Requirement for the Phospho-H2AX Binding Module of Crb2 in Double-Strand Break Targeting and Checkpoint Activation

Activation of DNA damage checkpoints requires the rapid accumulation of numerous factors to sites of genomic lesions, and deciphering the mechanisms of this targeting is central to our understanding of DNA damage response. Histone modification has recently emerged as a critical element for the correct localization of damage response proteins, and one key player in this context is the fission yeast checkpoint mediator Crb2. Accumulation of Crb2 at ionizing irradiation-induced double-strand breaks (DSBs) requires two distinct histone marks, dimethylated H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX). A tandem tudor motif in Crb2 directly binds H4K20me2, and this interaction is required for DSB targeting and checkpoint activation. Similarly, pH2AX is required for Crb2 localization to DSBs and checkpoint control. Crb2 can directly bind pH2AX through a pair of C-terminal BRCT repeats, but the functional significance of this binding has been unclear. Here we demonstrate that loss of its pH2AX-binding activity severely impairs the ability of Crb2 to accumulate at ionizing irradiation-induced DSBs, compromises checkpoint signaling, and disrupts checkpoint-mediated cell cycle arrest. These impairments are similar to that reported for abolition of pH2AX or mutation of the H4K20me2-binding tudor motif of Crb2. Intriguingly, a combined ablation of its two histone modification binding modules yields a strikingly additive reduction in Crb2 activity. These observations argue that binding of the Crb2 BRCT repeats to pH2AX is critical for checkpoint activity and provide new insight into the mechanisms of chromatin-mediated genome stability.DNA damage response is an essential cellular guard that protects the genetic material from a constant barrage of genotoxic agents. To ensure their survival after genomic insult, cells orchestrate a signaling cascade that leads to checkpoint-mediated cell cycle arrest and the repair of damaged DNA (16, 35). A failure in this process can have catastrophic cellular consequences leading to the development of numerous disorders such as cancer (18, 30, 32). Because of its intimate connection with human health, deciphering the molecular mechanisms of DNA damage response is of high interest (16, 20).Recently, histone posttranslational modification has emerged as one element that is critical for ensuring a faithful response to genomic challenge (7, 31). An octamer of the four core histones, H3, H4, H2A, and H2B, forms the core protein component of chromatin, and cells possess a considerable number of enzymes that target histones for posttranslation modification (21). These marks can impinge upon many aspects of DNA biology by acting to directly alter chromatin structure or by serving as a binding scaffold for the recruitment of regulatory factors (24).In the context of DNA damage response, one factor that is intimately linked with histone modification is the fission yeast DNA damage checkpoint protein Crb2. After genomic insult, DNA damage checkpoints function to halt cell cycle progression, ensuring sufficient time for lesion repair (16, 35). In the fission yeast Schizosaccharomyces pombe, regulating the transition from G₂ to mitosis (G₂/M) represents the major DNA damage checkpoint and Crb2 is essential for this activity (4, 34). Crb2 is a member of a family of checkpoint regulators that have been termed mediators because they are thought