可溶性环氧化物水解酶抑制剂对脂肪细胞胆固醇流出的影响

目的 观察可溶性环氧化合物水解酶抑制剂(sEHi)tAUCB对脂肪细胞胆固醇流出的影响,并探讨其机制.方法 测定脂肪细胞过氧化物酶体增殖物激活受体γ(PPARγ)及三磷酸腺苷结合盒转运体A1(ABCA1)mRNA及蛋白的表达,检测细胞内胆固醇流出.结果 tAUCB呈剂量依赖性促进载脂蛋白(Apo)A1介导的胆固醇流出,1、10、50、100 μmol/L浓度的tAUCB干预后,胆固醇流出率分别为(5.93±0.66)%,(7.40±0.43)%,(8.30±0.34)%和(9.77±0.42)%.加入10～100μmol/L tAUCB干预组与空白组(5.67±0.17)%比较,胆固醇流出差异有统计学意义(P＜0.05).同时发现随着tAUCB干预浓度的增加,脂肪细胞ABCA1、PPARy mRNA及蛋白的表达也呈剂量依赖性地升高,而GW9662明显抑制tAUCB对脂肪细胞胆固醇流出及ABCA1和PPARγ表达的促进作用.结论 tAUCB可通过上调PPARy的表达,促进脂肪细胞Apo A1-ABCA1通路加速细胞内胆固醇流出,抑制脂肪细胞内胆固醇过度蓄积.

Abstract：

Objective To observe the effects of soluble epoxide hydrolase inhibitors tAUCB on cholesterol efflux in adipocytes. Methods 3T3-L1 preadipocytes were induced to differentiation and maturation. Cells were stimilated with 100μg/L LPS after starved for 24 hours, then tAUCB in various concentrations(1 ,10,50,100 μmol/L)were added for 24 h, or incubated with the peroxisome proliferator activated receptor gamma (PPARy) antagonist GW9662 (5 μmol/L).0μmol/L tAUCB treated group was taken as empty control. After then, the mRNA expression of PPARγ and adenosine triphosphate binding cassette transporter Al (ABCA1) in cells were determined via realtime-PCR, the amounts of protein expression of PPARγand ABCA1 in cells were detected by Western blot, the efflux rates of 3H-cholesterol in cells were detected by means of liquid scintillation counter. Results tAUCB could dose-dependently increase the apolipoprotein A1 (apoA1)-mediated cholesterol efflux in adipocytes. After stimulated by 1, 10,50,100 μmol/L tAUCB, cholesterol efflux rates were (5.93±0.66) %, (7.40 ± 0. 43) %, (8. 30 ±0. 34)% ,(9.77±0.42)% respectively, there were significant difference after treated by 10-100 μmol/L tAUCB compared with control(5.67±0.17)%(P＜0.05). With the concentration of tAUCB increased,ABCA1, PPAR mRNA and protein expression were also dose-dependently up-regulated. GW9662 could significantly inhibit the effects of tAUCB, and then reduce the cholesterol efflux and the expression of PPARγ and ABCA1 in adipocytes. Conclusions tAUCB could up-regulate PPARγ expression in adipocytes, and promote the cholesterol efflux of adipocytes via apoA1-ABCA1 pathway, which might decrease the cellular cholesterol accumulation in adipocytes.     

