文蛤过氧化氢酶的生物信息学分析

基于NCBI数据库,对文蛤过氧化氢酶基因(MmeCAT)进行生物信息学分析,旨在为文蛤过氧化氢酶的结构与功能的研究提供理论基础。结果表明,该基因编码511个氨基酸。文蛤过氧化氢酶分子量为58 181.29 Da,分子式为C_(2588)H_(3928)O_(767)N_(730)S_(20),理论等电点为8.05,属亲水蛋白。带负电荷氨基酸残基数(Asp+Glu)为63个,带正电荷氨基酸残基数(Arg+Lys)为65个。假设所有半胱氨酸全部形成胱氨酸,其消光系数为63 175 mol/L,相应的吸光度为1.086;假设所有的半胱氨酸均未形成胱氨酸时,消光系数为62 800 mol/L,相应的吸光度为1.079;其半衰期为30 h,脂肪族氨基酸指数为57.28,不稳定系数为27.77(40),可知文蛤过氧化氢酶为稳定蛋白质。亚细胞定位于过氧化物酶体,存在71个磷酸化位点和51个糖基化位点,无信号肽。二级结构以无规卷曲和α-螺旋为主,在物种进化上具有高度的保守性保守结构域预测表明,该基因编码蛋白可能属于典型单功能过氧化氢酶的第三分支。研究文蛤过氧化氢酶结构,能够为文蛤抗逆性品种选育提供理论基础。     

改造大肠杆菌卟啉代谢途径对重组过氧化物酶活性的影响

【目的】原核表达某些需辅因子的外源蛋白时往往酶活偏低,为提高酶活和减少外加辅因子的成本,我们尝试在大肠杆菌中表达外源过氧化氢-过氧化物酶的同时提高大肠杆菌中与该酶辅因子相关的合成代谢。【方法】本研究克隆了中度嗜盐菌Halomonas elongata DSM2581的过氧化氢-过氧化物酶CAT-POD(catalase-peroxidase)编码基因kat G的ORF,构建原核表达载体p ET28a-kat G,实现了CAT-POD在大肠杆菌中的重组表达。由于CAT-POD活性依赖其活性中心血红素,而血卟啉是血红素的骨架,通过构建原核表达载体p UC19-tac-hem A,将编码5-氨基乙酰丙酸合成酶的hem A基因在大肠杆菌中过量表达,提高卟啉的含量,从而提高重组蛋白CAT-POD的酶活。【结果】最终的CAT酶活达到了377 U/m L,为对照组的7.5倍。【结论】本研究为工业生产高活性CAT-POD提供了有效的方案,也为体外重组表达含辅因子的蛋白提供可借鉴的思路。     

融合蛋白M-IL-2(~(88)Arg,~(125)Ala)在毕赤酵母中的表达及抗肿瘤活性研究

构建融合基因蜂毒肽(melittin)与人变构白介素2(M-IL-2(88Arg,125Ala))毕赤酵母分泌型表达载体pPICZαA/M-IL-2(88Arg,125Ala),转化毕赤酵母菌株KM71H,甲醇诱导筛选出的多拷贝阳性菌株表达融合蛋白,纯化融合蛋白,并进行初步的抗肿瘤活性研究。经测序及PCR鉴定,M-IL-2(88Arg,125Ala)正确插入pPICZαA中,电转化后,重组载体通过同源重组整合到酵母基因组中,SDS-PAGE检测在26 ku左右有明显目的条带,与理论相符。经Western blot鉴定具有较高抗原性及特异性。BCA法测定甲醇诱导96 h融合蛋白表达量达315.2 mg/L。CCK-8法检测融合蛋白对人卵巢癌细胞SKOV3细胞及Hela细胞生长抑制作用,表明纯化的融合蛋白在体外能抑制人卵巢癌细胞SKOV3细胞及Hela细胞的生长增殖。该研究为融合蛋白M-IL-2(88Arg,125Ala)大规模制备和动物实验以及临床前期研究奠定了基础。     

单功能过氧化氢酶的高级结构

单功能过氧化氢酶是在生物界广泛分布的抗氧化酶.近年来,人们在对单功能过氧化氢酶一级结构与生化性质深入研究的基础上,针对十几种单功能过氧化氢酶的高度保守的空间结构开展了研究.认识了其活性中心的血红素及周围保守残基,发现了酶的许多特殊结构,如方向不同的血红素,增强酶抗氧化性的侧链残基的共价键和保证酶高效催化的分子内通道等.本文综述了单功能过氧化氢酶空间结构研究现状,概括了酶构象的基本特点,分析了关注和争议较多的酶血红素、肽链侧链共价键及酶分子内通道等重点问题.深入研究单功能过氧化氢酶空间结构是一挑战性的课题,它将推动酶蛋白高级结构形成和酶催化模式等基础理论研究.     

