Calf Thymus Histone Research Articles

Combining SDS-PAGE to capillary zone electrophoresis-tandem mass spectrometry for high-resolution top-down proteomics analysis of intact histone proteoforms.

Mass spectrometry (MS)-based top-down proteomics (TDP) analysis of histone proteoforms provides critical information about combinatorial post-translational modifications (PTMs), which is vital for pursuing a better understanding of epigenetic regulation of gene expression. It requires high-resolution separations of histone proteoforms before MS and tandem MS (MS/MS) analysis. In this work, for the first time, we combined SDS-PAGE-based protein fractionation (passively eluting proteins from polyacrylamide gels as intact species for mass spectrometry, PEPPI-MS) with capillary zone electrophoresis (CZE)-MS/MS for high-resolution characterization of histone proteoforms. We systematically studied the histone proteoform extraction from SDS-PAGE gel and follow-up cleanup as well as CZE-MS/MS, to determine an optimal procedure. The optimal procedure showed reproducible and high-resolution separation and characterization of histone proteoforms. SDS-PAGE separated histone proteins (H1, H2, H3, and H4) based on their molecular weight and CZE provided additional separations of proteoforms of each histone protein based on their electrophoretic mobility, which was affected by PTMs, for example, acetylation and phosphorylation. Using the technique, we identified over 200 histone proteoforms from a commercial calf thymus histone sample with good reproducibility. The orthogonal and high-resolution separations of SDS-PAGE and CZE made our technique attractive for the delineation of histone proteoforms extracted from complex biological systems.

Covalent adduct formation of histone with organophosphorus pesticides in vitro

It is established that organophosphorus pesticide (OPP) toxicity results from modification of amino acids in active sites of target proteins. OPPs can also modify unrelated target proteins such as histones and such covalent histone modifications can alter DNA-binding properties and lead to aberrant gene expression. In the present study, we report on non-enzymatic covalent modifications of calf thymus histones adducted to selected OPPs and organophosphate flame retardants (OPFRs) in vitro using a bottom-up proteomics method approach. Histones were not found to form detectable adducts with the two tested OPFRs but were avidly modified by a few of the seven OPPs that were tested in vitro. Dimethyl phosphate (or diethyl phosphate) adducts were identified on Tyr, Lys and Ser residues. Most of the dialkyl phosphate adducts were identified on Tyr residues. Methyl and ethyl modified histones were also detected. Eleven amino residues in histones showed non-enzymatic covalent methylation by exposure of dichlorvos and malathion. Our bottom-up proteomics approach showing histone-OPP adduct formation warrants future studies on the underlying mechanism of chronic illness from exposure to OPPs.

Fibrinogen binding to histones in circulation protects against adverse cellular and clinical outcomes

BackgroundCirculating histones are released by extensive tissue injury or cell death and play important pathogenic roles in critical illnesses. Their interaction with circulating plasma components and the potential roles in the clinical setting are not fully understood. ObjectivesWe aimed to characterize the interaction of histones with fibrinogen and explore its roles in vitro, in vivo, and in patient samples. MethodsHistone–fibrinogen binding was assessed by electrophoresis and enzyme-linked immunosorbent assay-based affinity assay. Functional significance was explored using washed platelets and endothelial cells in vitro and histone-infusion mouse models in vivo. To determine clinical translatability, a retrospective single-center cohort study was conducted on patients requiring intensive care admission (n = 199) and validated in a cohort of hospitalized patients with COVID-19 (n = 69). ResultsFibrinogen binds histones through its D-domain with high affinity (calf thymus histones, KD = 18.0 ± 5.6 nM; histone 3, KD = 2.7 ± 0.8 nM; and histone 4, KD = 2.0 ± 0.7 nM) and significantly reduces histone-induced endothelial damage and platelet aggregation in vitro and in vivo in a histone-infusion mouse model. Physiologic concentrations of fibrinogen can neutralize low levels of circulating histones and increase the cytotoxicity threshold of histones to 50 μg/mL. In a cohort of patients requiring intensive care, a histone:fibrinogen ratio of ≥6 on admission was associated with moderate–severe thrombocytopenia and independently predicted mortality. This finding was validated in a cohort of hospitalized patients with COVID-19. ConclusionFibrinogen buffers the cytotoxic properties of circulating histones. Detection and monitoring of circulating histones and histone:fibrinogen ratios will help identify critically ill patients at highest risk of adverse outcomes who might benefit from antihistone therapy.