to transmit the checkpoint signal from damage-sensing ATM/ATR-related kinases to effector kinases, such as Chk1, that trigger cell cycle arrest (11, 25). Crb2 is closely related to budding yeast Rad9 and mammalian p53 binding protein 53BP1, which all share two distinct domains, a tandem tudor motif and a pair of C-terminal BRCT repeats (Fig. ​(Fig.1A)1A) (11, 25). Besides 53BP1, Crb2 also shares some functional similarities with other mammalian BRCT-containing checkpoint regulators, such as MDC1 and BRCA1 (11, 25). In response to ionizing irradiation (IR), the rapid accumulation of Crb2 and other checkpoint proteins can be readily visualized as nuclear foci that mark sites of double-strand breaks (DSBs) (9, 25). Understanding the mechanisms that govern this targeting has been an area of intense interest, and for Crb2 this accumulation requires two distinct histone marks: dimethylation of histone H4 lysine 20 (H4K20me2) and phosphorylated H2AX (pH2AX) (27, 36).Open in a separate windowFIG. 1.Crb2 pH2AX-binding mutations. (A) Top, schematic representation of Crb2 (not drawn to scale) with relevant mutations indicated. Bottom, protein sequence alignment of a portion of the BRCT phospho-binding motifs from Schizosaccharomyces pombe (sp) Crb2, human (h) 53BP1, human MDC1, and Saccharomyces cerevisiae (sc) Rad9. Identical residues are shaded black; similar residues are shaded gray. *, Crb2 phospho-binding residues. (B) The Crb2 BRCT domains specifically interact with pH2AX. Peptide pulldowns were performed as described in the text with C-terminal fission yeast H2A.1 peptides either unmodified or phosphorylated at Ser129 (see − or + pH2AX) and increasing amounts of the indicated recombinant Crb2 BRCT domain fragments (∼0.1 and 0.3 μM). After binding and washing, SDS-PAGE and Coomassie staining were used to visualize peptide-bound protein. A fraction of the total protein used for binding was also visualized (Input).Mono-, di-, and trimethyl H4K20 are conserved chromatin marks that are readily detectable in fission yeast and mammalian cells (29, 36). In fission yeast, the Kmt5 methylase catalyzes all three H4K20 methyl modifications and its inactivation, or mutation of its H4K20 substrate, severely diminishes Crb2 accumulation at DSBs and compromises checkpoint activity (10, 36). Note that as outlined by the unified nomenclature for the naming of histone lysine methyltransferases (2), the fission yeast H4K20 methylase previously known as Set9 (36) is now termed Kmt5. The requirement for H4K20 methylation is mediated by the tandem tudor domains of Crb2 that preferentially bind H4 tail peptides dimethylated at lysine 20 (3, 14). Tudor motif mutations impair Crb2 DSB targeting and genome integrity in a manner analogous to loss of Kmt5 activity, and dimethylation of H4K20, but not trimethylation, is required for Crb2 activity (10, 14, 42). The tudor domain of 53BP1 can also directly bind H4K20me2, and this recognition event is required for its accumulation at IR-induced DSBs (3, 23, 45).After DNA damage, serine 139 phosphorylation in the mammalian H2A variant H2AX, or a homologous site in canonical yeast H2A, specifically marks sites of genomic lesions (7, 12). The fission yeast genome encodes two H2A proteins, H2A.1 and H2A.2, which differ slightly in their primary amino acid sequence. Phosphorylation of S129 in H2A.1 and S128 in H2A.2 is collectively referred to as phosphorylated H2AX (pH2AX). The ATM/ATR family of PI3-like kinases that includes the fission yeast Rad3 and Tel1 enzymes