水稻CAT与逆境应答关系及酶活性分析

过氧化氢酶(catalase,CAT)是一种四聚体血红素酶,主要存在于过氧化物酶体与乙醛酸循环体及相关细胞区域内.水稻中含有CAT1、CAT2和CAT3三种主要同工酶.它们在各组织不同的发育阶段,发挥着重要作用.在本研究解析了水稻过氧化氢酶同工酶基因在NaCl、NaHCO3、Na2CO3、PEG6000、H2O2、低温等逆境处理下mRNA表达特性.结果表明在不同的逆境下过氧化氢酶同工酶在表达上存在差异性.同时利用蛋白表达系统,对水稻过氧化氢酶CAT1和CAT3蛋白进行了纯化实验,对纯化的蛋白的酶活性分析的结果表明,CAT1和CAT3的酶活性没有十分显著的差别.     

实验性糖尿病大鼠肝过氧化物酶体脂肪酸β-氧化及D-双功能蛋白活性变化的初步研究

为研究过氧化物酶体脂肪酸β-氧化及D-双功能蛋白在糖尿病脂代谢紊乱中所起的作用,分析了链脲佐菌素诱导的糖尿病大鼠肝脏过氧化物酶体数量和过氧化物酶体脂肪酸β-氧化及D-双功能蛋白活性的改变,并用蛋白质印迹检测过氧化氢酶和D-双功能蛋白的表达量.发现糖尿病大鼠肝脏过氧化物酶体增殖,过氧化氢酶蛋白量和酶活性显著增加,过氧化物酶体脂肪酸β-氧化增强,D-双功能蛋白含量和酶活性显著降低,脂酰CoA氧化酶、L-3-羟脂酰CoA脱氢酶活性显著增加.对过氧化物酶体脂肪酸β-氧化增强和D-双功能蛋白活性降低与糖尿病脂代谢紊乱的关系进行了初步探讨.     

过氧化氢酶在豌豆蚜抵御藤黄微球菌和大肠杆菌感染引起的氧化胁迫中的作用

【目的】研究细菌-过氧化氢酶-豌豆蚜Acyrthosiphon pisum三者之间的关系,探索过氧化氢酶在豌豆蚜免疫防御反应中的作用。【方法】用体外培养结合菌落计数的方法检测藤黄微球菌Micrococcus luteus和大肠杆菌Escherichia coli感染豌豆蚜后在蚜虫体内的增殖;分别用微孔板荧光检测和实时定量PCR的方法检测藤黄微球菌和大肠杆菌感染豌豆蚜之后,蚜虫体内H_2O_2水平和过氧化氢酶基因的转录水平;通过RNA干扰技术对豌豆蚜过氧化氢酶基因(Cat)进行沉默,并检测沉默后感染藤黄微球菌和大肠杆菌的豌豆蚜体内H_2O_2浓度和细菌数目以及豌豆蚜存活率。【结果】藤黄微球菌感染12 h后,豌豆蚜体内H_2O_2水平以及过氧化氢酶基因表达水平显著升高;大肠杆菌感染12 h后,豌豆蚜过氧化氢酶基因表达水平上升,但H_2O_2水平无明显变化。沉默过氧化氢酶基因后,感染藤黄微球菌导致豌豆蚜体内H_2O_2含量在12 h显著上升,细菌数目在24 h约为对照组的一半,但对豌豆蚜存活率无影响;而过氧化氢酶基因沉默并未引起感染大肠杆菌的豌豆蚜体内H_2O_2水平、细菌数目以及豌豆蚜存活率发生明显变化。【结论】过氧化氢酶参与豌豆蚜对藤黄微球菌的防御过程,但不是该防御反应过程中的关键酶;而针对大肠杆菌感染,豌豆蚜可能通过其他途径来应对。     

Arg缓解MSTN对猪肌肉间充质干细胞成脂-分化的抑制

间充质干细胞可分化为脂肪细胞并沉积脂质,从而增加动物脂肪沉积.因此,猪肌肉间充质干细胞被认为是肌内脂肪的重要来源之一.本研究主要在体外探讨了肌肉生长抑制素（MSTN）对猪肌肉来源间充质干细胞成脂分化的影响及Arg的调控作用.结果表明,外源添加MSTN活性蛋白极显著地降低了胞内甘油三酯水平,而添加Arg或MSTN抗体则表现相反的作用（P〈0.01）.同时添加Arg可缓解MSTN对间充质干细胞脂质沉积的抑制作用（P〈0.01）.生脂转录因子表达模式分析表明,外源添加MSTN抑制了细胞PPARγ2和aP2的表达,添加Arg和MSTN抗体增强了细胞ADD1的表达（P〈0.01）.此外,同时添加MSTN蛋白和Arg较仅添加MSTN蛋白极显著增强了细胞ADD1和PPARδ的表达（P〈0.01）,同时添加MSTN和Arg抗体较仅添加MSTN抗体显著增强了细胞ADD1,PPARδ,C/EBPα,PPARγ2和LPL的表达（P〈0.05）.由此可见,MSTN抑制了猪肌肉来源间充质干细胞成脂分化,添加Arg至少部分地通过上调ADD1和PPARδ的表达来缓解MSTN对成脂分化的抑制作用.     