M6229 Protects against Extracellular-Histone-Induced Liver Injury, Kidney Dysfunction, and Mortality in a Rat Model of Acute Hyperinflammation.

Extracellular histones have been shown to act as DAMPs in a variety of inflammatory diseases. Moreover, they have the ability to induce cell death. In this study, we show that M6229, a low-anticoagulant fraction of unfractionated heparin (UFH), rescues rats that were challenged by continuous infusion of calf thymus histones at a rate of 25 mg histones/kg/h. Histone infusion by itself induced hepatic and homeostatic dysfunction characterized by elevated activity of hepatic enzymes (ASAT and ALAT) and serum lactate levels as well as by a renal dysfunction, which contributed to the significantly increased mortality rate. M6229 was able to restore normal levels of both hepatic and renal parameters at 3 and 9 mg M6229/kg/h and prevented mortality of the animals. We conclude that M6229 is a promising therapeutic agent to treat histone-mediated disease.

Protein arginine N-methyltransferase 2 plays a noncatalytic role in the histone methylation activity of PRMT1

Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.

Urinary liver‐type fatty acid binding protein is increased in the early stages of the disease with a risk of acute kidney injury induced by histone

Urinary liver-type fatty acid binding protein (L-FABP) has potential utility as an early prognostic biomarker ahead of traditional severity scores in coronavirus disease 2019 and sepsis, however, the mechanism of elevated urinary L-FABP in the disease has not been clearly elucidated. We investigated the background mechanisms of urinary L-FABP excretion through non-clinical animal model focusing on histone, which is one of the aggravating factors in these infectious diseases. Male Sprague-Dawley rats were placed in central intravenous catheters, and these rats were given a continuous intravenous infusion of 0.25 or 0.5 mg/kg/min calf thymus histones for 240 min from caudal vena cava. After the administration of histone, urinary L-FABP and gene expression of an oxidative stress marker in the kidney increased in a histone dose-dependent manner before increased serum creatinine. Upon further investigation, fibrin deposition in the glomerulus was observed and it tended to be remarkable in the high dose administrated groups. The levels of coagulation factor were significantly changed after the administration of histone, and these were significantly correlated with the levels of urinary L-FABP. Firstly, it was suggested that histone is one of the causative agents for the urinary L-FABP increase at an early stage of the disease with a risk of acute kidney injury. Secondly, urinary L-FABP could be a marker reflecting the changes of coagulation system and microthrombus caused by histone in the early stage of acute kidney injury before becoming severely ill and maybe a guide to early treatment initiation.

Enhanced Characterization of Histones Using 193 nm Ultraviolet Photodissociation and Proton Transfer Charge Reduction

Top-down characterization of histones, proteins that are critical participants in an array of DNA-dependent processes, offers the potential to examine the relationship between histone structure and mechanisms of genetic regulation. Mapping patterns of post-translational modifications (PTMs) of histones requires extensive backbone cleavages to bracket the sites of mass shifts corresponding to specific PTMs. Ultraviolet photodissociation (UVPD) causes substantial fragmentation of proteins, which is well-suited for PTM localization, but the resulting spectra are congested with fragment ions that may have overlapping isotopic distributions that confound deconvolution. Gas-phase proton transfer charge reduction (PTCR) decreases the charge states of highly charged ions, thus alleviating this congestion and facilitating the identification of additional sequence-determining and PTM-localizing fragment ions. By integrating UVPD with PTCR for histone proteoform analyses, sequence coverages up to 91% were achieved for calf thymus histone H4 containing acetylation marks at the N-terminus and Lys12 as well as a dimethylation at Arg3. UVPD-PTCR exhibited large gains in characterization for other histones, such as histone H2A, increasing the sequence coverage from 59 to 77% for monoacetylated H2A.

