Characterization of the Murine SIRT3 Mitochondrial Localization Sequence and Comparison of Mitochondrial Enrichment and Deacetylase Activity of Long and Short SIRT3 Isoforms

Translational Medicine Branch, NHLBI, NIH, Bethesda, Maryland, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.26). 05/2010; 110(1):238-47. DOI: 10.1002/jcb.22531
Source: PubMed


SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity. In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. The long isoform is generated via intra-exon splicing creating a frame-shift to expose a novel upstream translation start site. Mitochondrial localization is significantly more robust following transfection of the long compared with the short isoform. Insertion of this alternatively spliced novel 5' sequence upstream of a GFP-reporter plasmid shows greater than 80% enrichment in mitochondria, confirming this region as a legitimate mitochondrial localization sequence. Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.

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    • "The sirtuin family of NAD+-dependent deacetylases (Sirt proteins) catalyzes a deacetylase reaction. Of seven members of the sirtuin protein families, Sirt3 is the predominant deacetylase in mitochondria [15] [16] [17] [18]. "
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    ABSTRACT: Malnutrition during the fetal growth period increases risk for later obesity and type 2 diabetes mellitus (T2DM). We have shown that a prenatal low protein (8% protein; LP) diet followed by postnatal high fat (45% fat; HF) diet results in offspring propensity for adipose tissue catch-up growth, obesity and T2DM in Sprague–Dawley rats. Skeletal muscle is the major tissue for insulin-mediated glucose uptake. Dysfunctional skeletal muscle mitochondrial function, particularly reduction in expression of mitochondrial protein sirtuin protein 3 (Sirt3) contributes to development of T2DM by reducing mitochondrial respiration. Therefore, we hypothesized that maternal LP and postnatal HF diet would increase T2DM risk due Sirt3 dysfunction within skeletal muscle mitochondria. Using our maternal LP and postnatal HF diet model, we showed that skeletal muscle mitochondrial oxygen consumption rate (OCR) was decreased by maternal LP diet. Mitochondria copy number, mitochondrial thermogenesis (UCP-1) expression, and mitochondrial biogenic factors including nuclear respiratory factor 1 (NRF1) and cytochrome c oxidase 1 and 4 (COX-1 and 4) were unaffected by maternal LP and postnatal HF diets. Skeletal muscle Sirt3 mRNA decreased with maternal LP diet. A mitochondrial substrate of Sirt3, succinate dehydrogenase (SDH), is regulated by Sirt3 via lysine residue acetylation status of SDH. Acetylated SDH protein (inactive form) levels were moderately decreased by maternal LP diet. Taken together these data suggest that maternal LP and postnatal HF diets may increase the risk for T2D by decreasing skeletal muscle oxidative respiration via increased Sirt3 and possibly by decreased amounts of the active form of SDH enzyme.
    Full-text · Article · Nov 2014 · The Journal of Nutritional Biochemistry
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    • "Moreover, Sirt3 deacetylates transcription factor Foxo3a, which increases the expression of manganese-dependent superoxide dismutase (MnSOD, Sod2) that, in turn, diminishes the accumulation of superoxide in mitochondria (Sundaresan et al., 2009). Data notwithstanding, it is still debatable whether Sirt3 is a bona fide regulator of Ku70 and Foxo3a, or whether enforced expression of Sirt3 accounts for the observed extra-mitochondrial activity (Bao et al., 2010). Furthermore, a quick survey in the acetylome database ( "
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    ABSTRACT: Lysine modifications have been studied extensively in the nucleus, where they play pivotal roles in gene regulation and constitute one of the pillars of epigenetics. In the cytoplasm, they are critical to proteostasis. However, in the last decade we have also witnessed the emergence of mitochondria as a prime locus for post-translational modification (PTM) of lysine thanks, in large measure, to evolving proteomic techniques. Here, we review recent work on evolving set of PTM that arise from the direct reaction of lysine residues with energized metabolic thioester-coenzyme A intermediates, including acetylation, succinylation, malonylation, and glutarylation. We highlight the evolutionary conservation, kinetics, stoichiometry, and cross-talk between members of this emerging family of PTMs. We examine the impact on target protein function and regulation by mitochondrial sirtuins. Finally, we spotlight work in the heart and cardiac mitochondria, and consider the roles acetylation and other newly-found modifications may play in heart disease.
    Full-text · Article · Sep 2014 · Frontiers in Physiology
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    • "Sirt3 is commonly considered as a mitochondrial protein; however, its presence in the nucleus and cytoplasm has also been reported [49]–[53]. Analysis of mitochondrial acetylome by Fritz et al. identified a number of peroxisomal proteins including ECHD and ACOX1 [35]. Acetylation states of these peroxisomal proteins are affected by alcohol treatment as well as the expression of Sirt3 [35]. "
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    Full-text · Article · Jan 2014 · PLoS ONE
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