RNA干扰下调可溶性环氧化物水解酶表达对异丙肾上腺素诱导心肌细胞凋亡的影响

目的:构建含有可溶性环氧化物水解酶(sEH)基因特异性的RNA干扰序列质粒,选择性下调小鼠心肌细胞sEH的表达,筛选出抑制sEH效果最明显的表达质粒;观察其对异丙基肾上腺素(ISO)诱导的心肌细胞的凋亡及相关基因的影响.方法:构建2条靶向sEH基因的特异性小干扰RNA(siRNA)质粒 EH-1和 EH-2,以含非特异性siRNA编码序列的质粒(PCN)为阴性对照,用FuGENE HD转染原代培养的心肌细胞,通过半定量RT-PCR和Western blot法检测sEH的mRNA和蛋白的表达情况,筛选出抑制sEH效果最明显的表达质粒,命名为EH-R.采用浓度为10μmol*L-1ISO诱导心肌凋亡.分为正常对照组、ISO组、PCN+ISO组和EH-R+ISO组.利用EH-R下调心肌细胞sEH基因的表达,观察其对ISO诱导的心肌细胞凋亡的影响,流式细胞仪检测各组细胞凋亡的发生率,Western blot法检测各组Bcl-2和Bax蛋白的表达变化.结果:质粒EH-R组心肌细胞sEH的mRNA和蛋白质表达较其余组明显降低,差异有统计学意义(P<0.01).与正常对照组相比,应用ISO各组,心肌细胞凋亡率明显升高,心肌细胞的Bax表达升高,而Bcl-2表达下降(P<0.01).然而EH-R+ISO组与ISO组和PCN+ISO组相比心肌细胞凋亡率明显降低;心肌Bax表达降低,Bcl-2表达升高(P<0.01).结论:构建了特异性小干扰RNA质粒,利用RNAi技术成功下调了原代培养的心肌细胞中sEH的表达,从而增加了抑凋亡基因Bcl-2的表达,减轻了ISO诱导心肌细胞的凋亡.为进一步进行心肌细胞的RNA干扰研究奠定了基础.     

中国北方汉族人微粒体环氧化物水解酶基因多态性与慢性阻塞性肺疾病易感性的关系

目的 探讨中国北方汉族人微粒体环氧化物水解酶基因多态性与慢性阻塞性肺疾病（COPD）易感性的关系。方法 应用聚合酶链反应限制性片段长度多态性法,检测微粒体环氧化物水解酶基因型在55例中国北方汉族人吸烟COPD患者和52例健康吸烟者中频率的分布。结果 55斧正吸烟COPD患者微粒体环氧化物水解酶外显子3的同源野生型,杂合型,同源突变型频率分布分别为27.3%(15/55)、27.3%(15/55),45.5%(25/55）,外显子4的同源野生型,杂合型,同源突变型频率分布分别为72.7%(40/55),18.2%(10/55),9.1%(5/55),与52例健康吸烟者的分布比较差异无显著性。结论 微粒体环氧化物水解酶基因与中国北方汉族人COPD易感性无关。     

Association between polymorphisms of microsomal epoxide hydrolase and COPD: results from meta-analyses

Background and objective: COPD is a complex polygenic disease in which gene–environment interactions are very important. The gene encoding microsomal epoxide hydrolase (EPHX1) is one of several candidate loci for COPD pathogenesis and is highly polymorphic. Based χ on the polymorphisms of EPHX1 gene (tyrosine/histidine 113, histidine/arginine 139), the population can be classified into four groups of putative EPHX1 phenotypes (fast, normal, slow and very slow). A number of studies have investigated the association between the genotypes and phenotypes of EPHX1 and COPD susceptibility in different populations, with inconsistent results. A systematic review and meta‐analysis of the published data was performed to gain a clearer understanding of this association. Methods: The MEDLINE database was searched for case–control studies published from 1966 to August 2007. Data were extracted and pooled odds ratios (OR) with 95% confidence intervals (CI) were calculated. Results: Sixteen eligible studies, comprising 1847 patients with COPD and 2455 controls, were included in the meta‐analysis. The pooled result showed that the EPHX1 113 mutant homozygote was significantly associated with an increased risk of COPD (OR 1.59, 95% CI: 1.14–2.21). Subgroup analysis supported the result in the Asian population, but not in the Caucasian population. When the analysis was limited to only the larger‐sample‐size studies, studies in which controls were in Hardy–Weinberg equilibrium and studies in which controls were smokers/ex‐smokers, the pooled results supported the conclusion. The EPHX1 139 heterozygote protected against the development of COPD in the Asian population, but not in the Caucasian population. The other gene types of EPHX1 113 and EPHX1 139 were not associated with an increased risk of COPD. The slow activity phenotype of EPHX1 was associated with an increased risk of COPD. The fast activity phenotype of EPHX1 was a protective factor for developing COPD in the Asian population, but not in the Caucasian population. However, the very slow activity phenotype of EPHX1 was a risk for developing COPD in the Caucasian population, but not in the Asian population. Conclusions: The polymorphisms of EPHX1 113 and EPHX1 139 are genetic contributors to COPD susceptibility in Asian populations. The phenotypes of EPHX1 were contributors to overall COPD susceptibility.     

