H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD(+)-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H(2)O(2) and examined DNA cleavage, depletion of intracellular NAD(+), expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated beta-galactosidase (SA-beta-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H(2)O(2), though no cell death was observed. NAD(+) levels in TIG-3 cells treated with H(2)O(2) were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD(+) levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H(2)O(2) treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H(2)O(2) induced cell cycle arrest at 24h and increased SA-beta-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.     

Reactivation of mutant p53 through interaction of a C-terminal peptide with the core domain

A synthetic 22-mer peptide (peptide 46) derived from the p53 C-terminal domain can restore the growth suppressor function of mutant p53 proteins in human tumor cells (G. Selivanova et al., Nat. Med. 3:632-638, 1997). Here we demonstrate that peptide 46 binds mutant p53. Peptide 46 binding sites were found within both the core and C-terminal domains of p53. Lys residues within the peptide were critical for both p53 activation and core domain binding. The sequence-specific DNA binding of isolated tumor-derived mutant p53 core domains was restored by a C-terminal polypeptide. Our results indicate that C-terminal peptide binding to the core domain activates p53 through displacement of the negative regulatory C-terminal domain. Furthermore, stabilization of the core domain structure and/or establishment of novel DNA contacts may contribute to the reactivation of mutant p53. These findings should facilitate the design of p53-reactivating drugs for cancer therapy.     

Aurora-A abrogation of p53 DNA binding and transactivation activity by phosphorylation of serine 215

The tumor suppressor p53 is important in the decision to either arrest cell cycle progression or induce apoptosis in response to a variety of stimuli. p53 posttranslational modifications and association with other proteins have been implicated in the regulation of its stability and transactivation activity. Here we show that p53 is phosphorylated by the mitotic kinase Aurora-A at serine 215. Unlike most identified phosphorylation sites of p53 that positively associate with p53 function (Brooks, C. L., and Gu, W. (2003) Curr. Opin. Cell Biol. 15, 164-171), the phosphorylation of p53 by Aurora-A at Ser-215 abrogates p53 DNA binding and transactivation activity. Downstream target genes of p53, such as p21Cip/WAF1 and PTEN, were inhibited by Aurora-A in a Ser-215 phosphorylation-dependent manner (i.e. phosphomimic p53-S215D lost and non-phosphorylatable p53-S215A retained normal p53 function). As a result, Aurora-A overrides the apoptosis and cell cycle arrest induced by cisplatin and gamma-irradiation, respectively. However, the effect of Aurora-A on p53 DNA binding and transactivation activity was not affected by phosphorylation of Ser-315, a recently identified Aurora-A phosphorylation site of p53 (Katayama, H., Sasai, K., Kawai, H., Yuan, Z. M., Bondaruk, J., Suzuki, F., Fujii, S., Arlinghaus, R. B., Czerniak, B. A., and Sen, S. (2004) Nat. Genet. 36, 55-62). Our data indicate that phosphorylation of p53 at Ser-215 by Aurora-A is a major mechanism to inactivate p53 and can provide a molecular insight for Aurora-A function.     

Substrate binding stabilizes S-adenosylhomocysteine hydrolase in a closed conformation

Comparison of crystal structures of S-adenosylhomocysteine (AdoHcy) hydrolase in the substrate-free, NAD(+) form [Hu, Y., Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (1999) Biochemistry 38, 8323-8333] and a substrate-bound, NADH form [Turner, M. A., Yuan, C.-S., Borchardt, R. T., Hershfield, M. S., Smith, G. D., and Howell, P. L. (1998) Nat. Struct. Biol. 5, 369-376] indicates large differences in the spatial arrangement of the catalytic and NAD(+) binding domains. The substrate-free, NAD(+) form exists in an "open" form with respect to catalytic and NAD(+) binding domains, whereas the substrate-bound, NADH form exists in a closed form with respect to those domains. To address whether domain closure is induced by substrate binding or its subsequent oxidation, we have measured the rotational dynamics of spectroscopic probes covalently bound to Cys(113) and Cys(421) within the catalytic and carboxyl-terminal domains. An independent domain motion is associated with the catalytic domain prior to substrate binding, suggesting the presence of a flexible hinge element between the catalytic and NAD(+) binding domains. Following binding of substrates (i.e., adenosine or neplanocin A) or a nonsubstrate (i.e., 3'-deoxyadenosine), the independent domain motion associated with the catalytic domain is essentially abolished. Likewise, there is a substantial decrease in the average hydrodynamic volume of the protein that is consistent with a reduction in the overall dimensions of the homotetrameric enzyme following substrate binding and oxidation observed in earlier crystallographic studies. Thus, the catalytic and NAD(+) binding domains are stabilized to form a closed active site through interactions with the substrate prior to substrate oxidation.     

Simultaneous liquid chromatographic assessment of thiamine,thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics

An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (P<0.001) and TDP=127.4+/-62.5 nmol/l (P<10(-5)). The diagnostic role of TDP was evaluated and a significant role for thiamine was established in the study of alcohol related problems.     

