前列腺素E2合酶的研究进展

目录 一、前列腺素E2合酶的分类 二、胞质型前列腺素E2合酶 三、膜结合型前列腺素E2合酶-1（mPGES-1） （一）mPGES-1的结构及酶学特性 （二）mPGES-1与COXs偶联 （三）mPGES-1的生理及病理生理作用 四、膜结合型前列腺素E2合酶-2 五、斗族谷胱甘肽S转移酶 六、展望[编者按]     

合酶与合成酶

合酶与合成酶是生化教学中经常提到的,同时也是比较容易混淆的两个概念。例如,对于三羧酸循环中催化乙酰ＣｏＡ与草酰乙酸反应生成柠檬酸的酶,国内教科书就出现了命名的混乱,有的书称为柠檬酸合酶,更多的书称为柠檬酸合成酶。此外,不少教科书把脂肪酸合酶、ＨＭＧＣｏＡ合酶、ＡＬＡ合酶及前列腺内过氧化物合酶误译成脂肪酸合成酶、ＨＭＧＣｏＡ合成酶、ＡＬＡ合成酶及前列腺内过氧化物合成酶等。可见,合酶与合成酶误用存在一定的普遍性。这种状况不仅使青年教师茫然,更使学生无所适从,实在有必要澄清。出现这种错误的可能原因一是名词的错…     

前列腺素E2在癌症发生发展中的研究进展

环氧合酶2、微粒体前列腺素E合酶1催化产生的前列腺素E2在肿瘤的发展过程中具有重要作用。COX2/PEG2途径的失调通过多种机制影响癌症的发生和发展。如促进肿瘤细胞的增殖和存活、抗凋亡,在肿瘤微环境中,前列腺素E2的升高促进血管再生、肿瘤细胞的粘附和迁移,促进癌症的转移。因此,研发在前列腺素E2合成过程中关键酶的抑制剂是治疗前列腺素E2相关癌症的策略之一。     

巨噬细胞炎症状态与过氧化物酶体增殖的相关性研究

肥胖引起巨噬细胞浸润机体组织,诱发慢性低度炎症反应,是形成胰岛素抵抗的重要诱因。因此研究影响巨噬细胞炎症状态的因素,有助于深入了解胰岛素抵抗的形成机理。该文通过免疫荧光、Real-time PCR等技术,研究巨噬细胞炎症状态与胞内过氧化物酶体数量之间的关系。结果表明,当巨噬细胞极化为炎症状态的M1型,胞内过氧化物酶体数量变化不显著;当巨噬细胞极化为抗炎状态的M2型,胞内过氧化物酶体数量显著升高。当饱和脂肪酸诱导巨噬细胞极化为炎症状态的类M1型,胞内过氧化物酶体数量变化不显著;当不饱和脂肪酸诱导的抗炎症状态的类M2型,胞内过氧化物酶体数量显著升高。此外,使用过氧化物酶体增殖缺陷型(Pex3~(–/–))巨噬细胞重复上述实验,也可以极化为M2/类M2型,呈现抗炎状态,但胞内过氧化物酶体数量无显著变化。综上所述,该研究发现巨噬细胞M2/类M2型极化能够诱导胞内过氧化物酶体增殖,但过氧化物酶体增殖不是M2/类M2型极化的必要条件。     

选择性co x 一2 抑制剂在胶质瘤放疗中的研究进展

环氧合酶（cyclooxygenase,COX）又名前列腺素内过氧化物合成酶,是前列腺素类似物合成的限速酶。COX-2是其诱导型酶。胶质瘤中COX-2的高表达被认为与肿瘤的侵袭性、预后相关。COX-2在胶质瘤的发生发展过程中发挥重要作用。选择性COX-2抑制剂通过直接和间接的作用机制而成为放射增敏剂。它们通过直接作用肿瘤细胞增强放射反应性,同时间接通过前列腺素影响肿瘤的血管形成抑制肿瘤生长。在体内和体外的研究表明选择性COX-2抑制剂可以增强胶质瘤对放射的反应性．降低恶性胶质瘤患者术后放射的必需照射剂量。而且在提高肿瘤放射敏感性的同时不增加对正常组织的放射损伤,甚至对正常组织有放射保护作用。因此,放疗联合选择性COX-2抑制剂可能成为胶质瘤治疗的新的有效途径。     