Gene deficiency and pharmacological inhibition of soluble epoxide hydrolase confers resilience to repeated social defeat stress

Depression is a severe and chronic psychiatric disease, affecting 350 million subjects worldwide. Although multiple antidepressants have been used in the treatment of depressive symptoms, their beneficial effects are limited. The soluble epoxide hydrolase (sEH) plays a key role in the inflammation that is involved in depression. Thus, we examined here the role of sEH in depression. In both inflammation and social defeat stress models of depression, a potent sEH inhibitor, TPPU, displayed rapid antidepressant effects. Expression of sEH protein in the brain from chronically stressed (susceptible) mice was higher than of control mice. Furthermore, expression of sEH protein in postmortem brain samples of patients with psychiatric diseases, including depression, bipolar disorder, and schizophrenia, was higher than controls. This finding suggests that increased sEH levels might be involved in the pathogenesis of certain psychiatric diseases. In support of this hypothesis, pretreatment with TPPU prevented the onset of depression-like behaviors after inflammation or repeated social defeat stress. Moreover, sEH KO mice did not show depression-like behavior after repeated social defeat stress, suggesting stress resilience. The sEH KO mice showed increased brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor TrkB in the prefrontal cortex, hippocampus, but not nucleus accumbens, suggesting that increased BDNF-TrkB signaling in the prefrontal cortex and hippocampus confer stress resilience. All of these findings suggest that sEH plays a key role in the pathophysiology of depression, and that epoxy fatty acids, their mimics, as well as sEH inhibitors could be potential therapeutic or prophylactic drugs for depression.Depression is the most severe and debilitating of the psychiatric illnesses. The World Health Organization estimates that more than 350 million individuals of all ages suffer from depression (1). Almost one million lives are lost annually because of suicide, which translates to 3,000 deaths daily (1). Although antidepressants are generally effective in the treatment of depression, it can still take weeks before patients feel the full antidepressant effects. However, approximately two-thirds of depressed patients fail to respond fully to pharmacotherapy. Furthermore, there is a high rate of relapse, and depressed patients have a high risk of committing suicide (2–4).Accumulating evidence suggests that inflammation plays a central role in the pathophysiology of depression (5–9). Meta-analyses showed higher blood levels of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6), in drug-free depressed patients compared with healthy controls (10–13). Studies using postmortem brain samples showed elevated gene expression of proinflammatory cytokines in the frontal cortex of people with a history of depression (14, 15). Taking these data together, we find that it is likely that both peripheral and central inflammations are associated with depression and that antiinflammatory drugs, such as cyclooxygenase inhibitors, could ameliorate depressive symptoms in depressed patients (16, 17).Epoxyeicosatrienoic acids (EETs), which are produced from arachidonic acid by the action of cytochrome P450s, have potent antiinflammatory actions. These mediators are broken down into the corresponding diols by soluble epoxide hydrolase (sEH), and inhibition of sEH enhances the beneficial effects of EETs (18–21). It is also reported that sEH inhibitors have potent antiinflammatory effects in a number of animal models (18–20, 22, 23). Although sEH has been associated with the onset of anorexia nervosa (24), the role of sEH in the pathophysiology of depression has not been studied to date.The purpose of this study was to examine the role of sEH in the pathophysiology of depression using a potent sEH inhibitor and sEH knockout (KO) mice. Furthermore, we examined the role of brain-derived neurotrophic factor (BDNF) and its receptor TrkB signaling in selected brain regions, because BDNF-TrkB signaling plays a key role in the pathophysiology of depression (25–30).     

Variations in the human soluble epoxide hydrolase gene and recurrence of atrial fibrillation after catheter ablation

Background

Single nucleotide polymorphisms (SNPs) of EPHX2 alter sEH activity and are associated with increased [rs41507953 (K55R)] or reduced [rs751141 (R287Q)] cardiovascular risk via modulation of fibrosis, inflammation or cardiac ion channels. This indicates an effect on development and therapy response of AF. This study tested the hypothesis that variations in the EPHX2 gene encoding human soluble epoxide hydrolase (sEH) are associated with atrial fibrillation (AF) and recurrence of atrial fibrillation after catheter ablation.

