Diabetes causes pancreatic beta cell failure through hyperglycemia-induced oxidative stress, or "glucose toxicity." We show that the forkhead protein FoxO1 protects beta cells against oxidative stress by forming a complex with the promyelocytic leukemia protein Pml and the NAD-dependent deacetylase Sirt1 to activate expression of NeuroD and MafA, two Insulin2 (Ins2) gene transcription factors. Using acetylation-defective and acetylation-mimicking mutants, we demonstrate that acetylation targets FoxO1 to Pml and prevents ubiquitin-dependent degradation. We show that hyperglycemia suppresses MafA expression in vivo and that MafA inhibition can be prevented by transgenic expression of constitutively nuclear FoxO1 in beta cells. The findings provide a mechanism linking glucose- and growth factor receptor-activated pathways to protect beta cells against oxidative damage via FoxO proteins.

SIRT1 is an NAD-dependent deacetylase critically involved in stress responses, cellular metabolism and, possibly, ageing [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . The tumour suppressor p53 represents the first non-histone substrate functionally regulated by acetylation and deacetylation 16,17 ; we and others previously found that SIRT1 promotes cell survival by deacetylating p53 (refs 4-6 ). These results were further supported by the fact that p53 hyperacetylation and increased radiation-induced apoptosis were observed in Sirt1-deficient mice 10 . Nevertheless, SIRT1-mediated deacetylase function is also implicated in p53-independent pathways under different cellular contexts, and its effects on transcriptional factors such as members of the FOXO family and PGC-1α directly modulate metabolic responses [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] . These studies validate the importance of the deacetylase activity of SIRT1, but how SIRT1 activity is regulated in vivo is not well understood. Here we show that DBC1 (deleted in breast cancer 1) acts as a native inhibitor of SIRT1 in human cells. DBC1-mediated repression of SIRT1 leads to increasing levels of p53 acetylation and upregulation of p53-mediated function. In contrast, depletion of endogenous DBC1 by RNA interference (RNAi) stimulates SIRT1-mediated deacetylation of p53 and inhibits p53-dependent apoptosis. Notably, these effects can be reversed in cells by concomitant knockdown of endogenous SIRT1. Our study demonstrates that DBC1 promotes p53-mediated apoptosis through specific inhibition of SIRT1.To understand the regulation of SIRT1-mediated deacetylation in vivo, biochemical purification was used to identify cellular factors that stably interact with SIRT1. We isolated physiologically formed protein complexes containing SIRT1 from cell extracts of native HeLa cells by conducting affinity chromatography with affinity-purified antisera raised against the carboxy (C) terminus (amino acids 480-737) of SIRT1 ( Supplementary Fig. 1a). As expected, we identified SIRT1 as the major component of the complexes, but several protein bands were also co-purified with SIRT1. Mass spectrometry of a prominent protein band of approximately 130 kilodaltons (kDa) from the SIRT1 complexes revealed peptide sequences corresponding to the DBC1 protein ( Supplementary Fig. 1b, Gi: 24432106). The DBC1 gene was initially identified as it is localized to a region of chromosome 8p21 that was homozygously deleted in human breast cancer; however, the molecular function of DBC1 is poorly understood 18,19 .To examine the interaction between endogenous DBC1 and SIRT1, cell extracts from human osteosarcoma U2OS cells were immunoprecipitated with the anti-SIRT1 antibody or with the

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Although it was originally thought of as a passive way to block the nuclear function of p53, accumulating evidence suggests that cytoplasmic localization of p53 plays an active role in p53-mediated functions such as apoptosis and autophagy. Previous studies by us and others demonstrated that Mdm2-mediated p53 ubiquitination induces both degradation and cytoplasmic localization. Here we describe MSL2, a novel E3 ligase for p53 that promotes ubiquitin-dependent cytoplasmic p53 localization. Unlike Mdm2 or most other p53 E3 ligases, MSL2-mediated p53 ubiquitination does not affect the stability of p53. Moreover, the MSL2-mediated effect on p53 is Mdm2-independent. Thus, our study identifies an important ubiquitin-ligase for modulating p53 subcellular localization.The function of the tumor suppressor p53 as a sequencespecific transcription factor controlling the expression of numerous target genes is critical for the regulation of cellular senescence, cell-cycle arrest, and apoptosis (1-3). During normal homeostasis, p53 is localized predominantly in the nucleus and is maintained at low levels via ubiquitination-mediated targeting for proteasomal degradation (4). Both protein levels and transcriptional activity increase dramatically in response to stress through an array of critical post-translational modifications (4, 5). Polyubiquitination of C-terminal lysines of p53 by Mdm2 (6 -8) and other ubiquitin-protein isopeptide ligases (E3) 2 such as Pirh2 (9), COP-1 (10), and Arf-BP1 (11) controls p53 levels by targeting p53 for proteasomal degradation in unstressed cells or after the cellular stress is resolved (4, 12). The stress-induced p53-dependent apoptotic response consists of transcription-dependent and -independent functions of p53 (13-17). Although transactivation of pro-apoptotic target genes such as PUMA, BAX, and PIG3 requires p53 to act as a transcription factor in the nucleus, cytoplasmic p53 can elicit an apoptotic response by localizing to the mitochondria and activating a direct mitochondrial death program (13, 18 -26). Mdm2-mediated p53 monoubiquitination, occurring when Mdm2 activity levels are low, promotes p53 nuclear export and the generation of a cytoplasmic p53 pools (27-32). Once p53 localizes to the mitochondria, both directly activated and enabled pathways are utilized to induce apoptosis (18). p53 interacts with anti-apoptotic members of the Bcl family such as BclXl and Bcl2 at the outer mitochondrial membrane to release and allow the oligomerization of the pro-apoptotic factors Bak and Bax. These in turn promote the formation of pores in the mitochondrial membrane resulting in the release of cytochrome c and other apoptotic activators from the mitochondria (21,22,(33)(34)(35). Alternatively, p53 can interact directly with Bak, releasing Bak from its inhibitory interaction with Mcl1 and thereby directly activating Bak-induced apoptosis (23, 36). Recently, a role for cytoplasmic p53 in autophagy was described, providing further evidence that changes on subcellular localization of p53 have pro...

In addition to its role as the central regulator of the cellular stress response, p53 can regulate aerobic respiration via the novel transcriptional target SCO2, a critical regulator of the cytochrome c oxidase complex (Matoba et al., 2006). Loss of p53 results in decreased oxygen consumption and aerobic respiration and promotes a switch to glycolysis, thereby reducing endurance during physical exercise.

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