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Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

ABSTRACT: 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.

SUBMITTER: Bao J

PROVIDER: S-EPMC2858784 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

Bao Jianjun J Lu Zhongping Z Joseph Joshua J JJ Carabenciov Darin D Dimond Christopher C CC Pang Liyan L Samsel Leigh L McCoy J Philip JP Leclerc Jaime J Nguyen Phuongmai P Gius David D Sack Michael N MN

Journal of cellular biochemistry 20100501 1

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 ami ...[more]

PMID: 20235147

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Project description:RationaleHypertension represents a major risk factor for stroke, myocardial infarction, and heart failure and affects 30% of the adult population. Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear. The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level.ObjectiveWe hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in hypertension, but increased Sirt3 protects vascular function and decreases hypertension.Methods and resultsTo test the therapeutic potential of targeting Sirt3 expression, we developed new transgenic mice with global Sirt3OX (Sirt3 overexpression), which protects from endothelial dysfunction, vascular oxidative stress, and hypertrophy and attenuates Ang II (angiotensin II) and deoxycorticosterone acetate-salt induced hypertension. Global Sirt3 depletion in Sirt3-/- mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM [vascular cell adhesion molecule-1], ICAM [intercellular adhesion molecule-1], and MCP1 [monocyte chemoattractant protein 1]), increases inflammatory cell infiltration in the kidney, reduces telomerase expression, and accelerates vascular senescence and age-dependent hypertension; conversely, increased Sirt3 expression in Sirt3OX mice prevents these deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with normotensive subjects.ConclusionsWe suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.

Dataset Information

Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

Publications

Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms.

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