Upon DNA damage and other types of stress, p53 induces either cell-cycle arrest or apoptosis depending on the cellular context. However, the molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis are not well understood. Here, we show that Tip60 is required for both cell growth arrest and apoptosis mediated by p53 and also induces its acetylation specifically at lysine 120 (K120) within the DNA-binding domain. Interestingly, this modification is crucial for p53-dependent apoptosis but is dispensable for its mediated growth arrest. K120 is a recurrent site for p53 mutation in human cancer, and the corresponding acetylation-defective tumor mutant (K120R) abrogates p53-mediated apoptosis, but not growth arrest. Thus, our study demonstrates that Tip60-dependent acetylation of p53 at K120 modulates the decision between cell-cycle arrest and apoptosis, and it reveals that the DNA-binding core domain is an important target for p53 regulation by posttranslational modifications.

The positively charged lysine residue plays an important role in protein folding and functions. Neutralization of the charge often has a profound impact on the substrate proteins. Accordingly all the known post-translational modifications at lysine have pivotal roles in cell physiology and pathology. Here we report the discovery of two novel, in vivo lysine modifications in histones, lysine propionylation and butyrylation. We confirmed, by in vitro labeling and peptide mapping by mass spectrometry, that two previously known acetyltransferases, p300 and CREB-binding protein, could catalyze lysine propionylation and lysine butyrylation in histones.

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

Marrow-resident mesenchymal stem cells (MSCs) serve as a functional component of the perivascular niche that regulates hematopoiesis. They also represent the main source of bone formed in adult bone marrow, and their bifurcation to osteoblast and adipocyte lineages plays a key role in skeletal homeostasis and aging. Although the tumor suppressor p53 also functions in bone organogenesis, homeostasis, and neoplasia, its role in MSCs remains poorly described. Herein, we examined the normal physiological role of p53 in primary MSCs cultured under physiologic oxygen levels. Using knockout mice and gene silencing we show that p53 inactivation downregulates expression of TWIST2, which normally restrains cellular differentiation to maintain wild-type MSCs in a multipotent state, depletes mitochondrial reactive oxygen species (ROS) levels, and suppresses ROS generation and PPARG gene and protein induction in response to adipogenic stimuli. Mechanistically, this loss of adipogenic potential skews MSCs toward an osteogenic fate, which is further potentiated by TWIST2 downregulation, resulting in highly augmented osteogenic differentiation. We also show that p53 −/− MSCs are defective in supporting hematopoiesis as measured in standard colony assays because of decreased secretion of various cytokines including CXCL12 and CSF1. Lastly, we show that transient exposure of wild-type MSCs to 21% oxygen upregulates p53 protein expression, resulting in increased mitochondrial ROS production and enhanced adipogenic differentiation at the expense of osteogenesis, and that treatment of cells with FGF2 mitigates these effects by inducing TWIST2. Together, these findings indicate that basal p53 levels are necessary to maintain MSC bi-potency, and oxygen-induced increases in p53 expression modulate cell fate and survival decisions. Because of the critical function of basal p53 in MSCs, our findings question the use of p53 null cell lines as MSC surrogates, and also implicate dysfunctional MSC responses in the pathophysiology of p53-related skeletal disorders.

Selective autophagy involves the recognition and targeting of specific cargo, such as damaged organelles, misfolded proteins, or invading pathogens for lysosomal destruction1–4. Yeast genetic screens have identified proteins required for different forms of selective autophagy, including cytoplasm-to-vacuole targeting, pexophagy, and mitophagy, and mammalian genetic screens have identified proteins required for autophagy regulation5. However, there have been no systematic approaches to identify molecular determinants of selective autophagy in mammalian cells. To identify mammalian genes required for selective autophagy, we performed a high-content, image-based, genome-wide siRNA screen to detect genes required for the colocalization of Sindbis virus capsid protein with autophagolysosomes. We identified 141 candidate genes required for viral autophagy, which were enriched for cellular pathways related to mRNA processing, interferon signaling, vesicle trafficking, cytoskeletal motor function, and metabolism. Ninety-six of these genes were also required for Parkin-mediated mitophagy, indicating that common molecular determinants may be involved in autophagic targeting of viral nucleocapsids and autophagic targeting of damaged mitochondria. Murine embryonic fibroblasts lacking one of these gene products, the C2-domain containing protein, Smurf1, are deficient in the autophagosomal targeting of Sindbis and herpes simplex viruses and in the clearance of damaged mitochondria. Moreover, Smurf1-deficient mice display an accumulation of damaged mitochondria in heart, brain, and liver. Thus, our study identifies candidate determinants of selective autophagy, and defines Smurf1 as a newly recognized mediator of both viral autophagy and mitophagy.

No abstract

p53 promotes tumor suppression through its ability to function as a transcriptional factor and is activated by posttranslational modifications that include acetylation. Our earlier study demonstrated that p53 acetylation can enhance its sequence-specific DNA binding in vitro, and this notion was later confirmed in several other studies. However, a recent study has reported that in vitro acetylation of p53 fails to stimulate its DNA binding to large DNA fragments, raising an important issue that requires further investigation. Here, we show that unacetylated p53 is able to bind weakly to its consensus site within the context of large DNA fragments, although it completely fails to bind the same site within short oligonucleotide probes. Strikingly, by using highly purified and fully acetylated p53 proteins obtained from cells, we show that acetylation of the C-terminal domain can dramatically enhance site-specific DNA binding on both short oligonucleotide probes and long DNA fragments. Moreover, endogenous p53 apparently can be fully acetylated in response to DNA damage when both histone deacetylase complex 1 (HDAC1)-and Sir2-mediated deacetylation are inhibited, indicating dynamic p53 acetylation and deacetylation events during the DNA damage response. Finally, we also show that acetylation of endogenous p53 indeed significantly augments its ability to bind an endogenous target gene and that p53 acetylation levels correlate well with p53-mediated transcriptional activation in vivo. Thus, our results clarify some of the confusion surrounding acetylation-mediated effects on p53 binding to DNA and suggest that acetylation of p53 in vivo may contribute, at least in part, to its transcriptional activation functions.ChIP ͉ transcription ͉ CBP͞p300

Lysine propionylation and butyrylation are protein modifications that were recently identified in histones. The molecular components involved in the two protein modification pathways are unknown, hindering further functional studies. Here we report identification of the first three in vivo non-histone protein substrates of lysine propionylation in eukaryotic cells: p53, p300, and CREB-binding protein. We used mass spectrometry to map lysine propionylation sites within these three proteins. We also identified the first two in vivo eukaryotic lysine propionyltransferases, p300 and CREB-binding protein, and the first eukaryotic depropionylase, Sirt1. p300 was able to perform autopropionylation on lysine residues in cells. Our results suggest that lysine propionylation, like lysine acetylation, is a dynamic and regulatory post-translational modification. Based on these observations, it appears that some enzymes are common to the lysine propionylation and lysine acetylation regulatory pathways. Our studies therefore identified first several important players in lysine propionylation pathway. Molecular & Cellular Proteomics 8:45-52, 2009.