前列腺素在胚胎着床和蜕膜化中的作用

前列腺素在哺乳动物的雌性生殖过程中起着十分重要的作用.环氧合酶-2 (cyclooxygenase-2, COX-2)主要在子宫着床位点处胚胎周围的基质细胞中表达, 介导着床和蜕膜化过程.由COX-2和微粒体型前列腺素E合成酶-1途径来源的前列腺素E 2 (prostaglandin E2, PGE2)在胚胎着床和蜕膜化过程中起重要作用.子宫中产生的前列腺素I 2 (prostaglandin I2, PGI2)通过核受体过氧化物酶体增殖因子活化受体δ(peroxisome proliferator-activated receptorδ,PPARδ)在胚胎着床过程中起关键作用.质膜上的前列腺素转运蛋白(prostaglandin transporter, PGT)通过转运新合成的前列腺素, 来满足胚胎着床和蜕膜化过程中对前列腺素的需求, 并维持前列腺素的代谢平衡.     

TassC结合过氧化物酶体对早期受染巨噬细胞内的鼠伤寒沙门菌的影响

探讨鼠伤寒沙门菌在感染鼠巨噬细胞早期与细胞器的相互作用。用pTassC-GFP质粒转染鼠巨噬细胞RAW264.7,结合多抗的溶酶体标志物溶酶体相关膜蛋白-1用键合了Alexa594的羊抗鼠二抗显色,以观察标记了绿色荧光蛋白的TassC与溶酶体的关系;用pTassC-GFP和pDsRed2-Perxi质粒共转染RAW264.7细胞,以观察TassC-GFP与过氧化物酶体的关系;用SYTO42标记鼠伤寒沙门菌,感染用pTassC-GFP和pDsRed2-Perxi质粒共转染的RAW264.7细胞,以观察细菌与TassC和过氧化物酶体的关系。免疫荧光显示TassC-GFP不与鼠巨噬细胞RAW264.7中的溶酶体结合,但与标记了红色荧光的过氧化物酶体共定位;感染1 h的RAW264.7胞内SYTO42标记的鼠伤寒沙门菌吞噬泡可招募TassC-GFP和过氧化物酶体。这些发现提示在鼠伤寒沙门菌感染早期过氧化物酶体携带杀菌成分通过TassC介导可参与发挥一定的杀菌作用。     

猪前列腺素内过氧化物酶2(PTGS2)基因的遗传变异及其与繁殖性状相关性研究

前列腺素内过氧化物酶2是花生四烯酸合成前列腺素的限速酶,在包括排卵、受精、着床、分娩等一系列生殖过程中起着重要作用,因而编码该酶的基因是影响繁殖性状的重要候选基因。通过PCR—RFLP分析前列腺索内过氧化物酶2基因在15个中外不同繁殖性能猪种中的遗传变异,结果表明,不同类型中国地方猪种和外来商业猪种往此摹因位点上存在丰富的多态性,繁殖性能相埘较好的江海型、华北型和华中型猪种中A等位基因表现为优势等位基因,卡方检验显示其基因频率分布与西方商业猪种及繁殖性能较低的高原型藏猪和华南型猪种差异均极为显著（P〈0．001）。利用二花脸x杜洛克资源家系F2群体分析该基因与繁殖性状的相关性,在180头F2代母猪群体中,未能进一步证实该基凶位点对总产仔数、产活仔数和死胎数3个繁殖性状存在显著影响（P〉0．05）,但携带优势等位基因4的个体趋向于拥有较高的总产仔数、产活仔数和偏低的死胎数,鉴于该基因的重要作用,基于全基因序列的SNP扫描和大样本群体的相关性分析仍很有必要。     

木枣叶片再生植株及其变异系过氧化物酶同工酶分析

用聚丙烯酰胺凝胶电泳分析了木枣叶片再生植株及其5种变异系过氧化物酶(POD)同工酶。结果表明：木枣叶片再生植株过氧化物酶同工酶由3个基因位点编码,位点1编码的是杂合三聚体酶,位点2和位点3编码的是纯合二聚体酶,其变异系中有不能表达位点2和位点3的植株,变异较大。也有完整表达3个基因位点的植株,且酶活性高。     

前列腺素E2途径与动脉导管闭合的关系

动脉导管开放是胎儿时期的重要交通,其及时闭合对出生后生理功能同等重要,否则将导致动脉导管未闭,严重威胁患者健康甚至生命,其发生率在超低体重儿中高达60%.目前研究认为出生前后这两个生理过程主要与前列腺素E2(prostaglandin E2,PGE2)的浓度有关,因此参与PGE2合成与代谢的酶,如环氧酶(COXs)、PGE2合酶(PGESs)和前列腺素15-羟基脱氢酶(PGDH),以及四种PGE2受体及该途径相关的药物在动脉导管的结构和功能中的作用成为近年来研究的热点.     

