O-GlcNAc modification affects the ATM-mediated DNA damage response

Background

O-Linked β-N-acetylglucosamine (O-GlcNAc) is a reversible, post-translational, and regulatory modification of nuclear, mitochondrial, and cytoplasmic proteins that is responsive to cellular stress. The role of O-GlcNAcylation in the ataxia-telangiectasia mutated (ATM)-mediated DNA damage response is unknown. It is unclear whether ATM, which is an early acting and central component of the signal transduction system activated by DNA double strand breaks, is an O-GlcNAc-modified protein.

Methods

The effect of O-GlcNAc modification on ATM activation was examined using two inhibitors, PUGNAc and DON that increase and decrease, respectively, levels of protein O-GlcNAcylation. To assess O-GlcNAcylation of ATM, immunoprecipitation and immunoblot analyses using anti-ATM or anti-O-GlcNAc antibody were performed in HeLa cells and primary cultured neurons. Interaction of ATM with O-GlcNAc transferase (OGT), the enzyme that adds O-GlcNAc to target proteins, was examined by immunoprecipitation and immunoblot analyses using anti-ATM.

Results

Enhancement of protein O-GlcNAcylation increased levels of X-irradiation-induced ATM activation. However, decreases in protein O-GlcNAcylation did not affect levels of ATM activation, but these decreases did delay ATM activation and ATM recovery processes based on assessment of de-phosphorylation of phospho-ATM. Thus, activation and recovery of ATM were affected by O-GlcNAcylation. ATM was subjected to O-GlcNAcylation, and ATM interacted with OGT. The steady-state O-GlcNAc level of ATM was not significantly responsive to X-irradiation or oxidative stress.

General significance

ATM is an O-GlcNAc modified protein, and dynamic O-GlcNAc modification affects the ATM-mediated DNA damage response.     

Grain of environment explains variation in the strength of genotype × environment interaction

Theory predicts that genetic variation in phenotypic plasticity (genotype × environment interaction or G × E) should be eroded by selection acting across environments. However, it appears that G × E is often maintained under selection, although not universally. This variation in the presence and strength of G × E requires explanation. Here I ask whether the explanation may lie in the grain of the environment at which G × E is expressed. The grain (or grain size) of the environment refers to the scale of environmental heterogeneity relative to generation time – that is, relative to the window of operation of selection – with higher rates of heterogeneity occurring in finer‐grained environments. The hypothesis that the grain of the environment explains variation in the expression of G × E encapsulates variation in the power of selection to shape reaction norms: selection should be able to erode G × E in fine‐grained environments but lose its power as the grain becomes coarser. I survey studies of G × E in sexual traits and demonstrate that the strength of G × E varies with the grain of the environment across which it is expressed, with G × E being stronger in coarser‐grained environments. This result elucidates when G × E is most likely to be sustained in the reaction norms of fitness‐related traits and when its evolutionary consequences will be most pronounced.     

Characterization of BARD1 targeting and dynamics at the centrosome: the role of CRM1, BRCA1 and the Q564H mutation

BARD1 heterodimerizes with BRCA1, forming an E3 ubiquitin ligase that functions at nuclear foci to repair DNA damage and the centrosome to regulate mitosis. We compared BARD1 recruitment at these structures using fluorescence recovery after photobleaching assays to measure YFP-BARD1 dynamics in live cells. In nuclei at ionizing radiation-induced foci, 20% of the BARD1 pool was immobile and 80% of slow mobility exhibiting a recovery time > 500 s. In contrast, at centrosomes 83% of BARD1 was rapidly mobile with extremely fast turnover (recovery time ~ 20 s). The ~ 25-fold faster exchange of BARD1 at centrosomes correlated with BRCA1-independent recruitment. We mapped key targeting sequences to a combination of the N and C-termini, and showed that mutation of the nuclear export signal reduced centrosome localization by 50%, revealing a role for CRM1. Deletion of the sequence 128-550 increased BARD1 turnover at the centrosome, consistent with a role in transient associations. Conversely, the cancer mutation Q564H reduced turnover by 25%. BARD1 is one of the most highly mobile proteins yet detected at the centrosome, and in contrast to its localization at DNA repair foci, which requires dimerization with BRCA1, targeting of BARD1 to the centrosome occurs prior to heterodimerization and its rapid turnover may provide a mechanism to regulate dimer formation.     

棕榈酸的组织吸收分布及对骨骼肌胰岛素抵抗的影响

脂肪酸代谢紊乱是Ⅱ型糖尿病的主要致病因素之一。棕榈酸是血液中含量最高的游离脂肪酸。我们建立了大鼠颈静脉置管输注棕榈酸的模型,发现血液中的大部分棕榈酸被骨骼肌组织所吸收。以棕榈酸处理的C2C12骨骼肌细胞为实验模型发现,棕榈酸进入骨骼肌细胞后的中间代谢产物(磷脂和甘油二酯)的累积,会造成内质网应激及胰岛素抵抗。提示血液中棕榈酸含量的升高可能通过骨骼肌的胰岛素抵抗机制,影响Ⅱ型糖尿病的发生和发展。     