Methods and results

A total of 218 consecutive patients who underwent catheter ablation for drug refractory AF and 268 controls were included. Two SNPs, rs41507953 and rs751141, were genotyped by direct sequencing. In the ablation group, holter recordings 3, 12 and 24 months after ablation were used to detect AF recurrence. No significant association of the SNPs and AF at baseline was detected. In the ablation group, recurrence of AF occurred in 20% of the patients 12 months after ablation and in 35% 24 months after ablation. The presence of the rs751141 polymorphism significantly increased the risk of AF recurrence 12 months (odds ratio [OR]: 3.2, 95% confidence interval [CI]: 1.237 to 8.276, p = 0.016) and 24 months (OR: 6.076, 95% CI: 2.244 to 16.451, p < 0.0001) after catheter ablation.

Conclusions

The presence of rs751141 polymorphism is associated with a significantly increased risk of AF recurrence after catheter ablation. These results point to stratification of catheter ablation by genotype and differential use of sEH-inhibitory drugs in the future.     

Evaluation of the effects of urotensin II and soluble epoxide hydrolase inhibitor on skin microvessel tone in healthy controls and heart failure patients

Introduction: Urotensin II (UII) is a potent vasoactive peptide that exerts differential effects on heart failure (HF) patients compared to health controls. However, the mechanism of action remains unclear. The role of soluble epoxide hydrolase (sEH) as a mediator of UII in the vasculature has not been explored. Aims: The aim of this study was to examine the effect of UII in the presence and absence of sEH inhibitor AUDA on skin microvessel tone in HF patients and healthy controls using iontophoresis and laser Doppler velocimetry. UII (10^?7 M) and AUDA (10^?10, 10^?7, and 10^?5 M) were administered to the forearm of participants by iontophoresis for 30 seconds. Laser Doppler velocimetry was performed for 5 minutes to measure flux through the subcutaneous blood vessels. Response (flux) was measured for 5 minutes per concentration with 25 continuous scans. Results: UII increased flux in healthy controls by 39% (P < 0.05) and increased flux in HF patients by 6% (ns). AUDA (10^?10 and 10^?7 M) administration further decreased flux by 115% (P < 0.05) and 255% (P < 0.0001), respectively in healthy controls. In HF patients, AUDA (10^?10, 10^?7, and 10^?5 M) further increased flux by 77% (P < 0.05), 67% (P < 0.01), and 100% (P < 0.05), respectively. AUDA alone at 10^?7 M increased flux in both groups by 31% (healthy controls, P < 0.05) and 36% (HF, P < 0.01). Conclusion: Taken together, the presence of HF appeared to abrogate the vasodilator responsiveness of sEH inhibitor. These results suggest an important role for both UII and sEH in vascular regulation and that sEH may be involved in mediating UII effects. Furthermore, the study highlights the therapeutic potential of sEH inhibitors for the treatment of HF.     

Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A₄ hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB₄. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA₄ hydrolase. Here, we determined the crystal structure of LTA₄ hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA₄ hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB₄ synthesis in human neutrophils (IC₅₀ of ∼0.5 μM) and conversion of LTA₄ into LTB₄ by purified LTA4H with a K_i of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA₄ hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA₄, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA₄ into LTB₄, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA₄ hydrolase inhibitor that holds promise for improved anti-inflammatory properties.Leukotriene (LT) A₄ hydrolase/aminopeptidase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the formation of the potent chemotactic agent LTB₄, a key lipid mediator in the innate immune response (1, 2). Previous work has shown that LTA₄ hydrolase (LTA4H) is an aminopeptidase with high affinity for N-terminal arginines of various synthetic tripeptides (3, 4). The two enzyme activities of LTA4H are exerted via distinct but overlapping active sites and depend on the catalytic zinc, bound within the signature HEXXH-(X)₁₈-E, typical of M1 metallopeptidases (5–7). In LTA4H, His295, His299, and Glu318 are the zinc-binding ligands, whereas Glu296 is the general base catalyst for peptide hydrolysis (8, 9).LTA4H’s crystal structure has been determined (10). The enzyme folds into an N-terminal domain, a catalytic domain, and a C-terminal domain, each with ∼200 amino acids. The interface of the domains forms a cavity, where the active site is located (Fig. 1). The cavity narrows at the zinc-binding site, forming a tunnel into the catalytic domain. The opening and wider parts of the cavity are highly polar; the tunnel is more hydrophobic. The cavity is mostly defined by the catalytic and C-terminal domains; part of the tunnel is defined by the N-terminal domain. Bound substrate is in contact with all three domains.Open in a separate windowFig. 1.Position and extension of the active center in LTA4H. Cartoon representation of the structure of LTA4H with a tunnel for LTA₄ (red mesh) and peptide substrates (blue mesh). The catalytic zinc (yellow sphere) is located in a wide section of the active site from which a narrow, L-shaped, hydrophobic tunnel protrudes ∼15 Å deeper into the protein. LTA₄ is believed to bind with its ω-end at the end of the hydrophobic tunnel. The volume of the active center was calculated in CAVER (31).Recently, it was discovered that LTA4H cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro, thus identifying a previously unrecognized endogenous, physiologically significant aminopeptidase substrate (11). Inasmuch as Pro-Gly-Pro attracts neutrophils and promotes inflammation, these data also suggest that LTA4H plays dual and opposite roles during an inflammatory response (i.e., production of chemotactic LTB₄, as well as inactivation of chemotactic Pro-Gly-Pro). Previous efforts to develop inhibitors of LTA4H have used the aminopeptidase activity for screening purposes, and these molecules therefore block both catalytic activities of LTA4H (12).Here, we used crystallography, MS, and a stable peptide analog to determine the binding mode of Pro-Gly-Pro at the active site of LTA4H, as well as the mechanism of peptide cleavage. Based on the structure, we also designed a lead compound that selectively blocks the conversion of LTA₄ into LTB₄, although sparing the hydrolysis of Pro-Gly-Pro.     

Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: Role for omega-3 epoxides

Soluble epoxide hydrolase (sEH) is an emerging therapeutic target in a number of diseases that have inflammation as a common underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxides derived from omega-6 and omega-3 polyunsaturated fatty acids (PUFA) by converting these antiinflammatory mediators into their less active diols. Here, we explored the metabolic effects of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression of an omega-3 desaturase capable of enriching tissues with endogenous omega-3 PUFA. These mice exhibited increased CYP1A1, CYP2E1, and CYP2U1 expression and abundant levels of the omega-3–derived epoxides 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin-sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP levels and reinforced the omega-3–dependent reduction observed in tissue inflammation and lipid peroxidation. t-TUCB also produced a more intense antisteatotic action in obese fat-1 mice, as revealed by magnetic resonance spectroscopy. Notably, t-TUCB skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose tissue volume. Moreover, t-TUCB restored hepatic levels of Atg12-Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced endoplasmic reticulum stress demonstrated by the attenuation of IRE-1α and eIF2α phosphorylation. These actions were recapitulated in vitro in palmitate-primed hepatocytes and adipocytes incubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less pronounced actions were observed with the omega-6 epoxide, 14,15-EET, and nonoxidized DHA. Together, these findings identify omega-3 epoxides as important regulators of inflammation and autophagy in insulin-sensitive tissues and postulate sEH as a druggable target in metabolic diseases.Cytochrome P450 (CYP) epoxygenases represent the third branch of polyunsaturated fatty acid (PUFA) metabolism (1). CYP epoxygenases add oxygen across one of the four double bonds of PUFA to generate three-membered ethers known as epoxides (1). In the case of arachidonic acid, CYP epoxygenases convert this omega-6 PUFA into epoxyeicosatrienoic acids (EETs), which act as autocrine or paracrine factors in the regulation of vascular tone, inflammation, hyperalgesia, and organ and tissue regeneration (2, 3). In addition to omega-6s, CYP epoxygenases also convert the omega-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into novel epoxyeicosatetraenoic (EEQs) and epoxydocosapentaenoic (EDPs) acids, respectively (4, 5). These omega-3–derived epoxides also exert salutary actions and are even more effective and potent than omega-6–derived EETs (4–8).Because the predicted in vivo half-lives of fatty acid epoxides (EpFA) are in the order of seconds (9), drugs that stabilize their levels by targeting the enzyme soluble epoxide hydrolase (sEH) are currently under investigation. sEH is a cytosolic enzyme with epoxide hydrolase and lipid phosphatase activities that catalyzes the rapid hydrolysis of EETs, EEQs and EDPs by adding water to these EpFA and converting them into inactive or less active 1,2-diols (10). Accordingly, inhibition of sEH exerts beneficial actions in controlling vascular tone, inflammation, and pain, and this strategy has shown its therapeutic potential for long-term use in hypertension, diabetes, renal disease, organ damage, and vascular remodeling (6, 9–12).The aim of the present study was to investigate the potential metabolic benefits of sEH inhibition in obesity. Specifically, this study addresses the question as to whether sEH inhibition increases the effectiveness of omega-3–derived epoxides in obese adipose tissue and liver in the context of enriched omega-3 tissue content. fat-1 mice with transgenic expression of the Caenorhabditis elegans omega-3 fatty acid desaturase gene represent a useful model to address this question because these mice have abundant tissue omega-3 distribution from their embryonic stage and throughout their lives (13, 14). This study builds on previous work by our laboratory demonstrating that fat-1 mice replicate the protection against insulin resistance and hepatic inflammation and steatosis observed in obese mice nutritionally enriched with exogenous omega-3 PUFA (13, 15). The results of the present investigation indicate that inhibition of sEH when there is an increased content of omega-3 PUFA exerts a more favorable role in counteracting the metabolic disorders associated with obesity. In addition, our findings expand focus to include EpFA to the protective actions described for those lipid mediators derived from omega-3s through lipoxygenase- and cycloxygenase-initiated pathways (i.e., resolvins, protectins, and maresins) (16, 17).     