Changes in phosphorylation of histone H2A.X and p53 in response of peripheral blood lymphocytes to gamma irradiation

The main aim of this study was to compare the reaction of quiescent and proliferating, i.e. phytohemagglutinin (PHA)-stimulated, human peripheral blood mononuclear cells (PBMCs) to gamma-radiation, and analyse changes of proteins related to repair of DNA damage and apoptosis, such as gammaH2A.X, p53, p53 phosphorylation at serines-15 and -392, and p21 and their dose dependence. Freshly isolated PBMCs in peripheral blood are predominantly quiescent, in G(0) phase, and with very low amounts of proteins p53 and p21. Using confocal microscopy we detected dose dependent (0.5-5 Gy) induction of foci containing gammaH2A.X (1 h after gamma-ray exposure), which are formed around radiation-induced double strand breaks of DNA. Apoptosis was detected from 24 h after irradiation by the dose of 4 Gy onwards by Annexin V binding and lamin B cleavage. Seventy two hours after irradiation 70% of CD3(+) lymphocytes were A(+). Neither increase in p53 nor its phosphorylation on serine-392 after irradiation was detected in these cells. However, massive increase in p21 (cyclin-dependent kinase inhibitor 1A) was detected after irradiation, which can be responsible for late occurrence of apoptosis in these quiescent cells. PHA-stimulation itself (72 h) caused an increase in early apoptosis (A(+)PI(-)) in comparison to non-stimulated PBMCs (38% A(+) resp. 13.4%). After PHA-stimulation also the amount of gammaH2A.X, p53, and p21 increased, but no phosphorylation of p53 on serine-392 or -15 was detected. Reaction to gamma-radiation was different in PHA-stimulated lymphocytes: the p53 pathway was activated and p53 was phosphorylated on serines-15 and -392 4 h after irradiation by the dose of 4 Gy. Phosphorylation of p53 at serine-15 increased in a dose-dependent manner in the studied dose range 0.2-7.5 Gy. Also the amount of p21 increased after irradiation. Seventy two hours after irradiation of PHA-stimulated CD3(+) T lymphocytes by the dose of 4 Gy 65% of cells were A(+).     

An essential role for REV3 in mammalian cell survival: absence of REV3 induces p53-independent embryonic death

The REV3 gene of budding yeast encodes the catalytic subunit of DNA polymerase zeta that carries out translesion DNA synthesis. While REV3-null yeast mutants are viable and exhibit normal growth, Rev3-deficient mice die around midgestation of embryogenesis, which is accompanied by massive apoptosis of cells within the embryo proper. We have investigated whether REV3 is required for the survival of mouse cells and whether the embryonic lethality caused by REV3 deficiency can be rescued by introduction of a Rev3 transgene or by inactivation of p53, the cellular gatekeeper that regulates DNA damage-induced apoptosis. We show that Rev3(-/-) blastocysts were unable to survive and grow in culture but expression of a Rev3 transgene restored their outgrowth. Moreover, Rev3 transgene expression suppressed the apoptosis in E7.5 Rev3(-/-) embryos. The Rev3(-/-) embryonic lethality, however, was not rescued by either Rev3 transgene expression or p53 deficiency. These results reveal an essential role for REV3 in the survival and growth of mammalian cells and suggest that Rev3(-/-) embryonic death occurs in a p53-independent pathway.     

Involvement of mouse Rev3 in tolerance of endogenous and exogenous DNA damage

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.     

DNA excision repair and DNA damage-induced apoptosis are linked to Poly(ADP-ribosyl)ation but have different requirements for p53

Poly(ADP-ribose) polymerase (PARP) is a DNA binding zinc finger protein that catalyzes the transfer of ADP-ribose residues from NAD(+) to itself and different chromatin constituents, forming branched ADP-ribose polymers. The enzymatic activity of PARP is induced upon DNA damage and the PARP protein is cleaved during apoptosis, which suggested a role of PARP in DNA repair and DNA damage-induced cell death. We have generated transgenic mice that lack PARP activity in thymocytes owing to the targeted expression of a dominant negative form of PARP. In the presence of single-strand DNA breaks, the absence of PARP activity correlated with a strongly increased rate of apoptosis compared to cells with intact PARP activity. We found that blockage of PARP activity leads to a drastic increase of p53 expression and activity after DNA damage and correlates with an accelerated onset of Bax expression. DNA repair is almost completely blocked in PARP-deficient thymocytes regardless of p53 status. We found the same increased susceptibility to apoptosis in PARP null mice, a similar inhibition of DNA repair kinetics, and the same upregulation of p53 in response to DNA damage. Thus, based on two different experimental in vivo models, we identify a direct, p53-independent, functional connection between poly(ADP-ribosyl)ation and the DNA excision repair machinery. Furthermore, we propose a p53-dependent link between PARP activity and DNA damage-induced cell death.     

Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+.

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.     

Design, synthesis and biological evaluation of potent NAD+-dependent DNA ligase inhibitors as potential antibacterial agents. Part I: aminoalkoxypyrimidine carboxamides

A series of 2,6-disubstituted aminoalkoxypyrimidine carboxamides (AAPCs) with potent inhibition of bacterial NAD(+)-dependent DNA ligase was discovered through the use of structure-guided design. Two subsites in the NAD(+)-binding pocket were explored to modulate enzyme inhibitory potency: a hydrophobic selectivity region was explored through a series of 2-alkoxy substituents while the sugar (ribose) binding region of NAD(+) was explored via 6-alkoxy substituents.     

Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer

It has been postulated that a segment (residues 594-645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca(2+)-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Delta45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Delta45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N-terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert-containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Delta594-604, Delta605-612, Delta613-625, Delta626-634, Delta632-639, and Delta640-645 mutants were further characterized. The crude lysate of mutants Delta613-625 and Delta632-639 did not show activity in the presence of Ca(2+)/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Delta594-604 and Delta605-612 proteins had a 3-5-fold higher affinity for Ca(2+)/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca(2+)/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Delta594-604) or slightly enhanced by (Delta605-612) the exogenous CaM. In contrast, activities of Delta626-634 and Delta640-645 like those of WTeNOS were largely Ca(2+)/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca(2+)/CaM binding.