Bottom-up proteomics analysis for adduction of the broad-spectrum herbicide atrazine to histone

Histones are the major proteinaceous components of chromatin in eukaryotic cells and an important part of the epigenome. The broad-spectrum herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1, 3, 5-triazine) and its metabolites are known to form protein adducts, but the formation of atrazine–histone adducts has not been studied. In this study, a bottom-up proteomics analysis method was optimized and applied to identify histone adduction by atrazine in vitro. Whole histones of calf thymus or human histone H3.3 were incubated with atrazine. After solvent-based protein precipitation, the protein was digested by trypsin/Glu-C and the resulting peptides were analyzed by high-resolution mass spectrometry using an ultra-high-performance liquid chromatograph interfaced with a quadrupole Exactive-Orbitrap mass spectrometer. The resulting tryptic/Glu-C peptide of DTNLCAIHAK from calf thymus histone H3.1 or human histone H3.3 was identified with an accurate mass shift of +179.117 Da in atrazine incubated samples. It is deduced that a chemical group with an elemental composition of C8H13N5 (179.1171 Da) from atrazine adducted with calf thymus histone H3.1 or human histone H3.3. It was confirmed by MS/MS analysis that the adduction position was at its cysteine 110 residue. Time- and concentration-dependent assays also confirmed the non-enzymatic covalent modification of histone H3.3 by atrazine in vitro. Thus, the potential exists that atrazine adduction may lead to the alteration of histones that subsequently disturbs their normal function.Graphical abstract

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively-charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively-charged surface of ACE2, and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

The study on the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis

Objective: To explore the role of extracellular histones in the pathogenesis of coal worker's pneumoconiosis (CWP) , the relationship of extracellular histones in plasma with pulmonary fibrosis caused by coal mine dust was analyzed, and the stimulating effect of extracellular histones on fibroblast proliferation was studied. Methods: In May 2019, a total of 220 coal mine dust exposure workers (including coal miners and CWP patients) who visited the occupational disease outpatient department of Peking University Third Hospital from 2012 to 2015 were enrolled in the study. According to the classification of small opacity profusion (SOP) in chest radiograph for pneumoconiosis diagnosis (category 0, 1, 2, 3) , 61 coal miners were in category 0 SOP, 65 coal miners were in category 1 SOP, 56 coal miners were in category 2 SOP and 38 coal miners were in category 3 SOP. The plasma levels of extracellular histone H4 and platelet-derived growth factor (PDGF) were measured by enzyme-linked immunosorbent assay (ELISA) kit. The stimulating effects of CWP patients' plasma and calf thymus histones (CTHs) on fibroblast and the antagonizing effect of anti-H4 antibody were investigated by fibroblast proliferation experiment in vitro. Results: Among the study subjects, there were 195 males (88.6%, 195/220) and 25 females (11.4%, 25/220) , age (55.1±7.2) years, coal mine dust exposure time (16.3±4.4) years. The plasma concentrations of histone H4 in the coal miners with category 0, 1, 2 and 3 SOP were (3.92±1.75) 、(9.84±4.17) 、(14.35±5.52) and (17.83±7.69) μg/ml, respectively. There were significant differences among the four groups (P<0.01) . The plasma level of histone H4 was positively correlated with the plasma level of PDGF in the coal miners (r=0.769, P<0.01) . Compared with healthy control plasma group, the cell proliferation percentages of patients' plasma group (272%±87%) and CTH group (283%±84%) were significantly increased (P<0.05) . Compared with patients' plasma group, the cell proliferation percentage of patients' plasma+anti-H4 antibody group (185%±66%) was significantly decreased (P<0.05) . Compared with CTH group, the cell proliferation percentage of CTH+anti-H4 antibody group (167%±59%) was significantly decreased (P<0.05) . Conclusion: Extracellular histones in plasma are associated with pulmonary fibrosis in patients with CWP. Studies in vitro have shown that extracellular histones can promote proliferation of pulmonary fibroblasts. It is suggested that extracellular histones can be important biomarkers for pulmonary fibrosis caused by coal mine dust.