Cytochrome P450 epoxygenases, soluble epoxide hydrolase, and the regulation of cardiovascular inflammation

The cytochrome P450 (CYP) epoxygenase enzymes CYP2J and CYP2C catalyze the epoxidation of arachidonic acid to epoxyeicosatrienoic acids (EETs), which are rapidly hydrolyzed to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). It is well-established that CYP epoxygenase-derived EETs possess potent vasodilatory effects; however, the cellular effects of EETs and their regulation of various inflammatory processes have become increasingly appreciated in recent years, suggesting that the role of this pathway in the cardiovascular system extends beyond the maintenance of vascular tone. In particular, CYP epoxygenase-derived EETs inhibit endothelial activation and leukocyte adhesion via attenuation of nuclear factor-kappaB activation, inhibit hemostasis, protect against myocardial ischemia-reperfusion injury, and promote endothelial cell survival via modulation of multiple cell signaling pathways. Thus, the CYP epoxygenase pathway is an emerging target for pharmacological manipulation to enhance the cardiovascular protective effects of EETs. This review will focus on the role of the CYP epoxygenase pathway in the regulation of cardiovascular inflammation and (1) describe the functional impact of CYP epoxygenase-derived EET biosynthesis and sEH-mediated EET hydrolysis on key inflammatory process in the cardiovascular system, (2) discuss the potential relevance of this pathway to pathogenesis and treatment of cardiovascular disease, and (3) identify areas for future research.     

Role of epoxide hydrolase 1 gene polymorphisms in esophageal cancer in a high‐risk area in India

Background and Aim: Microsomal epoxide hydrolase 1 (EPHX1) is involved in the metabolism of environmental and tobacco carcinogens. Tobacco smoking, betel quid chewing, and alcohol consumption are the major known risk factors for esophageal cancer. The present case‐control study evaluated the influence of EPHX1 genetic variations on esophageal cancer susceptibility in 142 patients and 185 healthy controls from a high‐incidence region of India where tobacco use and alcohol consumption are widespread and the users of these two substances are also betel quid chewers. Methods: EPHX1 polymorphic alleles (exon 3, Tyr113His and exon 4, His139Arg) were genotyped by polymerase chain reaction–restriction fragment length polymorphism method and direct sequencing. The results were analyzed using logistic regression to calculate odds ratios (OR) and confidence intervals (CI). Results: Patients with exon 4 genotypes (139His/Arg, 139Arg/Arg) and the 139Arg allele were significantly associated with a risk of esophageal cancer (OR_His139Arg 1.887, 95% CI = 1.112–3.201, P = 0.019; OR_Arg139Arg 7.140, 95% CI = 1.276–393.953, P = 0.025 and OR_Arg 1.83, 95% CI = 1.19–2.82, P = 0.003). The 139His/Arg genotype was a significant risk factor for esophageal cancer in tobacco chewers and betel quid chewers. Patients with the 139Arg/Arg genotype were at significantly higher risk for developing a well‐differentiated and moderately‐differentiated grade of tumor. In contrast, the 113His/His genotype of exon 3 was a significant protective factor for esophageal cancer in tobacco smokers (OR 0.291, 95% CI = 0.138–0.616, P = 0.001), betel quid chewers (OR 0.434, 95% CI = 0.236–0.797, P = 0.007), and alcohol users. Conclusion: EPHX1 exon 4 139His/Arg, and 139Arg/Arg genotypes were associated with a higher risk of esophageal cancer in a high‐risk area of India.     