Genetic and biochemical identification of the glutamate synthase structural gene in Neurospora crassa.

Neurospora crassa cells require glutamate synthase activity for growth under ammonium-limiting conditions. Despite the physiological importance of glutamate synthase, little is known about the genetics of its expression. To identify the glutamate synthase structural gene, we isolated three new mutants lacking this activity. All mutations are recessive to the wild-type allele and belong to the same complementation group as the previously described en(am)-2 (C24) mutation. Two lines of evidence indicate that en(am)-2 is the structural gene for glutamate synthase in N. crassa. The en(am)-2+ gene shows a gene dosage effect on enzyme activity, and some mutants lacking glutamate synthase activity have cross-reacting material. These data suggest that the mutations are located in the structural gene for N. crassa glutamate synthase.     

Extramitochondrial citrate synthase activity in bakers'' yeast.

We isolated the gene for citrate synthase (citrate oxaloacetate lyase; EC 4.1.3.7) from Saccharomyces cerevisiae and ablated it by inserting the yeast LEU2 gene within its reading frame. This revealed a second, nonmitochondrial citrate synthase. Like the mitochondrial enzyme, this enzyme was sensitive to glucose repression. It did not react with antibodies against mitochondrial citrate synthase. Haploid cells lacking a gene for mitochondrial citrate synthase grew somewhat slower than wild-type yeast cells, but exhibited no auxotrophic growth requirements.     

Studies of propionate toxicity in Salmonella enterica identify 2-methylcitrate as a potent inhibitor of cell growth

Salmonella enterica serovar Typhimurium LT2 showed increased sensitivity to propionate when the 2-methylcitric acid cycle was blocked. A derivative of a prpC mutant (which lacked 2-methylcitrate synthase activity) resistant to propionate was isolated, and the mutation responsible for the newly acquired resistance to propionate was mapped to the citrate synthase (gltA) gene. These results suggested that citrate synthase activity was the source of the increased sensitivity to propionate observed in the absence of the 2-methylcitric acid cycle. DNA sequencing of the wild-type and mutant gltA alleles revealed that the ATG start codon of the wild-type gene was converted to the rare GTG start codon in the revertant strain. This result suggested that lower levels of this enzyme were present in the mutant. Consistent with this change, cell-free extracts of the propionate-resistant strain contained 12-fold less citrate synthase activity. This was interpreted to mean that, in the wild-type strain, high levels of citrate synthase activity were the source of a toxic metabolite. In vitro experiments performed with homogeneous citrate synthase enzyme indicated that this enzyme was capable of synthesizing 2-methylcitrate from propionyl-CoA and oxaloacetate. This result lent further support to the in vivo data, which suggested that citrate synthase was the source of a toxic metabolite.     

Citrate synthase encoded by the CIT2 gene of Saccharomyces cerevisiae is peroxisomal.

The product of the CIT2 gene has the tripeptide SKL at its carboxyl terminus. This amino acid sequence has been shown to act as a peroxisomal targeting signal in mammalian cells. We examined the subcellular site of this extramitochondrial citrate synthase. Cells of Saccharomyces cerevisiae were grown on oleate medium to induce peroxisome proliferation. A fraction containing membrane-enclosed vesicles and organelles was analyzed by sedimentation on density gradients. In wild-type cells, the major peak of citrate synthase activity was recovered in the mitochondrial fraction, but a second peak of activity cosedimented with peroxisomes. The peroxisomal activity, but not the mitochondrial activity, was inhibited by incubation at pH 8.1, a characteristic of the extramitochondrial citrate synthase encoded by the CIT2 gene. In a strain in which the CIT1 gene encoding mitochondrial citrate synthase had been disrupted, the major peak of citrate synthase activity was peroxisomal, and all of the activity was sensitive to incubation at pH 8.1. Yeast cells bearing a cit2 disruption were unable to mobilize stored lipids and did not form stable peroxisomes in oleate. We conclude that citrate synthase encoded by CIT2 is peroxisomal and participates in the glyoxylate cycle.     

