High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.     

Ubiquitinated Sirtuin 1 (SIRT1) Function Is Modulated during DNA Damage-induced Cell Death and Survival

Downstream signaling of physiological and pathological cell responses depends on post-translational modification such as ubiquitination. The mechanisms regulating downstream DNA damage response (DDR) signaling are not completely elucidated. Sirtuin 1 (SIRT1), the founding member of Class III histone deacetylases, regulates multiple steps in DDR and is closely associated with many physiological and pathological processes. However, the role of post-translational modification or ubiquitination of SIRT1 during DDR is unclear. We show that SIRT1 is dynamically and distinctly ubiquitinated in response to DNA damage. SIRT1 was ubiquitinated by the MDM2 E3 ligase in vitro and in vivo. SIRT1 ubiquitination under normal conditions had no effect on its enzymatic activity or rate of degradation; hypo-ubiquitination, however, reduced SIRT1 nuclear localization. Ubiquitination of SIRT1 affected its function in cell death and survival in response to DNA damage. Our results suggest that ubiquitination is required for SIRT1 function during DDR.     

Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan,HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation

During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W^sash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.     

Cholinergic anti-inflammatory pathway activity and High Mobility Group Box-1 (HMGB1) serum levels in patients with rheumatoid arthritis

High Mobility Group Box-1 (HMGB1) is a cytokine implicated in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. The cholinergic anti-inflammatory pathway, a vagus nerve-dependent mechanism, inhibits HMGB1 release in experimental disease models. Here, we examine the relationship between vagus nerve activity and HMGB1 in patients with RA. We compared RR interval variability, an index of cardiac vagal modulation, HMGB1 and hsCRP serum levels, and disease activity scores in thirteen RA patients and eleven age- and sex-matched controls. In RA patients, serum levels of HMGB1 and hsCRP were elevated as compared with controls (HMGB1=71 ng/mL [45-99] vs. 18 ng/mL [0-40], P<0.0001; hsCRP=14.5 mg/L [0.7-59] vs. 1 mg/L [0.4-2.9], P<0.001). RR interval variability in RA patients was significantly decreased as compared with controls (HF=38 msec2 [14-80] vs. 288 msec2 [38-364], P<0.0001; rMSSD=20.9+/-9.79 msec, 52.6+/-35.3 msec, P<0.01). HMGB1 levels and RR interval variability were significantly related (rho=-0.49, P<0.01). HMGB1 serum levels significantly correlated with disease activity scores (DAS-28) in patients with RA (P=0.004). The study design does not enable a determination of causality, but the results are consistent with the hypothesis that decreased cholinergic anti-inflammatory pathway activity is associated with increased HMGB1 levels in patients with RA.     

SPASM and Twitch Domains in S-Adenosylmethionine (SAM) Radical Enzymes

S-Adenosylmethionine (SAM, also known as AdoMet) radical enzymes use SAM and a [4Fe-4S] cluster to catalyze a diverse array of reactions. They adopt a partial triose-phosphate isomerase (TIM) barrel fold with N- and C-terminal extensions that tailor the structure of the enzyme to its specific function. One extension, termed a SPASM domain, binds two auxiliary [4Fe-4S] clusters and is present within peptide-modifying enzymes. The first structure of a SPASM-containing enzyme, anaerobic sulfatase-maturating enzyme (anSME), revealed unexpected similarities to two non-SPASM proteins, butirosin biosynthetic enzyme 2-deoxy-scyllo-inosamine dehydrogenase (BtrN) and molybdenum cofactor biosynthetic enzyme (MoaA). The latter two enzymes bind one auxiliary cluster and exhibit a partial SPASM motif, coined a Twitch domain. Here we review the structure and function of auxiliary cluster domains within the SAM radical enzyme superfamily.     

Structural Basis for Lack of ADP-ribosyltransferase Activity in Poly(ADP-ribose) Polymerase-13/Zinc Finger Antiviral Protein

