Regulation of p53 Translation and Induction after DNA Damage by Ribosomal Protein L26 and Nucleolin

SummaryIncreases in p53 protein levels after DNA damage have largely been attributed to an increase in the half-life of p53 protein. Here we demonstrate that increased translation of p53 mRNA is also a critical step in the induction of p53 protein in irradiated cells. Ribosomal protein L26 (RPL26) and nucleolin were found to bind to the 5′ untranslated region (UTR) of p53 mRNA and to control p53 translation and induction after DNA damage. RPL26 preferentially binds to the 5′UTR after DNA damage, and its overexpression enhances association of p53 mRNA with heavier polysomes, increases the rate of p53 translation, induces G1 cell-cycle arrest, and augments irradiation-induced apoptosis. Opposite effects were seen when RPL26 expression was inhibited. In contrast, nucleolin overexpression suppresses p53 translation and induction after DNA damage, whereas nucleolin downregulation promotes p53 expression. These findings demonstrate the importance of increased translation of p53 in DNA-damage responses and suggest critical roles for RPL26 and nucleolin in affecting p53 induction

Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks

We show that DNA double-strand breaks (DSBs) induce complex subcompartmentalization of genome surveillance regulators. Chromatin marked by γ-H2AX is occupied by ataxia telangiectasia–mutated (ATM) kinase, Mdc1, and 53BP1. In contrast, repair factors (Rad51, Rad52, BRCA2, and FANCD2), ATM and Rad-3–related (ATR) cascade (ATR, ATR interacting protein, and replication protein A), and the DNA clamp (Rad17 and -9) accumulate in subchromatin microcompartments delineated by single-stranded DNA (ssDNA). BRCA1 and the Mre11–Rad50–Nbs1 complex interact with both of these compartments. Importantly, some core DSB regulators do not form cytologically discernible foci. These are further subclassified to proteins that connect DSBs with the rest of the nucleus (Chk1 and -2), that assemble at unprocessed DSBs (DNA-PK/Ku70), and that exist on chromatin as preassembled complexes but become locally modified after DNA damage (Smc1/Smc3). Finally, checkpoint effectors such as p53 and Cdc25A do not accumulate at DSBs at all. We propose that subclassification of DSB regulators according to their residence sites provides a useful framework for understanding their involvement in diverse processes of genome surveillance

Low dose chloroquine decreases insulin resistance in human metabolic syndrome but does not reduce carotid intima-media thickness

Background: Metabolic syndrome, an obesity-related condition associated with insulin resistance and low-grade inflammation, leads to diabetes, cardiovascular diseases, cancer, osteoarthritis, and other disorders. Optimal therapy is unknown. The antimalarial drug chloroquine activates the kinase ataxia telangiectasia mutated (ATM), improves metabolic syndrome and reduces atherosclerosis in mice. To translate this observation to humans, we conducted two clinical trials of chloroquine in people with the metabolic syndrome. Methods: Eligibility included adults with at least 3 criteria of metabolic syndrome but who did not have diabetes. Subjects were studied in the setting of a single academic health center. The specific hypothesis: chloroquine improves insulin sensitivity and decreases atherosclerosis. In Trial 1, the intervention was chloroquine dose escalations in 3-week intervals followed by hyperinsulinemic euglycemic clamps. Trial 2 was a parallel design randomized clinical trial, and the intervention was chloroquine, 80 mg/day, or placebo for 1 year. The primary outcomes were clamp determined-insulin sensitivity for Trial 1, and carotid intima-media thickness (CIMT) for Trial 2. For Trial 2, subjects were allocated based on a randomization sequence using a protocol in blocks of 8. Participants, care givers, and those assessing outcomes were blinded to group assignment. Results: For Trial 1, 25 patients were studied. Chloroquine increased hepatic insulin sensitivity without affecting glucose disposal, and improved serum lipids. For Trial 2, 116 patients were randomized, 59 to chloroquine (56 analyzed) and 57 to placebo (51 analyzed). Chloroquine had no effect on CIMT or carotid contrast enhancement by MRI, a pre-specified secondary outcome. The pre-specified secondary outcomes of blood pressure, lipids, and activation of JNK (a stress kinase implicated in diabetes and atherosclerosis) were decreased by chloroquine. Adverse events were similar between groups. Conclusions: These findings suggest that low dose chloroquine, which improves the metabolic syndrome through ATM-dependent mechanisms in mice, modestly improves components of the metabolic syndrome in humans but is unlikely to be clinically useful in this setting

A TSC signaling node at the peroxisome regulates mTORC1 and autophagy in response to ROS

Subcellular localization is emerging as an important mechanism for mTORC1 regulation. We report that the tuberous sclerosis complex (TSC) signaling node, TSC1, TSC2 and Rheb, localizes to peroxisomes, where it regulates mTORC1 in response to reactive oxygen species (ROS). TSC1 and TSC2 were bound by PEX19 and PEX5, respectively, and peroxisome-localized TSC functioned as a Rheb GAP to suppress mTORC1 and induce autophagy. Naturally occurring pathogenic mutations in TSC2 decreased PEX5 binding, abrogated peroxisome localization, Rheb GAP activity, and suppression of mTORC1 by ROS. Cells lacking peroxisomes were deficient in mTORC1 repression by ROS and peroxisome-localization deficient TSC2 mutants caused polarity defects and formation of multiple axons in neurons. These data identify a role for TSC in responding to ROS at the peroxisome, and identify the peroxisome as a signaling organelle involved in regulation of mTORC1