Cloning and sequencing of the yeast gene for dolichol phosphate mannose synthase, an essential protein

Dolichol phosphate mannose (Dol-P-Man) synthase (EC 2.4.1.83) catalyzes the formation of Dol-P-Man from Dol-P and GDP-Man. The structural gene for yeast Dol-P-Man synthase (DPM1) was isolated by screening a yeast genomic DNA library for colonies that overexpressed Dol-P-Man synthase activity. This approach relied on a method to screen for Dol-P-Man synthase activity in lysed yeast colonies and used a yeast mutant with very low Dol-P-Man synthase activity in colony lysates. Transformants isolated using this technique expressed Dol-P-Man synthase activity 9-14-fold higher than that of a wild type strain, and all seven plasmids conferring this overproduction had a common region in their yeast genomic DNA insert. DPM1 is the structural gene for yeast Dol-P-Man synthase since Escherichia coli transformants harboring this gene express Dol-P-Man synthase activity in vitro. DNA sequencing of the DPM1 gene revealed an open reading frame of 801 bases. The 30-kDa size of the predicted protein is in excellent agreement with the size of the purified yeast enzyme (Haselbeck, A., and Tanner, W. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1520-1524). Analysis of the predicted amino acid sequence reveals the protein has a potential membrane spanning domain of 25 amino acids at its COOH terminus. The protein's NH2 terminus, though not hydrophobic, meets existing criteria for yeast signal sequences, but there is no site for cleavage by signal peptidase. If the NH2 terminus is a functional signal sequence, the protein is predicted to be oriented toward the lumen of the endoplasmic reticulum with both NH2 and COOH termini serving as membrane anchors. If there is no signal sequence, the enzyme is predicted to face the cytoplasm and be anchored only by its COOH terminus. The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal. We suspect Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth. Instead, DPM1 may be an essential gene because its product is required for O-glycosylation in yeast or because Dol-P-Man synthase is needed in some unidentified pathway.     

Isolation of the GSY1 gene encoding yeast glycogen synthase and evidence for the existence of a second gene

Glycogen synthase preparations from Saccharomyces cerevisiae contained two polypeptides of molecular weights 85,000 and 77,000. Oligonucleotides based on protein sequence were utilized to clone a S. cerevisiae glycogen synthase gene, GSY1. The gene would encode a protein of 707 residues, molecular mass 80,501 daltons, with 50% overall identity to mammalian muscle glycogen synthases. The amino-terminal sequence obtained from the 85,000-dalton species matched the NH2 terminus predicted by the GSY1 sequence. Disruption of the GSY1 gene resulted in a viable haploid with glycogen synthase activity, and purification of glycogen synthase from this mutant strain resulted in an enzyme that contained the 77,000-dalton polypeptide. Southern hybridization of genomic DNA using the GSY1 coding sequence as a probe revealed a second weakly hybridizing fragment, present also in the strain with the GSY1 gene disrupted. However, the sequences of several tryptic peptides derived from the 77,000-dalton polypeptide were identical or similar to the sequence predicted by the GSY1 gene. The data are explained if S. cerevisiae has two glycogen synthase genes encoding proteins with significant sequence similarity The protein sequence predicted by the GSY1 gene lacks the extreme NH2-terminal phosphorylation sites of the mammalian enzymes. The COOH-terminal phosphorylated region of the mammalian enzyme over-all displayed low identity to the yeast COOH terminus, but there was homology in the region of the mammalian phosphorylation sites 3 and 4. Three potential cyclic AMP-dependent protein kinase sites are located in this region of the yeast enzyme. The region of glycogen synthase likely to be involved in covalent regulation are thus more variable than the catalytic center of the molecule.     

Two glycogen synthase isoforms in Saccharomyces cerevisiae are coded by distinct genes that are differentially controlled

In previous work, we identified a Saccharomyces cerevisiae glycogen synthase gene, GSY1, which codes for an 85-kDa polypeptide present in purified yeast glycogen synthase (Farkas, I., Hardy, T.A., DePaoli-Roach, A.A., and Roach, P.J. (1990) J. Biol. Chem. 265, 20879-20886). We have now cloned another gene, GSY2, which encodes a second S. cerevisiae glycogen synthase. The GSY2 sequence predicts a protein of 704 residues, molecular weight 79,963, with 80% identity to the protein encoded by GSY1. Amino acid sequences obtained from a second polypeptide of 77 kDa present in yeast glycogen synthase preparations matched those predicted by GSY2. GSY1 resides on chromosome VI, and GSY2 is located on chromosome XII. Disruption of the GSY1 gene produced a strain retaining about 85% of wild type glycogen synthase activity at stationary phase, while disruption of the GSY2 gene yielded a strain with only about 10% of wild type enzyme activity. The level of glycogen synthase activity in yeast cells disrupted for GSY1 increased in stationary phase, whereas the activity remained at a constant low level in cells disrupted for GSY2. Disruption of both genes resulted in a viable haploid that totally lacked glycogen synthase activity and was defective in glycogen deposition. In conclusion, yeast expresses two forms of glycogen synthase with activity levels that behave differently in the growth cycle. The GSY2 gene product appears to be the predominant glycogen synthase with activity linked to nutrient depletion.