可溶性环氧化物水解酶在疾病中的作用

花生四烯酸经过细胞色素P450(cytochrome P450,CYP)表氧化酶途径生成环氧二十碳三烯酸(epoxy eicosatrienoic acid,EETs),具有扩张血管、降低血压、抗炎等多种生物学功能。在哺乳动物系统中的可溶性环氧化物水解酶（soluble epoxide hydrolase,sEH)具有α/β水解酶折叠结构,对环氧脂肪酸具有高度的选择性。sEH能够快速水解EETs,增加患心血管疾病的风险。目前,研究发现sEH抑制剂具有抑制sEH活性、提高EETs的含量的重要功能。 在多种疾病动物模型中应用sEH抑制剂或sEH基因敲除,证实sEH在心肌肥厚、糖尿病、高血压和肾病等疾病中发挥重要的生理作用。因此,sEH已被作为疾病治疗的新靶点而进行研究。本文就sEH的分布、作用机制以及sEH与疾病的关系等方面进行了讨论。     

定点突变提高D-氨甲酰水解酶的可溶性表达

采用定点突变的方法对皮氏伯克霍尔德氏菌(Burkholderia pickettii)来源的D-氨甲酰水解酶(D-carbamoylase,DCase)编码基因的3个位点A18、Y30、K34进行突变,并将获得的突变体基因片段构建入高表达载体pET-28b中,转化E coli BL21( DE3),获得带有组合三突变(A18E/Y30D/K34E)的DCase-SM表达菌株BL21/pET-DCSM.当以IPTG诱导目的蛋白表达时,发现突变菌株(DCase-SM)与出发菌株(DCase)菌株相比,目的蛋白的可溶性表达显著提高,其可溶蛋白比例约为64％;与出发菌株相比,其单位菌体酶活增加427％;另外,与本实验室前期构建的高可溶性三叠加突变体菌株DCase-M3相比,单位菌体酶活亦增加7.9％.     

益生菌胆盐水解酶的研究进展

近些年来,肠道微生物越来越多地与新陈代谢、健康和疾病联系在一起。胆盐水解酶（bile salt hydrolase,BSH）是一类由肠道微生物合成的,可以将胆汁酸盐水解成胆汁酸和盐。这种解耦联作用不仅能帮助人体吸收和利用营养物质,而且直接或间接地参与宿主和肠道微生物的多种生理活动。因此,胆盐水解酶在人体内发挥着不可替代的作用。本文简要介绍了胆盐水解酶,总结了胆盐水解酶的底物特异性,并就胆盐水解酶对宿主、益生菌和有害微生物的影响进行讨论。

     

突变型环酰亚胺水解酶的底物专一性

摘要 目的：研究环酰亚胺水解酶（CIH293）C-末端区残基对其底物专一性的影响。方法：通过缺失或替代获得了环酰亚胺水解酶C-末端剔除2个或3个氨基酸残基及C-末端两个Lys替代为两个Glu的突变型酶CIH291、CIH290以及KK292,293EE,用比色法与高效液相色谱法分析了重组野生型酶与突变型酶的底物专一性和动力学参数。结果：突变型酶与野生型酶相比,底物专一性未发生显著改变,最适底物仍为琥珀酰亚胺,然突变型酶对最适底物的亲和力略有降低,导致反应速度减小。结论：环酰亚胺水解酶（CIH293）C-末端区残基的改变对其底物专一性的影响不大,但影响了酶对底物的亲和力。     

新型冠状病毒3CL水解酶的结构特征及其抗原表位分析

新型冠状病毒(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)含有的3CLpro (3C-like protease)是由306个氨基酸构成的亲水性蛋白酶.3CLpro序列在冠状病毒中高度保守,对SARS-CoV-2的正常功能至关重要.为了深入了解3...     

根表铁锰氧化物胶膜对不同品种水稻吸镉的影响

采用土培方法,研究了不同品种水稻吸镉的差异及其与根表铁锰氧化物胶膜的关系,结果表明：不同品种水稻其根膜,根部及地上部含镉量均存在显著性差异,且镉在不同水稻植株体内运输转移能力不同,不同水稻其根表淀积的铁锰氧化物数量也存在显著性差异,根膜及地上部的含镉量与极膜的含铁量均未达到显著性相关,但与根膜的含锰量相关性显著。     

花生四烯酸代谢与肝脏糖脂代谢稳态调控

花生四烯酸(arachidonic acid, AA)是一种ω-6多不饱和脂肪酸,在生物体内主要是以磷脂的形式存在于细胞膜上。AA在细胞内主要通过环氧合酶(cyclooxygenase, COX)途径、脂氧合酶(lipoxygenases, LOX)途径、细胞色素P450单氧化酶(cytochrome P450 monooxygenase, CYP450)途径等进行代谢。糖脂代谢的稳态调控是维持机体基本生命活动的基础,肝脏是糖脂代谢调控的中枢器官。肝脏糖脂代谢紊乱与2型糖尿病、非酒精性脂肪性肝病等代谢性疾病的发生和发展密切相关。已有研究表明AA代谢与肝脏糖脂代谢紊乱有密切的关系。本文就AA代谢在肝脏糖脂代谢稳态调控中的作用及其作为脂肪肝和胰岛素抵抗等代谢性疾病治疗靶点的价值作一综述。     