Genotoxic agents promote the nuclear accumulation of annexin A2: role of annexin A2 in mitigating DNA damage

Annexin A2 is an abundant cellular protein that is mainly localized in the cytoplasm and plasma membrane, however a small population has been found in the nucleus, suggesting a nuclear function for the protein. Annexin A2 possesses a nuclear export sequence (NES) and inhibition of the NES is sufficient to cause nuclear accumulation. Here we show that annexin A2 accumulates in the nucleus in response to genotoxic agents including gamma-radiation, UV radiation, etoposide and chromium VI and that this event is mediated by the nuclear export sequence of annexin A2. Nuclear accumulation of annexin A2 is blocked by the antioxidant agent N-acetyl cysteine (NAC) and stimulated by hydrogen peroxide (H2O2), suggesting that this is a reactive oxygen species dependent event. In response to genotoxic agents, cells depleted of annexin A2 show enhanced phospho-histone H2AX and p53 levels, increased numbers of p53-binding protein 1 nuclear foci and increased levels of nuclear 8-oxo-2'-deoxyguanine, suggesting that annexin A2 plays a role in protecting DNA from damage. This is the first report showing the nuclear translocation of annexin A2 in response to genotoxic agents and its role in mitigating DNA damage.Natural Sciences and Engineering Research Council of Canada (NSERC); European Union [PCOFUND-GA-2009-246542]; Foundation for Science and Technology of Portugal; Beatrice Hunter Cancer Research Institute; Terry Fox Foundationinfo:eu-repo/semantics/publishedVersio

Autophagy Links Inflammasomes to Atherosclerotic Progression

We investigated the role of autophagy in atherosclerosis. During plaque formation in mice, autophagic markers co-localized predominantly with macrophages (mϕ). Atherosclerotic aortas had elevated levels of p62, suggesting that dysfunctional autophagy is characteristic of plaques. To determine if autophagy directly influences atherogenesis, we characterized Beclin-1 heterozygous-null and mϕ-specific ATG5-null (ATG5-mϕKO) mice, commonly used models of autophagy haploinsufficiency and deficiency, respectively. Haploinsufficent Beclin-1 mice had no atherosclerotic phenotype, but ATG5-mϕKO mice had increased plaques suggesting an essential role for basal levels of autophagy in atheroprotection. Defective autophagy is associated with pro-atherogenic inflammasome activation. Classic inflammasome markers were robustly induced in ATG5-null mϕ, especially when co-incubated with cholesterol crystals. Moreover, cholesterol crystals appear to be increased in ATG5-mϕKO plaques, suggesting a potentially vicious cycle of crystal formation and inflammasome activation in autophagy-deficient plaques. These results show that autophagy becomes dysfunctional in atherosclerosis and its deficiency promotes atherosclerosis in part through inflammasome hyperactivation

Disruption of DNA methylation-dependent long gene repression in Rett syndrome

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain

Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication

Books in the News in Cromwellian England

This article offers detailed analysis of the patterns of book advertising in Marchamont Nedham’s government-sponsored newsbook, Mercurius Politicus. It contends that, for a brief period, Politicus was the nearest thing that the mid-seventeenth century had to a literary periodical and contests standard accounts that Politicus was only successful because government monopoly made it so. Instead I show that Politicus was instrumental in creating an image of the Commonwealth and Protectorate as a Republic of Letters; the cheap print of its small advertisements insisted that the publication of a book was an event, that London was a city of the book, and that its inhabitants might respond to the uncertainty of political revolution by eagerly imagining a future comprised of new books as yet unread

Analysis of variants in DNA damage signalling genes in bladder cancer

<p>Abstract</p> <p>Background</p> <p>Chemicals from occupational exposure and components of cigarette smoke can cause DNA damage in bladder urothelium. Failure to repair DNA damage by DNA repair proteins may result in mutations leading to genetic instability and the development of bladder cancer. Immunohistochemistry studies have shown DNA damage signal activation in precancerous bladder lesions which is lost on progression, suggesting that the damage signalling mechanism acts as a brake to further tumorigenesis. Single nucleotide polymorphisms (SNPs) in DSB signalling genes may alter protein function. We hypothesized that SNPs in DSB signalling genes may modulate predisposition to bladder cancer and influence the effects of environmental exposures.</p> <p>Methods</p> <p>We recruited 771 cases and 800 controls (573 hospital-based and 227 population-based from a previous case-control study) and interviewed them regarding their smoking habits and occupational history. DNA was extracted from a peripheral blood sample and genotyping of 24 SNPs in <it>MRE11, NBS1, RAD50, H2AX </it>and <it>ATM </it>was undertaken using an allelic discrimination method (Taqman).</p> <p>Results</p> <p>Smoking and occupational dye exposure were strongly associated with bladder cancer risk. Using logistic regression adjusting for age, sex, smoking and occupational dye exposure, there was a marginal increase in risk of bladder cancer for an <it>MRE11 </it>3'UTR SNP (rs2155209, adjusted odds ratio 1.54 95% CI (1.13–2.08, p = 0.01) for individuals homozygous for the rare allele compared to those carrying the common homozygous or heterozygous genotype). However, in the hospital-based controls, the genotype distribution for this SNP deviated from Hardy-Weinberg equilibrium. None of the other SNPs showed an association with bladder cancer and we did not find any significant interaction between any of these polymorphisms and exposure to smoking or dye exposure.</p> <p>Conclusion</p> <p>Apart from a possible effect for one MRE11 3'UTR SNP, our study does not support the hypothesis that SNPs in DSB signaling genes modulate predisposition to bladder cancer.</p