含Arg9的人抗HBsAg单链抗体/ EGFP融合蛋白基因的构建、表达和内化作用的研究

目的：构建、表达和纯化带有转膜结构域Arg9的ScFv14/EGFP融合蛋白, 并对纯化产物的亲和 活性和内化作用进行研究。 方法： 将Arg9的编码序列分别重组到ScFv14/EGFP基因的5'端或3'端或二者之间,将它们分别克隆入原核表达载体pET32a,转化大肠杆菌BL21(DE3)LysS进行诱导表达和纯化,用间接ELISA方法检测表达产物与HBsAg的亲和活性,并用间接免疫荧光检测纯化蛋白的内化活性。 结果：经测序及酶切鉴定证实四种融合基因序列完全正确.SDS-PAGE和Western blot证实四种融合基因成功表达和纯化.间接ELISA检测证实四种融合蛋白均具有HBsAg结合活性,间接免疫荧光检测显示N端带有Arg9的融合蛋白有较强的内化作用,而且不内化进入HBsAg非表达的细胞。 结论：成功构建、表达和纯化了ScFv14/EGFP融合蛋白和三种带有Arg9的ScFv14/EGFP融合蛋白,纯化产物均具有与抗原HBsAg亲和的活性,N端带有Arg9的融合蛋白与靶细胞作用有较强的内化作用。     

N—甲基吩嗪为介体辣根过氧化物酶传感器的研究

研究了将Ｎ－甲基吩嗪作为介体,通过牛血清白蛋白和戊二醛使其作为结合到玻碳电极上去的辣根过氧化物酶生物传感器。该酶电极对过氧化氢有良好的响应,Ｎ－甲基吩嗪还原电流的增值与过氧化氢浓度在１×１０－６～５×１０－４ｍｏｌ／Ｌ范围内有良好的线性关系,该传感器灵敏度高,检出限为１０－７ｍｏｌ／Ｌ,对过氧化氢的响应时间小于１０ｓ。     

Catalase activity is regulated by c-Abl and Arg in the oxidative stress response

The Abl family of mammalian non-receptor tyrosine kinases includes c-Abl and Arg. Recent studies have demonstrated that c-Abl and Arg are activated in the response of cells to oxidative stress. This work demonstrates that catalase, a major effector of the cellular defense against H2O2, interacts with c-Abl and Arg. The results show that H2O2 induced binding of c-Abl and Arg to catalase. The SH3 domains of c-Abl and Arg bound directly to catalase at a P293FNP site. c-Abl and Arg phosphorylated catalase at Tyr231 and Tyr386 in vitro and in the response of cells to H2O2. The functional significance of the interaction is supported by the demonstration that cells deficient in both c-Abl and Arg exhibit substantial increases in H2O2 levels. In addition, c-abl-/- arg-/- cells exhibited a marked increase in H2O2-induced apoptosis compared with that found in the absence of either kinase. These findings indicate that c-Abl and Arg regulate catalase and that this signaling pathway is of importance to apoptosis in the oxidative stress response.     

Bidirectional signaling links the Abelson kinases to the platelet-derived growth factor receptor

The c-Abl nonreceptor tyrosine kinase is activated by growth factor signals such as the platelet-derived growth factor (PDGF) and functions downstream of the PDGF-beta receptor (PDGFR) to mediate biological processes such as membrane ruffling, mitogenesis, and chemotaxis. Here, we show that the related kinase Arg is activated downstream of PDGFRs in a manner dependent on Src family kinases and phospholipase C gamma1 (PLC-gamma1)-mediated phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, as we showed previously for c-Abl. PIP2, a highly abundant phosphoinositide known to regulate cytoskeletal and membrane proteins, inhibits the tyrosine kinase activities of both Arg and c-Abl in vitro and in cells. We now demonstrate that c-Abl and Arg form inducible complexes with and are phosphorylated by the PDGFR tyrosine kinase in vitro and in vivo. Moreover, c-Abl and Arg, in turn, phosphorylate the PDGFR. We show that c-Abl and Arg exhibit nonredundant functions downstream of the activated PDGFR. Reintroduction of c-Abl into Arg-Abl double-null fibroblasts rescues the ability of PLC-gamma1 to increase PDGF-mediated chemotaxis, while reexpression of Arg fails to rescue the chemotaxis defect. These data show that, although both kinases are activated and form complexes with proteins in the PDGFR signaling pathway, only c-Abl functions downstream of PLC-gamma1 to mediate chemotaxis.     