The mammalian poly(ADP-ribose) polymerase (PARP) family includes ADP-ribosyltransferases with diphtheria toxin homology (ARTD). Most members have mono-ADP-ribosyltransferase activity. PARP13/ARTD13, also called zinc finger antiviral protein, has roles in viral immunity and microRNA-mediated stress responses. PARP13 features a divergent PARP homology domain missing a PARP consensus sequence motif; the domain has enigmatic functions and apparently lacks catalytic activity. We used x-ray crystallography, molecular dynamics simulations, and biochemical analyses to investigate the structural requirements for ADP-ribosyltransferase activity in human PARP13 and two of its functional partners in stress granules: PARP12/ARTD12, and PARP15/BAL3/ARTD7. The crystal structure of the PARP homology domain of PARP13 shows obstruction of the canonical active site, precluding NAD⁺ binding. Molecular dynamics simulations indicate that this closed cleft conformation is maintained in solution. Introducing consensus side chains in PARP13 did not result in 3-aminobenzamide binding, but in further closure of the site. Three-dimensional alignment of the PARP homology domains of PARP13, PARP12, and PARP15 illustrates placement of PARP13 residues that deviate from the PARP family consensus. Introducing either one of two of these side chains into the corresponding positions in PARP15 abolished PARP15 ADP-ribosyltransferase activity. Taken together, our results show that PARP13 lacks the structural requirements for ADP-ribosyltransferase activity.     

高迁移率族蛋白成员HMG20a/b的研究进展

高迁移率族(HMG)蛋白是一类广泛存在的非组蛋白型染色体蛋白,能通过诱导染色质结构的变化影响DNA表达。HMG20a和HMG20b是一对高度同源的HMG家族蛋白,均含有一个结构保守的HMG-box结构域和一个coiled-coil结构域,在生物体内广泛表达。HMG20a/b在细胞核内参与组蛋白去甲基酶复合物LSD1-Co REST的形成及一系列与细胞分裂分化相关的生理进程,如神经细胞核红细胞的分化、细胞质分裂以及EMT过程。研究发现,HMG20a/b一些功能的发挥是通过LSD1-Co REST复合物来实现的;在神经分化过程中,HMG20a、HMG20b具有相互拮抗的作用;而HMG20a促进EMT过程反映它很可能是一个促癌因子。本文对HMG20a/b的结构和体内分布及生物学功能进行综述。     

Extracellular High Mobility Group Box-1 (HMGB1) Inhibits Enterocyte Migration via Activation of Toll-like Receptor-4 and Increased Cell-Matrix Adhesiveness

Toll-like receptor-4 (TLR4) is the receptor for bacterial lipopolysaccharide, yet it may also respond to a variety of endogenous molecules. Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in newborn infants and is characterized by intestinal mucosal destruction and impaired enterocyte migration due to increased TLR4 signaling on enterocytes. The endogenous ligands for TLR4 that lead to impaired enterocyte migration remain unknown. High mobility group box-1 (HMGB1) is a DNA-binding protein that is released from injured cells during inflammation. We thus hypothesize that extracellular HMGB1 inhibits enterocyte migration via activation of TLR4 and sought to define the pathways involved. We now demonstrate that murine and human NEC are associated with increased intestinal HMGB1 expression, that serum HMGB1 is increased in murine NEC, and that HMGB1 inhibits enterocyte migration in vitro and in vivo in a TLR4-dependent manner. This finding was unique to enterocytes as HMGB1 enhanced migration of inflammatory cells in vitro and in vivo. In seeking to understand the mechanisms involved, TLR4-dependent HMGB1 signaling increased RhoA activation in enterocytes, increased phosphorylation of focal adhesion kinase, and increased phosphorylation of cofilin, resulting in increased stress fibers and focal adhesions. Using single cell force traction microscopy, the net effect of HMGB1 signaling was a TLR4-dependent increase in cell force adhesion, accounting for the impaired enterocyte migration. These findings demonstrate a novel pathway by which TLR4 activation by HMGB1 delays mucosal repair and suggest a novel potential therapeutic target in the amelioration of intestinal inflammatory diseases like NEC.     

Hepatic Apolipoprotein M (ApoM) Overexpression Stimulates Formation of Larger ApoM/Sphingosine 1-Phosphate-enriched Plasma High Density Lipoprotein

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3–5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.     

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

The components of the cellular protein translation machinery, such as ribosomal proteins and translation factors, are subject to numerous post-translational modifications. In particular, this group of proteins is frequently methylated. However, for the majority of these methylations, the responsible methyltransferases (MTases) remain unknown. The human FAM86A (family with sequence similarity 86) protein belongs to a recently identified family of protein MTases, and we here show that FAM86A catalyzes the trimethylation of eukaryotic elongation factor 2 (eEF2) on Lys-525. Moreover, we demonstrate that the Saccharomyces cerevisiae MTase Yjr129c, which displays sequence homology to FAM86A, is a functional FAM86A orthologue, modifying the corresponding residue (Lys-509) in yeast eEF2, both in vitro and in vivo. Finally, Yjr129c-deficient yeast cells displayed phenotypes related to eEF2 function (i.e. increased frameshifting during protein translation and hypersensitivity toward the eEF2-specific drug sordarin). In summary, the present study establishes the function of the previously uncharacterized MTases FAM86A and Yjr129c, demonstrating that these enzymes introduce a functionally important lysine methylation in eEF2. Based on the previous naming of similar enzymes, we have redubbed FAM86A and Yjr129c as eEF2-KMT and Efm3, respectively.     