catalyzes pH2AX (37). H2AX phosphorylation has a critical role in controlling both DNA repair and checkpoint activation in a variety of organisms from yeast to humans (7, 12). Central to its function is the ability of the pH2AX mark to coordinate the recruitment of a number of proteins to genomic lesions, and several factors can directly bind the modification (40). Serine-to-alanine substitutions at the H2AX phosphorylation site in fission yeast H2A (h2ax⁻) severely reduce Crb2 accumulation at IR-induced DSBs and compromise the ability of cells to maintain checkpoint cell cycle arrest in a manner very similar to loss of H4K20 methylation (10, 27).The mechanism underlying the control of Crb2 DSB targeting and checkpoint activation by pH2AX is not understood. Because BRCT domains are known phospho-binding motifs (13), the initial demonstration that pH2AX is required for Crb2 function suggested that direct binding to the modification by Crb2 is critical for checkpoint activity (27). Supporting this idea, it has been demonstrated that the Crb2 BRCT repeats directly and specifically bind pH2AX peptides (22). Structural and biochemical studies have also identified a conserved pH2AX-binding motif in the BRCT repeats of Crb2, budding yeast Rad9, and human MDC1 and 53BP1 (Fig. ​(Fig.1A)1A) (15, 22, 39). As would be expected, mutation of Crb2''s critical phospho-binding motif impairs cell survival after DNA damage (22). Unexpectedly though, loss of its pH2AX-binding activity did not significantly affect the ability of Crb2 to localize to IR-induced DSBs (22). Rather, mutation of the Crb2 pH2AX-binding motif altered the kinetics of Rad22 accumulation at DSBs and triggered a prolonged checkpoint arrest after IR exposure (22). From these observations it was suggested that binding of the Crb2 BRCT repeats to pH2AX is critical for aspects of DNA repair but is not central to Crb2 targeting and checkpoint activity (22).The apparent dispensability of its pH2AX-binding motif in controlling Crb2 localization to IR-induced DSBs (22) was a surprising observation because of the established requirement for the pH2AX modification (10, 27). The extended checkpoint delay seen in Crb2 pH2AX-binding mutants (22) was also unexpected because h2ax⁻ cells cannot maintain checkpoint-mediated cell cycle arrest (10, 27). The prolonged checkpoint arrest was also surprising because a defect in IR-induced Chk1 phosphorylation was observed in the same Crb2 pH2AX-binding mutants (22). For these reasons we sought to reevaluate the requirement for the pH2AX-binding module of Crb2 in controlling DNA damage checkpoint activity. We demonstrate that the critical phospho-coordinating residue of Crb2 is required for binding to pH2AX peptides, Crb2 accumulation at IR-induced DSBs, cell survival after DNA damage, and maintenance of checkpoint-mediated cell cycle arrest. The observed impairments are similar to that reported for abolishment of pH2AX or mutation of the H4K20me2 binding tudor motif of Crb2. Strikingly, a combined ablation of the two modification binding modules of Crb2 produces an additive impairment in checkpoint dysfunction and genome integrity. These results argue that recognition of pH2AX by its BRCT repeats is critical for Crb2 accumulation at genomic lesions and its subsequent checkpoint activity. These observations also corroborate the independent findings of Sofueva et al. (38), who have observed a similar requirement for Crb2 binding to pH2AX in controlling DSB targeting and checkpoint activity.     