Activation of lung endothelial cells by extracellular histone in mice with acute respiratory distress syndrome

Objective: To observe the changes of extracellular histones and pulmonary microvascular endothelial cells, and study the activating role of extracellular histones to pulmonary microvascular endothelial cells in the pathogenesis of acute respiratory distress syndrome (ARDS) . Methods: The correlation of the severity of acute lung injury with extracellular histones and pulmonary endothelial damage was studied through mice model, and acute lung injury was produced by aspiration of different concentrations of hydrochloric acid (0.01、0.1、0.3 and 0.5 mol/L, 2 ml/kg). Tumor necrosis factor-α (TNF-α), soluble thrombomodulin (sTM) and lung pathological change were measured. The pro-inflammatory role of extracellular histones was tested by injecting calf thymus histones (CTH) or specific anti-H4 antibody through tail vein. The direct activating role of extracellular histones to pulmonary microvascular endothelial cells was studied through pulmonary endothelial model. Results: The extracellular histones in plasma were increased obviously 6h after aspiration of different concentrations of hydrochloric acid in mice. A positive correlation was seen between extracellular histones and concentrations of aspirated hydrochloric acid (r=0.9180, P<0.05). The sTM in plasma also showed a positive correlation with concentrations of aspirated hydrochloric acid (r=0.8701, P<0.05). Merely administering CTH could not only increase TNF-α and sTM in plasma but also cause obvious lung injury, while specific anti-H4 antibody could relieve the inflammation and lung damage caused by CTH. Extracellular histones could directly damage pulmonary endothelial cells to release sTM in pulmonary endothelial model in vitro, while anti-H4 antibody could protect the endothelial cells. Conclusion: Extracellular histones are the key endogenic inflammatory mediators during the pathogenesis of ARDS caused by aspiration of hydrochloric acid, which could promote inflammation by directly activating pulmonary endothelial cells.

Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses.

Multiple organ dysfunction syndrome is characterized by simultaneous multiple organ failure, which is the leading cause of death in acute critically ill patients. However, what mediates multiple organ dysfunction syndrome is not fully understood. The discovery of toxic effects by extracellular histones on different individual organs strongly suggests their involvement in multiple organ dysfunction syndrome. In this study, we investigate whether circulating histones are major mediators of multiple organ dysfunction syndrome in acute critical illnesses. Combination of retrospective clinical studies and animal models with intervention. ICU in a tertiary hospital and research laboratories. Four hundred and twenty ICU patients, including sepsis (140), severe trauma (63), severe pancreatitis (89), and other admission diagnoses (128). Cells from major organs are treated with calf thymus histones or histone-containing sera. Animal models for sepsis, trauma, and acute pancreatitis are treated with antihistone reagents. Antihistone reagents in in vitro, ex vivo, and animal models. Retrospective analysis of a prospectively recruited ICU cohort demonstrated a strong correlation between circulating histones and organ injury markers and Sequential Organ Failure Assessment scores. Ex vivo experiments showed that patient sera containing high histone levels were toxic to cultured cells from different origins, suggesting their universal toxicity to multiple organs. Animal models of sepsis, trauma, and pancreatitis further demonstrated a temporal correlation between histone levels and disease severity and multiple organ injury. Importantly, antihistone reagents, that is, antihistone single-chain variable fragment and nonanticoagulant heparin, could dramatically reduce multiple organ injury, particularly of the heart and lungs, and improve survival in mouse models. High levels of circulating histones are major mediators of multiple organ dysfunction syndrome. Our results indicate that monitoring upon ICU admission could inform on disease severity and developing antihistone therapy holds great potential of reducing multiple organ dysfunction syndrome and improving survival of critically ill patients.

Unfractionated heparin attenuates histone-mediated cytotoxicity in vitro and prevents intestinal microcirculatory dysfunction in histone-infused rats.