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates

Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.     

Development of a generic adenovirus delivery system based on structure-guided design of bispecific trimeric DARPin adapters

Adenoviruses (Ads) have shown promise as vectors for gene delivery in clinical trials. Efficient viral targeting to a tissue of choice requires both ablation of the virus’ original tropism and engineering of an efficient receptor-mediated uptake by a specific cell population. We have developed a series of adapters binding to the virus with such high affinity that they remain fully bound for >10 d, block its natural receptor binding site and mediate interaction with a surface receptor of choice. The adapter contains two fused modules, both consisting of designed ankyrin repeat proteins (DARPins), one binding to the fiber knob of adenovirus serotype 5 and the other binding to various tumor markers. By solving the crystal structure of the complex of the trimeric knob with three bound DARPins at 1.95-Å resolution, we could use computer modeling to design a link to a trimeric protein of extraordinary kinetic stability, the capsid protein SHP from the lambdoid phage 21. We arrived at a module which binds the knob like a trimeric clamp. When this clamp was fused with DARPins of varying specificities, it enabled adenovirus serotype 5-mediated delivery of a transgene in a human epidermal growth factor receptor 2-, epidermal growth factor receptor-, or epithelial cell adhesion molecule-dependent manner with transduction efficiencies comparable to or even exceeding those of Ad itself. With these adapters, efficiently produced in Escherichia coli, Ad can be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.     

Soluble epoxide hydrolase: A potential target for metabolic diseases

Epoxyeicosatrienoic acids (EETs), important lipid mediators derived from arachidonic acid, have many beneficial effects in metabolic diseases, including atherosclerosis, hypertension, cardiac hypertrophy, diabetes, non‐alcoholic fatty liver disease, and kidney disease. Epoxyeicosatrienoic acids can be further hydrolyzed to less active diols by the enzyme soluble epoxide hydrolase (sEH). Increasing evidence suggests that inhibition of sEH increases levels of EETs, which have anti‐inflammatory effects and can prevent the development of hypertension, atherosclerosis, heart failure, fatty liver, and multiple organ fibrosis. Arachidonic acid is the most abundant omega‐6 polyunsaturated fatty acid (PUFA) and shares the same set of enzymes with omega‐3 PUFAs, such as docosahexaenoic acid and eicosapentaenoic acid. The omega‐3 PUFAs and metabolites, such as regioisomeric epoxyeicosatetraenoic acids and epoxydocosapentaenoic acids, have been reported to have strong vasodilatory and anti‐inflammatory effects. Therefore, sEH may be a potential therapeutic target for metabolic disorders. In this review, we focus on our and other recent studies of the functions of sEH, including the effects of its eicosanoid products from both omega‐3 and omega‐6 PUFAs, in various metabolic diseases. We also discuss the possible cellular and molecular mechanisms underlying the regulation of sEH.     