Developing dual functional allosteric modulators of GABA(A) receptors

Positive modulators at benzodiazepine sites of α2- and α3-containing GABA(A) receptors are believed to be anxiolytic. Negative allosteric modulators of α5-containing GABA(A) receptors enhance cognition. By oocyte two-electrode voltage clamp and subsequent structure-activity relationship studies, we discovered cinnoline and quinoline derivatives that were both positive modulators at α2-/α3-containing GABA(A) receptors and negative modulators at α5-containing GABA(A) receptors. In addition, these compounds showed no functional activity at α1-containing GABA(A) receptors. Such dual functional modulators of GABA(A) receptors might be useful for treating comorbidity of anxiety and cognitive impairments in neurological and psychiatric illnesses.     

Beta-arrestin2, a novel member of the arrestin/beta-arrestin gene family.

Homologous or agonist-specific desensitization of beta 2-adrenergic receptors (beta 2AR) is mediated by the beta-adrenergic receptor kinase (beta ARK) which specifically phosphorylates the agonist-occupied form of the receptor. However, the capacity of beta ARK-phosphorylated beta 2AR to stimulate Gs in a reconstituted system is only minimally impaired. Recently, a protein termed beta-arrestin, was cloned from a bovine brain cDNA library and found to quench phosphorylated beta 2AR-coupling to Gs. Utilizing a low stringency hybridization technique to screen a rat brain cDNA library, we have now isolated cDNA clones representing two distinct beta-arrestin-like genes. One of the cDNAs is the rat homolog of bovine beta-arrestin (beta-arrestin1). In addition, we have isolated a cDNA clone encoding a novel, beta-arrestin-related protein which we have termed beta-arrestin2. Overall, beta-arrestin2 exhibits 78% amino acid identity with beta-arrestin1. The primary structure of these proteins delineates a family of proteins that regulates receptor coupling to G proteins. The capacity of purified beta-arrestin1, beta-arrestin2, and arrestin to inhibit the coupling of phosphorylated receptors to their respective G proteins were assessed in a reconstituted beta 2AR-Gs system and in a reconstituted rhodopsin-GT system. beta-Arrestin2 was equipotent to beta-arrestin1 and specifically inhibited beta 2AR function. Conversely, arrestin inhibited rhodopsin coupling to GT, whereas beta-arrestin1 and beta-arrestin2 were at least 20-fold less potent in this system. beta-Arrestin1 and beta-arrestin2 are predominantly localized in neuronal tissues and in the spleen. However, low mRNA levels can be detected in most peripheral tissues. In the central nervous system, beta-arrestin2 appears to be even more abundant than beta-arrestin1. Immunohistochemical analysis of the tissue distribution of beta-arrestin1 and beta-arrestin2 in rat brain shows extensive, but heterogenous, neuronal labeling of the two proteins. They are found in several neuronal pathways suggesting that they have relatively broad receptor specificity regulating many G protein-coupled receptors. Furthermore, immunoelectron microscopy shows that the beta-arrestins are appropriately situated at postsynaptic sites to act in concert with beta ARK to regulate G protein-coupled neurotransmitter receptors.     

Alcohol Abuse Promotes Changes in Non-Synaptic Epileptiform Activity with Concomitant Expression Changes in Cotransporters and Glial Cells

Non-synaptic mechanisms are being considered the common factor of brain damage in status epilepticus and alcohol intoxication. The present work reports the influence of the chronic use of ethanol on epileptic processes sustained by non-synaptic mechanisms. Adult male Wistar rats administered with ethanol (1, 2 e 3 g/kg/d) during 28 days were compared with Control. Non-synaptic epileptiform activities (NEAs) were induced by means of the zero-calcium and high-potassium model using hippocampal slices. The observed involvement of the dentate gyrus (DG) on the neurodegeneration promoted by ethanol motivated the monitoring of the electrophysiological activity in this region. The DG regions were analyzed for the presence of NKCC1, KCC2, GFAP and CD11b immunoreactivity and cell density. The treated groups showed extracellular potential measured at the granular layer with increased DC shift and population spikes (PS), which was remarkable for the group E1. The latencies to the NEAs onset were more prominent also for the treated groups, being correlated with the neuronal loss. In line with these findings were the predispositions of the treated slices for neuronal edema after NEAs induction, suggesting that restrict inter-cell space counteracts the neuronal loss and subsists the hyper-synchronism. The significant increase of the expressions of NKCC1 and CD11b for the treated groups confirms the existence of conditions favorable to the observed edematous necrosis. The data suggest that the ethanol consumption promotes changes on the non-synaptic mechanisms modulating the NEAs. For the lower ethanol dosage the neurophysiological changes were more effective suggesting to be due to the less intense neurodegenertation.     

Arterial hypertension assessed “out-of-office” in a contemporary cohort of rheumatoid arthritis patients free of cardiovascular disease is characterized by high prevalence,low awareness,poor control and increased vascular damage-associated “white coat” phenomenon

Introduction

Rheumatoid arthritis (RA) is associated with a high cardiovascular disease (CVD) risk, whereas arterial hypertension is a major modifiable CVD risk factor with still unclear prevalence in RA disease. We conducted a comprehensive study on hypertension characteristics evaluating for the first time out-of-office blood pressure (BP) in a typical contemporary RA cohort.

Methods

Assessment of office and out-of-office BP (when office systolic/diastolic BP was >129/79) and vascular studies including evaluation of aortic stiffness, carotid hypertrophy/plaques and ankle-brachial index, were performed in 214 consecutive, consenting RA patients free of CVD (aged 58.4 ± 12.3 years, 82% women). As comparators regarding office hypertension measurements, data from 214 subjects (1:1 matched for age and gender with the RA patients) derived from a cohort designed to assess the prevalence of hypertension in the general population were used.