Extracellular histones are major mediators of organ dysfunction and death in sepsis, and they may cause microcirculatory dysfunction. Heparins have beneficial effects in sepsis and have been reported to bind to histones and neutralize their cytotoxicity. The aim of this study was to investigate the impact of histones on intestinal microcirculation and the intestinal endothelium and to discuss the protective effect of unfractionated heparin (UFH) on the endothelial cytotoxicity and microcirculatory dysfunction induced by histones. Anesthetized rats were infused with 30 mg/kg calf thymus histones, and UFH was administered intravenously at a concentration of 100 IU/kg per hour. The intestinal microcirculation was visualized and measured with incident dark field microscope. Plasma von Willebrand factor (vWF) and soluble thrombomodulin were detected, and structural changes in the rat intestinal microvascular endothelium were examined. The effects of histones and UFH on cell survival rates, vWF release and calcium influx were investigated in human intestinal microvascular endothelial cells (HIMECs). Histone infusion caused severe intestinal microcirculatory dysfunction in the absence of obvious hemodynamic changes, and UFH protected intestinal microcirculation in histone-infused rats. Concentrations of the plasma endothelial injury markers vWF and soluble thrombomodulin were elevated, and structural abnormalities were found in the intestinal microvascular endothelium in the histone-infused rats. These events were attenuated by UFH. In vitro, UFH significantly reduced the histone-induced cytotoxicity of HIMECs, reduced the release of vWF from the cytoplasm into the culture medium, and inhibited calcium influx into HIMECs. Histones induce intestinal microcirculatory dysfunction followed by direct injury to the endothelial cells; UFH protects the intestinal microcirculation partly by antagonizing the endothelial toxicity of histones.

Diiron Dithiolate Complex Induced Helical Structure of Histone and Application in Photochemical Hydrogen Generation.

Very-lysine-rich calf thymus histone proteins form disordered structure and hydrophobic interaction-driven aggregates in weakly acidic solution. We reported that the conjugation of diiron dithiolate complex to the lysine residues induced formation of helical conformation and condensed nanoassemblies with a high loading capacity up to 18.7 wt %. The incorporated diiron dithiolate complex showed photocatalytic activity for hydrogen evolution in aqueous solutions, with a turnover number (based on [FeFe] catalyst moiety) up to 359 that was more than 6 times that of the free catalyst. The increase of helical conformation in proteins was well correlated to the increasing enhancement of photocatalytic activity. We demonstrated that the [FeFe]-hydrogenase-mimic biohybrid system based on the photocatalyst-induced protein conformational conversion and reassembly is efficient for hydrogen generation regardless of the relatively large size.

Recombinant Thrombomodulin Reduce Histone-Induced Rat Lung Transplant Model of Primary Graft Dysfunction

Purpose In ISHLT 2018, we reported a rat lung transplant model of primary graft dysfunction (PGD) by histone administration. Recombinant thrombomodulin is clinically used antiinflammation and anticoagulation agent and previously reported that it reduces histone-induced endothelial damage and coagulation. We hypothesized that recombinant thrombomodulin administration reduced histone-induced PGD after lung transplantation. Methods Donor rats were randomly assigned to receive calf thymus histone (40 mg/kg) (Histone group), calf thymus histone (40 mg/kg) and recombinant thrombomodulin (3mg/kg) (Histone+rhTM group) or phosphate buffered saline (PBS) (control group). They were administered intravenously to the donor rats, then left lungs were explanted 3 hours after the administration. Immediately after the procurements of the donor lungs, orthotropic lung transplantations were performed. Donor and recipient rats were evaluated for lung functions and tissue damages by means of blood gas analysis, X-ray, real-time RT-PCR, plasma protein analysis and histopathological assessment. Results In the donor assessment, there were no significant differences in the blood gas analysis, chest X-ray nor histology between the groups. Gene expressions of inflammatory cytokines in lung tissue and plasma level of inflammation protein significantly increase in Histone group and Histone+rhTM group (figure A). However, there was marked worsening of gas exchanges in the Histone group after lung transplantation and recovery in Histone+rhTM group (figure B). Histopathologic evaluation revealed lung injury in Histone group and recovery in Histone+rhTM group (figure C). Radiograph taken after autopsy showed reduced radiolucency in Histone group and recovery in Histone+rhTM group (figure D). Conclusion Recombinant thrombomodulin administration reduced PGD like reaction in lung transplantation. It may indicate a possibility of novel treatment for PGD in lung transplantation.