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein–protein interactions involved in cellular sorting of AQP2. Two Cd²⁺-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.Water is the major ingredient of the human body, constituting 55–65% of our total body weight (1). Water homeostasis is maintained by the kidneys, which filter ∼180 L of primary urine every day. Although most water is constitutively reabsorbed in the proximal tubules and descending limbs of Henle (2), the body’s water balance is fine-tuned by regulated water reabsorption, which takes place in the kidney collecting duct. Water reabsorption is mediated by aquaporins, membrane-bound water channels, of which seven of the 13 human isoforms have been located in the human kidney (3). Of these, human aquaporin 2 (AQP2) is present in the principal cells of the collecting duct and is responsible for regulated water reabsorption.AQP2 is stored in intracellular vesicles under water-saturating conditions. When the levels of the pituitary antidiuretic hormone arginine vasopressin (AVP) are elevated in response to dehydration or hypernatremia, AVP binding to the vasopressin 2 receptor (V2R) in the basolateral membrane stimulates an increase in intracellular cAMP. This triggers the phosphorylation of Ser256 in the AQP2 C terminus by protein kinase A (PKA) and flags the protein for trafficking from storage vesicles to the apical membrane (4–6). AVP also triggers additional phosphorylation at Ser264 and Ser269 (7, 8), with all three sites being phosphorylated in AQP2s targeted to the plasma membrane (9). The resulting redistribution of AQP2 increases transcellular water permeability and concentrates urine (Fig. S1). Once correct water balance is restored, AQP2 is internalized through ubiquitin-mediated endocytosis and redirected to storage vesicles or targeted for degradation (10–12).Because of its central role in water homeostasis, dysregulation of AQP2 is implicated in several human disease states including congestive heart failure, liver cirrhosis, and preeclampsia (13). Failure to recruit AQP2 to the apical membrane underlies acquired and congenital nephrogenic diabetes insipidus (NDI), a water balance disorder in which patients lack the ability to concentrate urine, leading to severe dehydration (14). Although congenital NDI is most commonly caused by mutations in the gene for the V2R receptor, around 10% of NDI patients harbor AQP2 gene mutations. NDI-causing AQP2 mutations either interfere with its shuttling from storage vesicles to the apical membrane or, more frequently, cause misfolding and retention in the endoplasmic reticulum (ER) (15, 16).Here we present the X-ray structure of human AQP2 at 2.75 Å resolution, providing a structural framework that contributes to our understanding of AQP2 trafficking as well as gives insight into the mechanism of how AQP2 mutations induce NDI. This structure reveals significant positional variability of the short AQP2 C-terminal helix. In one protomer, this region interacts with the cytoplasmic surface of a symmetry-related AQP2 molecule, an interaction that may mimic protein–protein interactions involved in AQP2 cellular sorting. Moreover, two Cd²⁺ ions (presumably Ca²⁺ in vivo) are observed to bind the AQP2 tetramer and stabilize a specific conformation of the cytoplasmic loop D that is necessary for this interaction to occur. Radioligand binding studies show that oocytes expressing AQP2 bind Ca²⁺, supporting Ca²⁺ as the physiological ligand for these sites. Several interactions that lead to NDI when disrupted by mutation are also highlighted. Together, this structure provides insights into AQP2 mutations in NDI and opens new opportunities to study the structural mechanism behind membrane protein sorting, including ER recognition of misfolded proteins.     

Chemical synthesis and X-ray structure of a heterochiral {D-protein antagonist plus vascular endothelial growth factor} protein complex by racemic crystallography

Total chemical synthesis was used to prepare the mirror image (D-protein) form of the angiogenic protein vascular endothelial growth factor (VEGF-A). Phage display against D-VEGF-A was used to screen designed libraries based on a unique small protein scaffold in order to identify a high affinity ligand. Chemically synthesized D- and L- forms of the protein ligand showed reciprocal chiral specificity in surface plasmon resonance binding experiments: The L-protein ligand bound only to D-VEGF-A, whereas the D-protein ligand bound only to L-VEGF-A. The D-protein ligand, but not the L-protein ligand, inhibited the binding of natural VEGF₁₆₅ to the VEGFR1 receptor. Racemic protein crystallography was used to determine the high resolution X-ray structure of the heterochiral complex consisting of {D-protein antagonist + L-protein form ofVEGF-A}. Crystallization of a racemic mixture of these synthetic proteins in appropriate stoichiometry gave a racemic protein complex of more than 73 kDa containing six synthetic protein molecules. The structure of the complex was determined to a resolution of 1.6 Å. Detailed analysis of the interaction between the D-protein antagonist and the VEGF-A protein molecule showed that the binding interface comprised a contact surface area of approximately 800 Å² in accord with our design objectives, and that the D-protein antagonist binds to the same region of VEGF-A that interacts with VEGFR1-domain 2.     

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO₂ and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles.