SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress

Abstract

SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.Sirtuins (SIRTs) are mammalian NAD + -dependent histone deacetylases (HDACs) 1 . Of the seven SIRTs, SIRT1 is the closest homologue of yeast SIR2 (ref. 2,3), which has important roles in diverse cellular processes, including transcriptional silencing 4 , rDNA recombination 5 , glucose metabolism and energy homeostasis 6 , DNA repair and cell survival [7][8][9] . Because of its dependency on NAD + , the activity of SIRT1 is regulated by the NAD + :NADH ratio and, is therefore sensitive to the status of redox and cellular metabolism. Author Contributions: Y.Y. designed (with W.B.) and performed (with W.F.) the included studies. J.C., X.Z. and K.B. contributed scientifically to the revision. The manuscript was written by W.B., edited by N.O. and S.V.N. and read by all authors. The senior author (W.B.) designed the project, helped with the analyses and the organization of the data, and provided the financial support. All authors have discussed the data and had scientific input into the manuscript.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/ NIH Public Access Similar to SIR2 in lower organisms, SIRT1 is potentially a nutrient sensor that regulates the lifespan of mammals in response to caloric restriction or nutrient starvation [9][10][11][12][13] . By deacetylating and inactivating apoptotic proteins (such as the p53 tumour suppressor 14-17 ), SIRT1 also protects cancer cells from apoptosis induced by DNA damage. The expression of SIRT1 is known to be controlled at both transcriptional and post-transcriptional levels, but post-translational mechanisms that regulate SIRT1 activity have yet to be defined.Sumoylation is a reversible post-translational modification in which proteins termed small ubiquitin-related modifiers (SUMOs) are covalently linked to lysine residues of target proteins. Similar to ubiquitination, sumoylation is catalysed by a three-step enzymatic reaction involving an E1 activating enzyme, an E2 conjugating enzyme and E3 ligases. The reverse reaction is catalysed by SENP desumoylases, a famil...

SIRT1 stabilizes PML promoting its sumoylation

“…One potential mechanism could be that SIRT1 prevents the desumoylation of PML. As SIRT1 is also a substrate for SUMO modification (Supplementary Figure 6 and Yang et al 32 ) and the sumoylation of both proteins, PML and SIRT1, is regulated by the SUMO-specific protease SENP1, 32-34 SIRT1 could potentially compete with PML for the interaction with this desumoylase. However, we could not detect a reduction in SENP1-mediated desumoylation of PML in the presence of SIRT1 (Supplementary Figure 7), pointing to alternative explanations.…”

Methyltransferase Set7/9 regulates p53 activity by interacting with Sirtuin 1 (SIRT1)

“…For instance, SIRT1 is phosphorylated by JNK2 at Ser27, and depletion of JNK2 reduces the half-life of the SIRT1 protein (15). SIRT1 is also sumoylated at K734, which in turn increases its activity (16). With these posttranslational modifications, SIRT1 deacetylase activity is substantially altered.…”

Sir2-Related Protein 1 fromLeishmania amazonensisis a glycosylated NAD+-dependent deacetylase

“…In addition, LaSir2RP1 deacetylase activity was inhibited at high NAD + concentrations, probably due to the release of nicotinamide, a subproduct of the deacetylation reaction, and a known inhibitor of other Sir2-related proteins (Bitterman et al 2002;Avalos et al 2005). Since some members of the mammalian sirtuin family appear to be post-translationally modified (North and Verdin, 2007;Yang et al 2007), we considered the possibility that LaSir2RP1 may also be modified, and decided to analyse the Leishmania protein sequence with an algorithm that predicts possible types of post-translational modifications, such as glycosylation. As a matter of fact, by Western blot analysis, the apparent molecular mass of LaSir2RP1 from promastigotes and amastigotes was higher than that estimated based on its amino acid composition alone, suggesting that LaSir2RP1 could be post-translationally modified by the covalent attachment of one or more functional groups, which will increase its apparent molecular mass.…”

“…(c) Isolation of the microsomal fraction. Leishmania amazonensis promastigotes were submitted to subcellular fractioning in water/ice bath as previously described (Zaverucha do Valle et al 2007). Parasites were washed with PBS + 2% glucose, re-suspended with 20 mM TrisHCl, pH 7·4, 12·5 mM sucrose, incubated for 20 min, and mechanically disrupted by a Douncer homogenizer in the presence of 10% non-ionic detergent Lubrol PX to produce the total protein extract (ET).…”

“…Further debris was eliminated by a 2-step centrifugation at 10 000 g for 10 min and 27 000 g for 30 min. Supernatant (6 ml) was layered on top of 2 ml of buffer containing 20% sucrose, 10 mM MgCl 2 , 25 mM KCl , and 10 mM TrisHCl, pH 7·4, and centrifuged at 100 000 g for 30 min to produce the microsomal fraction (micF), which contains endoplasmic reticulum-derived vesicles, including ribosomes (Zaverucha do Valle et al 2007).…”