The comparison of heparan sulfate and its fragments on the protection against extracellular histones during the pathogenesis of acute respiratory distress syndrome

Objective: In order to explore the role of heparan sulfate (HS) during the pathogenesis of acute respiratory distress syndrome (ARDS) , the protective effect of HS and its fragments against extracellular histones was compared. Methods: Calf thymus histones (CTH) were injected via femoral vein to induce ARDS in rats. HS, HS fragments or saline was intraperitoneally injected (10mg/kg, Q6h, 24h) to test the protective effect against CTH. The ratio of wet/dry lung weight, protein content in bronchoalveolar lavage fluid (BALF) , total leukocyte and neutrophil count in BALF were measured. Results: After CTH injection, the ratio of wet/dry lung weight (5.7±0.95) was much higher than the saline control group (3.1±0.15). The protein content (0.47±0.086mg/ml) , total leukocyte[ (97.4±15.6l) ×10(4)/ml] and neutrophil (18±3.4/LPF) in BALF were obviously increased compared with the saline control group. The intervention of HS evidently decreased ratio of wet/dry lung weight (4.2±0.41) , protein content[ (0.26±0.019) mg/ml], leukocyte[ (61.3±5.74) ×10(4)/ml] and neutrophil (12±1.8/LPF) in BALF. HS fragments also decreased ratio of wet/dry lung weight, protein content, leukocyte and neutrophil count in BALF though the strength was much less than HS. Conclusion: HS and its fragments could provide protection against extracellular histones during the pathogenesis of ARDS. For the protective effect full length HS was much better than HS fragments.

Cytotoxicity of extracellular histones on vascular endothelial cells and the underlying mechanisms in sepsis

Objective To investigate the change of extracellular histone level as well as the mechanism of the cytotoxicity of extracellular histones on vascular endothelial cell in sepsis. Methods Septic children admitted to PICU in Shanghai Children′s Medical Center between January 2010 and December 2014 were included in the present study.According to the diagnostic criteria of sepsis, the patients were divided into the sepsis group(51 cases) and the severe sepsis group(79 cases), with healthy children as the control group(108 cases). Patients in the severe sepsis group were further divided into the survival group(45 cases) and the non-survival group(34 cases) based on 28-day mortality.The plasma concentration of extracellular histones in these children was determined and its correlation with the severity of sepsis was analyzed.Human umbilical vein endothelial cells(HUVECs) were incubated with calf thymus histone(CTH) at various concentrations(0, 50, 100, 200 and 300 μg/ml) or different time periods(200 μg/ml, 0, 5, 15, 30, 45 and 60 minutes). The treated cells were subject to flow cytometer to measure the cell survival rate and scanning/transmission electron microscopy to observe their morphological changes.Western blot was used to detect the expression of IκB and phosphor-p38/p38 in nuclear factor(NF)-κB and mitogen-activated protein kinase(MAPK)signaling pathways, while ELISA was used to determine the levels of tumor necrosis factor-α and interleukin-6. Results The levels of circulating histones in the septic children(2.29±1.00) and severe septic children(19.17±10.20) were significantly higher than that of healthy controls(0.23±0.26)(P<0.001), and the histone levels in the severe septic children were even higher(P<0.001). Among the children diagnosed as severe sepsis, the level of circulating histones in the non-survivors was significantly higher than that in the survivors(29.47±5.99 vs.10.94±2.68, P<0.001). The survival rate of HUVEC gradually decreased along with the increase of CTH concentration or the treatment period in vitro.Data from electron microscopy showed that CTH treatment could directly disrupt the plasma membrane of HUVEC.Histones could also activate NF-κB and MAPK pathways, leading to the release of large amount of tumor necrosis factor-α and interleukin-6. Conclusion The levels of extracellular histones in the septic children are correlated with the severity of sepsis.CTH can induce HUVEC death in a dose-and time-dependent manner.Extracellular histone-induced endothelial dysfunction may mediate the progression of sepsis and such cytotoxicity might be due to the destruction of endothelial cell membranes and activation of the NF-κB and MAPK signaling pathways. Key words: Sepsis; Extracellular histones; Endothelial cells; Multiple organ dysfunction syndrome; Inflammatory mediators

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2–p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53‐mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.