Results

The prevalence of declared known hypertension in the RA population was 44%. Of the remaining RA patients, 2 in every 5 individuals had abnormal office BP (systolic/diastolic >139/89 mmHg), contributing to almost double the prevalence of declared/office hypertension compared to the general matched population (67% vs. 34%). Out-of-office (home or ambulatory 24 hour) BP measurements revealed that: (i) a 54% prevalence of actual hypertension in RA, in other words almost 10% of the patients were unaware of having hypertension and (ii) 29% of the RA patients with known hypertension were not well controlled. Actual hypertension was positively associated with age and body mass index, and inversely with the use of biologic drugs. Overall, almost 1 out of 5 presented the ''white coat’ phenomenon. An intermediately compromised vascular phenotype was evident in this “white coat” subgroup (lying between patients with sustained normotension and sustained hypertension) in terms of aortic stiffness, carotid hypertrophy and ankle-brachial index, even after adjustment for confounders.

Conclusion

Beyond any doubt on the basis of out-of-office evaluation, arterial hypertension in RA has a high prevalence, low awareness and poor control, as well as substantial and vascular damage-associated “white coat” phenomenon. Thus, correct diagnosis and effective treatment of hypertension is of key importance in RA for CVD risk reduction.     

Effect of exhaustive ultra-endurance exercise in muscular glycogen and both Alpha1 and Alpha2 Ampk protein expression in trained rats

Glycogen is the main store of readily energy in skeletal muscle and plays a key role in muscle function, demonstrated by the inability to sustain prolonged high-intensity exercise upon depletion of these glycogen stores. With prolonged exercise, glycogen depletion occurs and 5′-AMP-activated protein kinase (AMPK), a potent regulator of muscle metabolism and gene expression, is activated promoting molecular signalling that increases glucose uptake by muscular skeletal cells. The aim of this study was primarily to determine the effect of ultra-endurance exercise on muscle glycogen reserves and secondly to verify the influence of this type of exercise on AMPK protein expression. Twenty-four male Wistar rats, 60 days old, were divided into four experimental groups: sedentary, sedentary exhausted (SE), endurance trained (T) and endurance trained exhausted (TE). The animals ran for 10 to 90 min/day, 5 days/week, for 12 weeks to attain trained status. Rats were killed immediately after the exhaustion protocol, which consisted of running on a treadmill (at approximately 60 % V _max until exhaustion). Optical density of periodic acid-Schiff was detected and glycogen depletion observed predominantly in type I muscle fibres of the TE group and in both type I and II muscle fibres in the SE group. Plasma glucose decreased only in the TE group. Hepatic glycogen was increased in T group and significantly depleted in TE group. AMPK protein expression was significantly elevated in TE and T groups. In conclusion, acute exhaustive ultra-endurance exercise promoted muscle glycogen depletion. It seems that total AMPK protein and gene expression is more influenced by status training.     

Differential effects of exercise on brain opioid receptor binding and activation in rats

Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short‐ (acute) or long‐term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute‐exercise animals and the opposite was found in the chronic‐exercise animals. The binding of [³⁵S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli.

     

Electrogenic uptake of gamma-aminobutyric acid by a cloned transporter expressed in Xenopus oocytes.

GAT-1, a gamma-aminobutyric acid (GABA) transporter cloned from rat brain, was expressed in Xenopus oocytes. Voltage-clamp measurements showed concentration-dependent, inward currents in response to GABA (K0.5 4.7 microM). The transport current required extracellular sodium and chloride ions; the Hill coefficient for chloride was 0.7, and that for sodium was 1.7. Correlation of current and [3H]GABA uptake measurements indicate that flux of one positive charge occurs per molecule of GABA transported. Membrane hyperpolarization from -40 to -100 mV increased the transport current approximately 3-fold. The results indicate that the transport of one molecule of GABA involves the co-transport of two sodium ions and one chloride ion.