with SIRT2. Moreover, immuno-gold electron microscopic images of mouse brains showed that SIRT2 associates with the inner and outer mitochondrial membrane in CNS cells. The loss of Sirt2 increased oxidative stress, decreased ATP levels, altered mitochondrial morphology at the cellular and tissue (i.e., brain) level. Furthermore, the autophagic/mitophagic processes were dysregulated in Sirt2-deficient neurons and MEFs. Together, these findings support that SIRT2 functions as a mitochondrial sirtuin, as well as a regulator of autophagy/mitophagy to maintain mitochondrial biology, thus facilitating cell survival.

Sirtuins participate in sensing nutrient availability and directing metabolic activity to match energy needs with energy production and consumption. However, the pivotal targets for sirtuins in cancer are mainly unknown. In this study, we identify the M2 isoform of pyruvate kinase (PKM2) as a critical target of the sirtuin SIRT2 implicated in cancer. PKM2 directs the synthesis of pyruvate and acetyl-CoA, the latter of which is transported to mitochondria for use in the Krebs cycle to generate ATP. Enabled by a shotgun mass spectrometry analysis founded on tissue culture models, we identified a candidate SIRT2 deacetylation target at PKM2 lysine 305 (K305). Biochemical experiments including site-directed mutants that mimicked constitutive acetylation suggested that acetylation reduced PKM2 activity by preventing tetramerization to the active enzymatic form. Notably, ectopic overexpression of a deacetylated PKM2 mutant in Sirt2-deficient mammary tumor cells altered glucose metabolism and inhibited malignant growth. Taken together, our results argued that loss of SIRT2 function in cancer cells reprograms their glycolytic metabolism via PKM2 regulation, partially explaining the tumor-permissive phenotype of mice lacking Sirt2. Cancer Res; 76(13); 3802–12. ©2016 AACR.