Factor VII-Activating Protease Is a Regulator of Histone Cytotoxicity

Background: During sepsis, ischemia-reperfusion injury and sterile inflammation, levels of circulating histones and nucleosomes have been found to correlate with disease severity. Although the pathophysiological effects of circulating nucleosomes are not fully understood, free histones have been described to mediate cytotoxicity and contribute to disease severity. Several plasma proteins have been identified that modulate these cytotoxic effects by neutralizing or degrading histones. We have previously described that the plasma serine protease Factor VII-activating protease (FSAP) is activated upon contact with late apoptotic or necrotic cells and induces release of nucleosomes from these cells. During this release, histone H1 is cleaved. In addition, it has been described that FSAP is (auto)activated upon contact with histones and subsequently degrades histone H3.Aims: To investigate the regulation of histone-induced cytotoxicity by FSAP. Moreover, we aimed to compare differences in the cytotoxicity of histones and nucleosomes (histone+DNA) and potential differences in their proteolysis by FSAP.Methods: To determine FSAP activation and proteolysis of histones in serum we incubated normal donor or FSAP-deficient serum with 100ug/ml calf thymus histones. Upon its activation FSAP is rapidly bound by plasma inhibitors and we measured complexes of FSAP and its plasma inhibitor alpha 2-antiplasmin as an indirect readout for FSAP activation. To detect FSAP mediated proteolysis of histones we performed immunoblotting for histone H3. Results were corroborated using plasma-purified or recombinant FSAP. To determine histone-mediated cytotoxicity, we measured LDH levels after overnight incubation of HEK293 or ECRF cells with 100ug/ml calf thymus histone that had undergone a pre-incubation step in the presence or absence of healthy donor serum, FSAP-deficient serum or FSAP-depleted serum. Moreover, we tested the cytotoxicity of purified nucleosomes that were pre-incubated in the presence or absence of benzonase to degrade DNA.Results: EndogenousFSAP in serum was activated upon contact with histones, and histone proteolysis was observed. Similarly, histones were proteolyzed upon incubation using active plasma-purified or pre-activated recombinant FSAP, whereas no histone proteolysis was observed using recombinant inactive FSAP. These results indicate that FSAP is responsible for the observed histone degradation. FSAP-mediated histone degradation was able to protect HEK293 and ECRF cells against histone-induced cytotoxicity, showing that the resultant histone breakdown products had lost their cytotoxic effects. Recombinant inactive FSAP did not affect histone-induced cytotoxicity, indicating that the catalytic activity of FSAP is required for the observed protective effect of FSAP on cytotoxicity. When FSAP-deficient or FSAP-depleted serum was used, this led to greatly diminished histone proteolysis and reduced histone-induced cytotoxicity. Using our assay, we found that nucleosomes did not show cytotoxic effects unless they had been pre-incubated with benzonase to release histones. Notably, whereas intact nucleosomes were resistant against degradation by FSAP, FSAP protected against the cytotoxicity induced by histones released from these nucleosomes.Conclusion: FSAP in serum is activated by histones and subsequently proteolyses histones ,resulting in protection of cells from histone-induced cytotoxicity. Histones in the form of nucleosomes did not induce cytotoxicity, however after DNA removal the histones regained cytotoxicity. FSAP efficiently protected against the cytotoxicity of the unbound histones. We predict that the form in which histones are released into the blood is highly relevant in the context of human health and disease. We speculate that when endogenous DNases in serum degrade the DNA in nucleosomes, or alternatively, when highly cytotoxic histones are inadvertently released from dead cells in their free form rather than in the form of nucleosomes, FSAP mediates their efficient degradation to protect against the damaging effects of free histones. Neutralization of histones by FSAP may be an effective therapeutic approach to treat systemic inflammation. DisclosuresZeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

3-Deoxyglucosone: a potential glycating agent accountable for structural alteration in H3 histone protein through generation of different AGEs.

Advanced glycation end-products (AGEs) are heterogeneous group of compounds, known to be implicated in diabetic complications. One of the consequences of the Maillard reaction is attributed to the production of reactive intermediate products such as α-oxoaldehydes. 3-deoxyglucosone (3-DG), an α-oxoaldehyde has been found to be involved in accelerating vascular damage during diabetes. In the present study, calf thymus histone H3 was treated with 3-deoxyglucosone to investigate the generation of AGEs (Nε-carboxymethyllysine, pentosidine), by examining the degree of side chain modifications and formation of different intermediates and employing various physicochemical techniques. The results clearly indicate the formation of AGEs and structural changes upon glycation of H3 by 3-deoxyglucosone, which may hamper the normal functioning of H3 histone, that may compromise the veracity of chromatin structures and function in secondary complications of diabetes.