Temperature explains global variation in biomass among humid old‐growth forests

Aim To develop and test a simple climate‐based ecophysiological model of above‐ground biomass – an approach that can be applied directly to predicting the effects of climate change on forest carbon stores. Location Humid lowland forests world‐wide. Methods We developed a new approach to modelling the aboveground biomass of old‐growth forest (AGB_max) based on the influences of temperature on gross primary productivity (GPP) and what we call total maintenance cost (TMC), which includes autotrophic respiration as well as leaf, stem and other plant construction required to maintain biomass. We parameterized the models with measured carbon fluxes and tested them by comparing predicted AGB_max with measured AGB for another 109 old‐growth sites. Results Our models explained 57% of the variation in GPP across 95 sites and 79% of the variation in TMC across 17 sites. According to the best‐fit models, the ratio of GPP to maintenance cost per unit biomass (MCB) peaks at 16.5 °C, indicating that this is the air temperature leading to the highest possible AGB_max when temperatures are constant. Seasonal temperature variation generally reduces predicted AGB_max, and thus maritime temperate climates are predicted to have the highest AGB_max. The shift in temperatures from temperate maritime to tropical climates increases MCB more than GPP, and thus decreases AGB_max. Overall, our model explains exactly 50% of the variation in AGB among humid lowland old‐growth forests. Main conclusions Temperature plays an important role in explaining global variation in biomass among humid lowland old‐growth forests, a role that can be understood in terms of the dual effects of temperature on GPP and TMC. Our simple model captures these influences, and could be an important tool for predicting the effects of climate change on forest carbon stores.     

Coenzyme Q10 as a potent compound that inhibits Cdt1–geminin interaction

A human replication initiation protein Cdt1 is a very central player in the cell cycle regulation of DNA replication, and geminin down-regulates Cdt1 function by directly binding to it. It has been demonstrated that Cdt1 hyperfunction resulting from Cdt1–geminin imbalance, for example by geminin silencing with siRNA, induces DNA re-replication and eventual cell death in some cancer-derived cell lines. In the present study, we first established a high throughput screening system based on modified ELISA (enzyme linked immunosorbent assay) to identify compounds that interfere with human Cdt1–geminin binding. Using this system, we found that coenzyme Q₁₀ (CoQ₁₀) can inhibit Cdt1–geminin interaction in vitro. CoQ compound is an isoprenoid quinine that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. CoQ₁₀, having a longer isoprenoid chain, was the strongest inhibitor of Cdt1–geminin binding in the tested CoQs, with 50% inhibition observed at concentrations of 16.2 μM. Surface plasmon resonance analysis demonstrated that CoQ₁₀ bound selectively to Cdt1, but did not interact with geminin. Moreover, CoQ₁₀ had no influence on the interaction between Cdt1 and mini-chromosome maintenance (MCM)4/6/7 complexes. These results suggested that CoQ₁₀ inhibits Cdt1–geminin complex formation by binding to Cdt1 and thereby could liberate Cdt1 from inhibition by geminin. Using three-dimensional computer modeling analysis, CoQ₁₀ was considered to interact with the geminin interaction interface on Cdt1, and was assumed to make hydrogen bonds with the residue of Arg243 of Cdt1. CoQ₁₀ could prevent the growth of human cancer cells, although only at high concentrations, and it remains unclear whether such an inhibitory effect is associated with the interference with Cdt1–geminin binding. The application of inhibitors for the formation of Cdt1–geminin complex is discussed.     

Size matters: non-LTR retrotransposable elements and ectopic recombination in Drosophila

The Drosophila melanogaster genome contains approximately 100 distinct families of transposable elements (TEs). In the euchromatic part of the genome, each family is present in a small number of copies (5-150 copies), with individual copies of TEs often present at very low frequencies in populations. This pattern is likely to reflect a balance between the inflow of TEs by transposition and the removal of TEs by natural selection. The nature of natural selection acting against TEs remains controversial. We provide evidence that selection against chromosome abnormalities caused by ectopic recombination limits the spread of some TEs. We also demonstrate for the first time that some TE families in the Drosophila euchromatin appear to be only marginally affected by purifying selection and contain many copies at high population frequencies. We argue that TEs in these families attain high population frequencies and even reach fixation as a result of low family-wide transposition rates leading to low TE copy numbers and consequently reduced strength of selection acting on individual TE copies. Fixation of TEs in these families should provide an upward pressure on the size of intergenic sequences counterbalancing rapid DNA loss through small deletions. Copy-number-dependent selection on TE families caused by ectopic recombination may also promote diversity among TEs in the Drosophila genome.     

Back signaling by the Nrg-1 intracellular domain

Transmembrane isoforms of neuregulin-1 (Nrg-1), ligands for erbB receptors, include an extracellular domain with an EGF-like sequence and a highly conserved intracellular domain (ICD) of unknown function. In this paper, we demonstrate that transmembrane isoforms of Nrg-1 are bidirectional signaling molecules in neurons. The stimuli for Nrg-1 back signaling include binding of erbB receptor dimers to the extracellular domain of Nrg-1 and neuronal depolarization. These stimuli elicit proteolytic release and translocation of the ICD of Nrg-1 to the nucleus. Once in the nucleus, the Nrg-1 ICD represses expression of several regulators of apoptosis, resulting in decreased neuronal cell death in vitro. Thus, regulated proteolytic processing of Nrg-1 results in retrograde signaling that appears to mediate contact and activity-dependent survival of Nrg-1-expressing neurons.