Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species

Abstract

Sirtuin 1 (SIRT1) is an NAD + -dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD + levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD + levels and its intracellular distribution remains unclear. Here, we show that SIRT1 determines the nuclear organization of protein-bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class, does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy in single living cells, we reveal that NAD + metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a connection between NAD + metabolism, NADH distribution, and SIRT1 activity in the nucleus of live cells and pave the way to decipher links between nuclear organization and metabolism.S irtuins (SIRTs) are a conserved family of deacetylases that target a variety of proteins located in virtually all cellular compartments (1). Deacetylation by SIRTs may control many functional aspects of target proteins (2). Because SIRT deacetylase activity depends on the energy carrier NAD + , these enzymes are thought to operate as cellular metabolic sensors. In addition, because histones are targeted by nuclear SIRT, these enzymes could link variations in cellular metabolism to chromatin function.There are seven mammalian SIRTs (SIRT1 to SIRT7) with distinct subcellular locations. SIRT2 is mainly cytoplasmic; SIRT3, SIRT4, and SIRT5 are found in the mitochondrial compartment; and SIRT1, SIRT6, and SIRT7 are located in the cell nucleus (1). In mammals, SIRT1 contributes to development and protects from metabolic and cardiovascular disease, neurodegeneration, and cancer (3). SIRT1 has been reported to promote healthy aging and regulate lifespan (4, 5). At the cellular level, SIRT1 regulates lipid and glucose homeostasis, apoptosis, DNA repair, and mitochondrial function. Variations in NAD + levels control SIRT1 activity (6-9), a relevant finding in the regulation of circadian rhythms (10). Circadian rhythms in NAD + levels have been observed (11,12), which lead to fluctuating SIRT1 deacetylase activity (9) that, in turn, results into cyclic acetylation of specific SIRT1 targets (6, 9, 13). SIRT1 and SIRT6 segregate circadian metabolism by driving transcription of a differential subset of circadian genes (14). SIRT6 is a chromatinbound protein that was first characterized as a regulator of genome stability (15). The other nuclear SIRT, SIRT7, appears to be highly localized in the nucleolus and possibly involved in Pol-I-dependent transcription (16), and it has been shown to regula...

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