小鼠可溶性ST2二级结构及B细胞表位的预测

目的:预测小鼠可溶性ST2的二级结构及B细胞表位。方法:采用SOPMA法预测小鼠可溶性ST2的二级结构;借助ProScale、Bcepred服务器,分析小鼠可溶性ST2的亲水性、可及性和柔韧性,并结合二级结构特征,预测小鼠可溶性ST2的B细胞表位。结果:小鼠可溶性ST2的二级结构由α螺旋(12.46%%)、无规卷曲(50.15%%)、β折叠(32.64%)和β转角(4.75%)组成。其B细胞表位可能位于N端第24～34、37～50、59～89、110～118、128～137、177～186、267～288和318～333氨基酸区段。结论:预测结果将有助于确定小鼠可溶性ST2的B细胞表位,为研制抗小鼠可溶性ST2抗体提供理论依据。     

表油菜素内酯对油菜幼苗生长及其可溶性糖和蛋白质含量的影响…

表油菜素内酯处理油菜幼苗,可明显促进下胚轴伸长生长,增加子叶面积,同时降低蛋白质含量及子叶中可溶性糖含量,ＳＤＳ－ＰＡＧＥ检测蛋白结果表明,ｅｐｉＢＲ处理后,下胚轴和子叶中的蛋白组分均发生明显的改变。     

乳酸菌胆盐水解酶和共轭脂肪酸产生及对宿主脂代谢影响的研究进展

乳酸菌是一类影响宿主脂代谢的人体肠道益生菌。乳酸菌对脂代谢的影响作用与其产生胆盐水解酶(bile salt hydrolase,EC3.5.1.24,BSH)及共轭转化多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)关系密切。菌株差异、菌群分布和饮食差异是影响BSH及共轭脂肪酸产生的重要因素。本文重点阐述了两类物质对宿主脂代谢的影响机制,以期为后续研究提供借鉴。BSH能够降解肝脏分泌的胆汁酸(bile acids,BAs),降低脂类物质的吸收。BAs的降解产物胆汁酸脱氧胆酸(deoxycholic acid,DCA)和石胆酸(lithocholic acid,LCA)能够通过机体信号通路法尼类X受体(farnesoid X receptor,FXR)、小异二聚体伴侣(small heterodimer partner,SHP)及肝脏X受体(liver X receptor,LXR)等信号通路进行调控,促进胆固醇转运及向BAs转化。此外,BSH还能够通过下调固醇调节元件结合蛋白1c (sterol regulatory element binding protein 1c,SREBP-1c)、上调5ʹ-腺苷单磷酸激活蛋白激酶α(5ʹ-AMP activated protein kinase,AMPKα)和过氧化物酶体增殖物激活受体α (peroxisome proliferator-activated receptor α,PPARα)抑制脂质合成,促进脂质的分解。PUFAs可被乳酸菌转化产生共轭脂肪酸,如共轭亚油酸(conjugated linoleic acid,CLA)和共轭亚麻酸(conjugated linolenic acid,CLNA),CLA/CLNA能够促进机体产生瘦素(leptin,LP),抑制食欲、促进能量消耗;CLA/CLNA还可以通过激活PPARα进行调控,促进人体脂质的氧化分解。乳酸菌通过以上多种途径共同作用调节宿主的脂代谢,对深入理解乳酸菌调控脂代谢机制及临床应用有着重要意义。     

Soluble epoxide hydrolase: Gene structure,expression and deletion

Mammalian soluble epoxide hydrolase (sEH) converts epoxides to their corresponding diols through the addition of a water molecule. sEH readily hydrolyzes lipid signaling molecules, including the epoxyeicosatrienoic acids (EETs), epoxidized lipids produced from arachidonic acid by the action of cytochrome p450s. Through its metabolism of the EETs and other lipid mediators, sEH contributes to the regulation of vascular tone, nociception, angiogenesis and the inflammatory response. Because of its central physiological role in disease states such as cardiac hypertrophy, diabetes, hypertension, and pain sEH is being investigated as a therapeutic target. This review begins with a brief introduction to sEH protein structure and function. sEH evolution and gene structure are then discussed before human small nucleotide polymorphisms and mammalian gene expression are described in the context of several disease models. The review ends with an overview of studies that have employed the sEH knockout mouse model.     

