Negative regulation of the deacetylase SIRT1 by DBC1

Abstract

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

SIRT1 stabilizes PML promoting its sumoylation

“…At 24 or 48 h after transfection with siRNA against SIRT1, PML levels were analyzed by western blot. As previously reported, transfection of siRNA against SIRT1 efficiently knocked down the protein, 23,24 leading to almost undetectable levels of SIRT1 protein expression ( Figure 1c). In agreement with our observations using genetically modified MEFs, we also detected an important reduction in PML protein levels after knockdown of SIRT1 (approximately 50% of the amount detected in the control cells; Figure 1c).…”

PINK1 as a Molecular Checkpoint in the Maintenance of Mitochondrial Function and Integrity

“…Therefore, PINK1 may directly phosphorylate and activate Sir2 in the cytoplasm. Alternatively, PINK1 may also indirectly regulate Sir2 via its upstream regulators such as active regulator of Sirt1 (AROS) (Kim et al, 2007b) or deleted in breast cancer 1 (DBC1) (Kim et al, 2008b;Zhao et al, 2008). This crosstalk between PINK1 and the Sir2-FOXO pathway suggests the novel role of PINK1 in inducing the expression of mitochondrial protective genes by transmitting signals to the cytosol and nucleus.…”

CCAR‐1 is a negative regulator of the heat‐shock response in Caenorhabditis elegans

“…Active regulator of SIRT1 (AROS) is a positive regulator of SIRT1 that promotes deacetylation of the SIRT1 substrates p53 and HSF1 (Kim, Kho, Kang, & Um, 2007; Raynes et al, 2013). CCAR2, also known as DBC1, is a negative regulator of SIRT1 that enhances acetylation of p53 and HSF1 (Kim, Chen, & Lou, 2008; Raynes et al, 2013; Zhao et al, 2008). The ability of AROS and CCAR2 to modulate SIRT1 activity, and thus impact the acetylated state of HSF1, allows these proteins to regulate the HSR (Raynes et al, 2013).…”