不同因素对人Ⅹ型磷脂酶A2包涵体重折叠的影响

人Ⅹ型磷脂酶A2 在大肠杆菌中的表达产物完全以不溶的包涵体形式存在. 用以前适用于人胰型(IB 型) 磷脂酶A2 的稀释重折叠方法,并不能使它有效重折叠. 以初步纯化的包涵体为对象,发现溶液的温度、pH 值和蛋白质浓度对Ⅹ型磷脂酶A2 体外重折叠有很大的影响. 研究了一些小分子化合物对Ⅹ型磷脂酶A2 ,在高蛋白质浓度(1 g/L)下重折叠的影响,发现1 mol/L 的L-精氨酸能提高其重折叠效率达6倍多. L-精氨酸的结构类似物L-瓜氨酸对Ⅹ型磷脂酶A2 的重折叠有较弱的改善,而L-赖氨酸和L-精氨酸甲基酯则降低了Ⅹ型磷脂酶A2 的活性恢复. 结果表明,L-精氨酸对蛋白质重折叠的帮助作用,可能是通过精氨酸与折叠中间物的结合产生的,精氨酸的胍基和羧基都是必需的,其侧链胍基以其特殊的带电方式阻遏了重折叠过程中的积聚和分子间二硫键形成的反应.     

人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

目的为了构建人分泌型磷脂酶A2(secretaryphospholipaseA2,sPLA2-IIA)的有效表达系统,从胎脾中提取总RNA。方法采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3′末端,构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导,hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达,表达产物以包涵体的形式存在。包涵体经8mol/L尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论以大肠杆菌为宿主,成功表达了hsPLA2-IIA蛋白,为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。     

人分泌型磷脂酶A2(hsPLA2-IIA)基因的克隆、表达及活性鉴定

目的 为了构建人分泌型磷脂酶A2(secretary phospholipase A2, sPLA2-IIA) 的有效表达系统,本文从胎脾中提取总RNA,采用RT-PCR方法扩增出编码sPLA2-IIA的基因定向地克隆于硫氧环蛋白基因融合表达载体pET32a的TrxA基因3’末端,构建符合读码框的融合表达载体pET32a-sPLA2-IIA。37℃下经IPTG诱导,hsPLA2-IIA融合蛋白在大肠杆菌BL21(DE3)中获得高效表达,表达产物以包涵体的形式存在。包涵体经8M尿素溶解、复性后检测结果显示具有较高的催化活性并呈现剂量依赖关系。结论：以大肠杆菌为宿主,成功表达了hsPLA2-IIA蛋白,为进一步进行hsPLA2-IIA的大量生产和功能研究奠定了基础。     

Ⅱ、Ⅳ、Ⅴ和Ⅹ型磷脂酶A2 mRNA在大鼠循环系统中的分布

目的：探讨Ⅱ、Ⅳ、Ⅴ和Ⅹ型磷脂酶A2（PLA2）mRNA在正常大鼠循环系统中的分布情况。方法：利用逆转录-聚合酶链反应（RT-PCR）扩增大鼠各型PLA2DNA。结果：在所检测循环系统的几个组织中均能测到Ⅱ、Ⅳ型PLA2mRNA;Ⅴ型PLA2mRNA在大鼠心肌、主动脉弓、下腔静脉近心段中可测到;Ⅹ型PLA2mRNA只在心肌中检测到。结论：Ⅱ、Ⅳ型PLA2广泛存在于循环系统中,在宿主防御细菌感染及炎症中发挥作用。Ⅴ型PLA2分布在心脏及近心脏的血管中,可能与心血管内皮细胞的病变有关。     

磷脂酶A2活性对中性粒细胞凋亡的影响

细胞凋亡（Ａｐｏｐｔｏｓｉｓ）是一种机体保持内环境稳定的特殊方式。正常情况下,中性粒细胞（ＰＭＮ）绝大部分通过凋亡而被清除,避免因坏死而造成组织损伤。我们在研究磷脂酶２（ＰＬＡ２）激活介导创伤和感染的机理时,发现其活性介导ＴＮＦ对ＰＭＮ的激发作用。其它证据也显示ＰＬＡ２及其代谢产物在细胞凋亡过程中发挥作用,我们推测ＰＬＡ２活性对ＰＭＮ凋亡或坏死的影响,可能是控制炎症反应的主要途径。这方面的工作尚少见,本文初步报告如下。１　材料和方法（１）材料和主要试剂　雄性Ｗｉｓｔａｒ大鼠由本院动物中心提供。Ｐ…     