Soluble epoxide hydrolase inhibition, epoxygenated fatty acids and nociception

The soluble epoxide hydrolase (sEH) enzyme regulates the levels of endogenous epoxygenated fatty acid (EFA) lipid metabolites by rapidly degrading these molecules. The EFAs have pleiotropic biological activities including the modulation of nociceptive signaling. Recent findings indicate that the EFAs, in particular the arachidonic acid (AA) derived epoxyeicosatrienoic acids (EETs), the docosahexaenoic acid (DHA) derived epoxydocosapentaenoic acids (EpDPEs) and eicosapentaenoic acid (EPA) derived epoxyeicosatetraenoic acids (EpETEs) are natural signaling molecules. The tight regulation of these metabolites speaks to their importance in regulating biological functions. In the past several years work on EFAs in regard to their activities in the nervous system evolved to demonstrate that these molecules are anti-inflammatory and anti-nociceptive. Here we focus on the recent advances in understanding the effects of sEH inhibition and increased EFAs on the nociceptive system and their ability to reduce pain. Evidence of their role in modulating pain signaling is given by their direct application and by inhibiting their degradation in various models of pain. Moreover, there is mounting evidence of EFAs role in the crosstalk between major nociceptive and anti-nociceptive systems which is reviewed herein. Overall the fundamental knowledge generated within the past decade indicates that orally bioavailable small molecule inhibitors of sEH may find a place in the treatment of a number of diverse painful conditions including inflammatory and neuropathic pain.     

Soluble epoxide hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory effects of a soluble epoxide hydrolase inhibitor

Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow-derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 μM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts.     

Cytosolic epoxide hydrolase

Epoxide hydrolase activity is recovered in the high-speed supernatant fraction from the liver of all mammals so far examined, including man. For some as yet unexplained reason, the rat has a very low level of this activity, so that cytosolic epoxide hydrolase is generally studied in mice. This enzyme selectively hydrolyzes trans epoxides, thereby complementing the activity of microsomal epoxide hydrolase, for which cis epoxides are better substrates. Cytosolic epoxide hydrolase has been purified to homogeneity from the livers of mice, rabbits and humans. Certain of the physicochemical and enzymatic properties of the mouse enzyme have been thoroughly characterized. Neither the primary amino acid, cDNA nor gene sequences for this protein are yet known, but such characterization is presently in progress. Unlike microsomal epoxide hydrolase and most other enzymes involved in xenobiotic metabolism, cytosolic epoxide hydrolase is not induced by treatment of rodents with substances such as phenobarbital, 2-acetylaminofluorene, trans-stilbene oxide, or butylated hydroxyanisole. The only xenobiotics presently known to induce cytosolic epoxide hydrolase are substances which also cause peroxisome proliferation, e.g., clofibrate, nafenopin and phthalate esters. These and other observations indicate that this enzyme may actually be localized in peroxisomes in vivo and is recovered in the high-speed supernatant because of fragmentation of these fragile organelles during homogenization, i.e., recovery of this enzyme in the cytosolic fraction is an artefact. The functional significance of cytosolic epoxide hydrolase is still largely unknown. In addition to deactivating xenobiotic epoxides to which the organism is exposed directly or which are produced during xenobiotic metabolism, primarily by the cytochrome P-450 system, this enzyme may be involved in cellular defenses against oxidative stress.     

Soluble epoxide hydrolase activity determines the severity of ischemia-reperfusion injury in kidney

Soluble epoxide hydrolase (sEH) in endothelial cells determines the plasma concentrations of epoxyeicosatrienoic acids (EETs), which may act as vasoactive agents to control vascular tone. We hypothesized that the regulation of sEH activity may have a therapeutic value in preventing acute kidney injury by controlling the concentration of EETs. In this study, we therefore induced ischemia-reperfusion injury (IRI) in C57BL/6 mice and controlled sEH activity by intraperitoneal administration of the sEH inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA). The deterioration of kidney function induced by IRI was partially moderated and prevented by AUDA treatment. In addition, AUDA treatment significantly attenuated tubular necrosis induced by IRI. Ischemic injury induced the down-regulation of sEH, and AUDA administration had no effect on the expression pattern of sEH induced by IRI. In vivo sEH activity was assessed by measuring the substrate epoxyoctadecenoic acid (EpOME) and its metabolite dihydroxyoctadec-12-enoic acid (DHOME). Ischemic injury had no effects on the plasma concentrations of EpOME and DHOME, but inhibition of sEH by AUDA significantly increased plasma EpOME and the EpOME/DHOME ratio. The protective effect of the sEH inhibitor was achieved by suppression of proinflammatory cytokines and up-regulation of regulatory cytokines. AUDA treatment prevented the intrarenal infiltration of inflammatory cells, but promoted endothelial cell migration and neovascularization. The results of this study suggest that treatment with sEH inhibitors can reduce acute kidney injury.     

Role of epoxide hydrolases in lipid metabolism

Epoxide hydrolases (EH), enzymes present in all living organisms, transform epoxide-containing lipids to 1,2-diols by the addition of a molecule of water. Many of these oxygenated lipid substrates have potent biological activities: host defense, control of development, regulation of blood pressure, inflammation, and pain. In general, the bioactivity of these natural epoxides is significantly reduced upon metabolism to diols. Thus, through the regulation of the titer of lipid epoxides, EHs have important and diverse biological roles with profound effects on the physiological state of the host organism. This review will discuss the biological activity of key lipid epoxides in mammals. In addition, the use of EH specific inhibitors will be highlighted as possible therapeutic disease interventions.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.     

Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.     

Soluble epoxide hydrolase homologs in Strongylocentrotus purpuratus suggest a gene duplication event and subsequent divergence

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.