Catalase is regulated by ubiquitination and proteosomal degradation. Role of the c-Abl and Arg tyrosine kinases

Catalase is a major effector in the defense of aerobic cells against oxidative stress. Recent studies have shown that catalase activity is stimulated by the c-Abl and Arg tyrosine kinases. Little, however, is otherwise known about the mechanisms responsible for catalase regulation. The present work demonstrates that mouse cells deficient in both c-Abl and Arg exhibit increased catalase stability. The results also show that catalase is subject to ubiquitination and degradation by the 26S proteosome. Significantly, ubiquitination of catalase is dependent on c-Abl- and Arg-mediated phosphorylation of catalase on both Y231 and Y386. In concert with these results, human 293 cells expressing catalase mutated at Y231 and Y386 exhibit attenuated levels of reactive oxygen species when exposed to hydrogen peroxide. These findings indicate that, in addition to stimulating catalase activity, c-Abl and Arg promote catalase degradation in the oxidative stress response.     

Two distinct phosphorylation pathways have additive effects on Abl family kinase activation

The activities of the related Abl and Arg nonreceptor tyrosine kinases are kept under tight control in cells, but exposure to several different stimuli results in a two- to fivefold stimulation of kinase activity. Following the breakdown of inhibitory intramolecular interactions, Abl activation requires phosphorylation on several tyrosine residues, including a tyrosine in its activation loop. These activating phosphorylations have been proposed to occur either through autophosphorylation by Abl in trans or through phosphorylation of Abl by the Src nonreceptor tyrosine kinase. We show here that these two pathways mediate phosphorylation at distinct sites in Abl and Arg and have additive effects on Abl and Arg kinase activation. Abl and Arg autophosphorylate at several sites outside the activation loop, leading to 5.2- and 6.2-fold increases in kinase activity, respectively. We also find that the Src family kinase Hck phosphorylates the Abl and Arg activation loops, leading to an additional twofold stimulation of kinase activity. The autoactivation pathway may allow Abl family kinases to integrate or amplify cues relayed by Src family kinases from cell surface receptors.     

Glycogen synthase kinase 3beta is tyrosine phosphorylated by PYK2

Glycogen synthase kinase 3beta (GSK3beta) is a Ser/Thr kinase that is involved in numerous cellular activities. GSK3beta is activated by tyrosine phosphorylation. However, very little is known about the tyrosine kinases that are responsible for phosphorylating GSK3beta. In this report, we investigated the ability of the calcium-dependent tyrosine kinase, proline-rich tyrosine kinase 2 (PYK2) to tyrosine phosphorylate GSK3beta. In transfected CHO cells, it was demonstrated that PYK2 tyrosine phosphorylates GSK3beta in situ. The two kinases also coimmunoprecipitated. Furthermore, GSK3beta was tyrosine phosphorylated in vitro by an active, wild type PYK2, but not by the inactive, kinase dead form of PYK2. Therefore, this study is the first to demonstrate that GSK3beta is a substrate of PYK2 both in vitro and in situ.     

A critical role for cortactin phosphorylation by Abl-family kinases in PDGF-induced dorsal-wave formation

Proper regulation of cell morphogenesis and migration by adhesion and growth-factor receptors requires Abl-family tyrosine kinases [1-3]. Several substrates of Abl-family kinase have been identified, but they are unlikely to mediate all of the downstream actions of these kinases on cytoskeletal structure. We used a human protein microarray to identify the actin-regulatory protein cortactin as a novel substrate of the Abl and Abl-related gene (Arg) nonreceptor tyrosine kinases. Cortactin stimulates cell motility [4-6], and its upregulation in several cancers correlates with poor prognosis [7]. Even though cortactin can be tyrosine phosphorylated by Src-family kinases in vitro [8], we show that Abl and Arg are more adept at binding and phosphorylating cortactin. Importantly, we demonstrate that platelet-derived growth-factor (PDGF)-induced cortactin phosphorylation on three tyrosine residues requires Abl or Arg. Cortactin triggers F-actin-dependent dorsal waves in fibroblasts after PDGF treatment and thus results in actin reorganization and lamellipodial protrusion [9]. We provide evidence that Abl/Arg-mediated phosphorylation of cortactin is required for this PDGF-induced dorsal-wave response. Our results reveal that Abl-family kinases target cortactin as an effector of cytoskeletal rearrangements in response to PDGF.     

Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum.

We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen. In vitro incubation of membrane fractions with [gamma-32P]ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein. Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody. In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo. These results demonstrate that membranes from P. solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein. This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.     

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.     

Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.