分泌型磷脂酶A_2家族及其受体

磷脂酶Ａ2 (ｐｈｏｓｐｈｏｌｉｐａｓｅＡ2 ,ＰＬＡ2 )的系统名为磷脂酰 2 脂酰水解酶 (ｐｈｏｓｐｈａｔｉｄｙｌ ｃｈｏｌｉｎｅ 2 ａｃｙｌｈｙｄｒｏｌａｓｅ)。根据其存在位置可分为细胞内型ＰＬＡ2 (ｉｎｔｒａｃｅｌｌｕｌａｒＰＬＡ2 )和分泌型ＰＬＡ2 (ｓｅｃｒｅｔｅｄＰＬＡ2 ,ｓＰＬＡ2 )两种。ｓＰＬＡ2 可以催化甘油磷酸酯的 2位酰键水解 ,生成游离脂肪酸和溶血酸脂。因为其产物在细胞信号转导及生物活性脂 (包括花生四烯酸、血小板活性因子等 )的生物合成中起重要作用 ,所以ＰＬＡ2 被普遍认为是控制脂介质前体释放的核心…     

人分泌型磷脂酶A2 GIIE的E54突变及其对底物选择性的影响

人分泌型磷脂酶A2GIIE(Human secreted phospholipase A2GIIE,hGIIE)通过发挥酶催化作用,参与炎症反应和脂代谢过程.为了揭示hGIIE的底物选择机制,文中对hGIIE进行了定点突变,采用毕赤酵母Pichiapastoris重组表达突变体蛋白,然后通过阳离子交换和分子排阻两步法纯...     

磷脂酶A2的生理机能新说

磷脂酶Ａ２的生理机能新说杜晓燕周元聪（中国科学院上海生物化学研究所,上海２０００３１）关键词磷脂酶Ａ２生理机能磷脂酶Ａ２（ｐｈｏｓｐｈｏｌｉｐａｓｅＡ２,ＥＣ３．１．１．４）,简称ＰＬＡ２。它能水解甘油磷脂的第二位酯酰键,生成溶血磷脂和脂肪酸,如下式...     

Group X Secretory Phospholipase A2 Regulates Insulin Secretion through a Cyclooxygenase-2-dependent Mechanism

Group X secretory phospholipase A₂ (GX sPLA₂) potently hydrolyzes membrane phospholipids to release arachidonic acid (AA). While AA is an activator of glucose-stimulated insulin secretion (GSIS), its metabolite prostaglandin E2 (PGE2) is a known inhibitor. In this study, we determined that GX sPLA₂ is expressed in insulin-producing cells of mouse pancreatic islets and investigated its role in beta cell function. GSIS was measured in vivo in wild-type (WT) and GX sPLA₂-deficient (GX KO) mice and ex vivo using pancreatic islets isolated from WT and GX KO mice. GSIS was also assessed in vitro using mouse MIN6 pancreatic beta cells with or without GX sPLA₂ overexpression or exogenous addition. GSIS was significantly higher in islets isolated from GX KO mice compared with islets from WT mice. Conversely, GSIS was lower in MIN6 cells overexpressing GX sPLA₂ (MIN6-GX) compared with control (MIN6-C) cells. PGE2 production was significantly higher in MIN6-GX cells compared with MIN6-C cells and this was associated with significantly reduced cellular cAMP. The effect of GX sPLA₂ on GSIS was abolished when cells were treated with NS398 (a COX-2 inhibitor) or L-798,106 (a PGE2-EP3 receptor antagonist). Consistent with enhanced beta cell function, GX KO mice showed significantly increased plasma insulin levels following glucose challenge and were protected from age-related reductions in GSIS and glucose tolerance compared with WT mice. We conclude that GX sPLA₂ plays a previously unrecognized role in negatively regulating pancreatic insulin secretion by augmenting COX-2-dependent PGE2 production.     

Phospholipase A2-Modified Low-Density Lipoprotein Activates Liver X Receptor in Human Macrophages

Macrophages respond to cholesterol accumulation by increasing cholesterol efflux, which is mediated by activation of the nuclear liver X receptor (LXR) and ATP binding cassette (ABC) transporters. In the present study, we investigated whether foam cell formation induced by phospholipase A(2)-modified low-density lipoprotein (PLA-LDL) influences LXR activity and cholesterol efflux in primary human monocyte-derived macrophages (MDMs). Macrophages were treated with PLA-LDL and expression of the LXR target genes ABCA1 and ABCG1 was analyzed by quantitative PCR and western blot. PLA-LDL time-dependently up-regulated ABCA1 and ABCG1 mRNA and protein. Removal of non-esterified fatty acids from PLA-LDL particles did not influence the induction of ABC transporters. A role of LXR in PLA-LDL-stimulated ABCG1 expression was verified by LXR-knockdown and luciferase reporter assays using a construct containing a LXR response element from the ABCG1 gene. Functionally, cholesterol efflux to apolipoprotein A-I and high-density lipoprotein was higher in PLA-LDL treated cells compared to controls. Together, these results demonstrate that in primary human MDMs PLA-LDL induces ABC transporter expression via LXR activation. A concomitantly increased cholesterol efflux may prevent excessive cholesterol accumulation and thus, attenuate foam cell formation.     

Molecular Properties of Purified Human Uncoupling Protein 2 Refolded from Bacterial Inclusion Bodies

One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (K _d) of 0.3–0.5 and 23–92 M. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a K _d of greater than 100 M, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-¹⁴C] ATP and [8-¹⁴C] ADP with K _ds of 4–5 and 16–18 M, respectively. Mg²⁺ (2 mM) reduced the apparent ATP affinity to 53 M, an effect entirely explained by chelation of ATP; with Mg²⁺, K _d using calculated free ATP was 3 M. A combination of gel filtration, Cu²⁺-phenanthroline cross-linking, and ultracentrifugation indicated that 75–80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg²⁺-free nucleotides: the K _d for ATP is about 3–5 M and for Mant-ATP it is about 10 times lower; and (c) in C₁₂E₉ detergent, the monodisperse protein may be in dimeric form.     

Protein Folding, Misfolding, and Aggregation. Formation of Inclusion Bodies and Aggresomes

In this review the mechanisms of protein folding, misfolding, and aggregation as well as the mechanisms of cell defense against toxic protein aggregates are considered. Misfolded and aggregated proteins in cells are exposed to chaperone-mediated refolding and are degraded by proteasomes if refolding is impossible. Proteolysis-stable protein aggregates accumulate, forming inclusion bodies. In eucaryotic cells, protein aggregates form structures in the pericentrosomal area that have been termed "aggresomes". Formation of aggresomes in cells is a general cellular response to the presence of misfolded proteins when the degrading capacity of the cells is exceeded. The role of aggresomes in disturbance of the proteasomal system operation and in cellular death, particularly in the so-called "protein conformational diseases", is discussed.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Group X Secretory Phospholipase A2 Regulates the Expression of Steroidogenic Acute Regulatory Protein (StAR) in Mouse Adrenal Glands

We developed C57BL/6 mice with targeted deletion of group X secretory phospholipase A₂ (GX KO). These mice have ∼80% higher plasma corticosterone concentrations compared with wild-type (WT) mice under both basal and adrenocorticotropic hormone (ACTH)-induced stress conditions. This increased corticosterone level was not associated with increased circulating ACTH or a defect in the hypothalamic-pituitary axis as evidenced by a normal response to dexamethasone challenge. Primary cultures of adrenal cells from GX KO mice exhibited significantly increased corticosteroid secretion compared with WT cells. Conversely, overexpression of GX secretory phospholipase A₂ (sPLA₂), but not a catalytically inactive mutant form of GX sPLA₂, significantly reduced steroid production 30–40% in Y1 mouse adrenal cell line. This effect was reversed by the sPLA₂ inhibitor, indoxam. Silencing of endogenous M-type receptor expression did not restore steroid production in GX sPLA₂-overexpressing Y1 cells, ruling out a role for this sPLA₂ receptor in this regulatory process. Expression of steroidogenic acute regulatory protein (StAR), the rate-limiting protein in corticosteroid production, was ∼2-fold higher in adrenal glands of GX KO mice compared with WT mice, whereas StAR expression was suppressed in Y1 cells overexpressing GX sPLA₂. Results from StAR-promoter luciferase reporter gene assays indicated that GX sPLA₂ antagonizes StAR promoter activity and liver X receptor-mediated StAR promoter activation. In summary, GX sPLA₂ is expressed in mouse adrenal glands and functions to negatively regulate corticosteroid synthesis, most likely by negatively regulating StAR expression.     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.