Post-translational Regulation of Mitogen-activated Protein Kinase Phosphatase (MKP)-1 and MKP-2 in Macrophages Following Lipopolysaccharide Stimulation: THE ROLE OF THE C TERMINI OF THE PHOSPHATASES IN DETERMINING THEIR STABILITY*

MAPK phosphatases (MKPs) are critical modulators of the innate immune response, and yet the mechanisms regulating their accumulation remain poorly understood. In the present studies, we investigated the role of post-translational modification in the accumulation of MKP-1 and MKP-2 in macrophages following LPS stimulation. We found that upon LPS stimulation, MKP-1 and MKP-2 accumulated with different kinetics: MKP-1 level peaked at ∼1 h, while MKP-2 levels continued to rise for at least 6 h. Accumulation of both MKP-1 and MKP-2 were attenuated by inhibition of the ERK cascade. Interestingly, p38 inhibition prior to LPS stimulation had little effect on MKP-1 and MKP-2 protein levels, but hindered their detection by an M-18 MKP-1 antibody. Studies of the epitope sequence recognized by the M-18 MKP-1 antibody revealed extensive phosphorylation of two serine residues in the C terminus of both MKP-1 and MKP-2 by the ERK pathway. Remarkably, the stability of both MKP-1 and MKP-2 was markedly decreased in macrophages in the presence of an ERK pathway inhibitor. Mutation of the two C-terminal serine residues in MKP-1 and MKP-2 to alanine decreased their half-lives, while mutating these residues to aspartate dramatically increased their half-lives. Deletion of the C terminus from MKP-1 and MKP-2 also considerably increased their stabilities. Surprisingly, enhanced stabilities of the MKP-1 and MKP-2 mutants were not associated with decreased ubiquitination. Degradation of both MKP-1 and MKP-2 was attenuated by proteasomal inhibitors. Our studies suggest that MKP-1 and MKP-2 stability is regulated by ERK-mediated phosphorylation through a degradation pathway independent of polyubiquitination.     

Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels

Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or “free HCs” at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.     

高迁移率族蛋白1拮抗剂BoxA对细菌性脑膜炎大鼠的治疗作用

目的:探究高迁移率族蛋白1(HMGB1)拮抗剂BoxA尾静脉注射对细菌性脑膜炎(BM)大鼠模型的临床体征改善和炎症抑制作用。方法:除外正常对照的雄性Sprague-Dawley(SD)大鼠设为对照组(n=20),另取60只大鼠行脑室立体定向注射20μL大肠杆菌Escherichia coli (DH5α1×10~7CFU/m L)建立BM模型,之后随机分为两组(各组n=30),一组尾静脉注射HMGB1拮抗剂BoxA,即BoxA组;一组麻醉后进行尾静脉注射无菌磷酸盐(PBS),即Vehicle组。造模3 d后,对各组大鼠的临床指标以及病理生理参数(颅内压和脑脊液白细胞(WBC)计数)进行评估,使用酶联免疫吸附实验(ELISA)法检测血清中HMGB1的相对含量,使用伊文思蓝染色观察血脑屏障(BBB)通透性,使用免疫荧光染色检测大脑皮层炎症因子(IL-1β和TNF-α)的表达水平。结果:相比Control组,Vehicle组临床指标,颅内压,WBC计数以及血清HMGB1含量明显提升(P0.05);而BoxA组相比Vehicle组,以上改变有部分减少(P0.05)。另外,Vehicle组较Control组EB渗漏增加且炎症因子(IL-1β和TNF-α)表达水平增高(P0.05)。与Vehicle组相比,BoxA组的这些变化亦被部分调节(P0.05)。结论:HMGB1抑制剂BoxA尾静脉注射能够下调HMGB1表达水平并同时缓解细菌性脑膜炎大鼠的临床症状和炎症反应。     

Phosphorylation of TET Proteins Is Regulated via O-GlcNAcylation by the O-Linked N-Acetylglucosamine Transferase (OGT)

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.     

Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations

Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2^n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2^n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia.