色氨酸操纵子研究进展

色氨酸只能由微生物和植物合成。催化色氨酸分支途径的酶由色氨酸操纵子编码。生物体内色氨酸合成受到严格调控,色氨酸操纵子发挥重要作用。本文综述色氨酸代谢途径及其调节,并对途径工程在色氨酸操纵子改造中的应用进行回顾。     

关于色氨酸操纵子的概念

关于色氨酸操纵子,在杨颐康先生主编的《微生物学》一书中是这样叙述的:“在色氨酸操纵子里,由调节基因产生的调节蛋白,本来不具阻遏蛋白活性,但与色氨酸结合后,由于构象变化,就具有与DNA的亲和力,而结合在DNA上,这样就使结构基因关闭,不可能产生色氨酸”。又如《基础微生物学专题选》(复旦大学“基础微生物学专题选”编写组)一书中用     

青霉素酰化酶操纵子为负调控模型

质粒pBR322用HindⅢ—BarnH Ⅰ双酶切作为载体,从质粒pPAd上克隆含有pac操纵基因的HindⅢ一。BglⅡl．65kb的DNA片段,得到质粒pPA41。质粒pBR322和pPA41转化染色体上含有完整pac操纵子的大肠杆菌D816,进行操纵基因滴定。大肠杆菌D816、D816(pPA4l)．D816(pBR322)在22℃摇瓶发酵96h,NIPAB法测定青霉素酰化酶活性。结果发现,在不加诱导剂时,D816(pPA41)产生的青霉素酰化酶是D816细胞的2．5倍,在加诱导剂时,D816(pPA41)产生的青霉素酰化酶是D816细胞的1．3倍。对照组D816(pBR322)细胞产生的青霉素酰化酶和：D816细胞产生的几乎一致,说明质粒pPA41的竞争滴定作用确是其上克隆的操纵基因引起的。高拷贝的操纵基因(pPA41)和pac操纵子竞争结合调节蛋白,使得pac操纵子表达增加,说明调节蛋白在pac操纵子表达过程中起阻遏作用,调节蛋白为阻遏蛋白,pac操纵子为负调控模型。R．NA—DNA点杂交检测pac基因表达,发现D816(pPA41)细胞转录产生的pac—mRNA量明显高于D816细胞,mRNA量和青霉素酰化酶活性呈现一致的趋势,证实了pac操纵子的负调控发生在转录水平。     

植物色氨酸一天冬氨酸重复序列蛋白响应逆境胁迫的调控作用

干旱、盐渍、低温等逆境胁迫会严重影响植物的正常生长发育,导致植物的许多响应基因被诱导表达,其蛋白质产物能够保护植物免受胁迫的伤害。色氨酸一天冬氨酸重复序列蛋白（wD40蛋自）在植物中广泛存在,参与植物体内众多代谢反应的调控,如花的发育、开花、花青素的生物合成、激素响应、渗透胁迫等。WD40蛋白含有40-60个氨基酸的保守的wD重复序列,其c末端为色氨酸．天冬氨酸（Trp-Asp,WD）,形成一个p螺旋桨（p—propeller）结构,通过调节多蛋白复合体的组装而影响蛋白质与蛋白质、蛋白质与DNA间的相互作用。本文综述植物WD40蛋白响应逆境胁迫的调控作用。     

大肠杆菌色氨酸生物合成途径关键酶的调控

为了通过基因工程手段提高大肠杆菌色氨酸产量, 对色氨酸生物合成途径中的关键基因trpR、tnaA、aroG和trpED进行了改造。首先通过敲除trpR基因解除了基因组上色氨酸合成和转运关键酶受到的反馈阻遏调控, 进而又敲除了tnaA基因, 阻断了色氨酸的分解代谢。然后, 将色氨酸合成途径的关键酶aroGfbr和trpEDfbr基因串联表达, 以去除色氨酸生物合成途径的瓶颈。与对照MG1655相比, trpR基因单敲菌色氨酸浓度提高了10倍, 双敲菌色氨酸浓度提高了约20倍。pZE12-trpEDfbr转入双敲菌后色氨酸浓度提高到168 mg/L, 而将aroGfbr和trpEDfbr转入双敲菌后, 色氨酸浓度提高到820 mg/L。为构建色氨酸高产菌奠定了基础。     

色氨酸2,3-双加氧酶调控炎症免疫反应在疾病中的作用

犬尿氨酸代谢通路(kynurenine pathway,KP)是色氨酸代谢的主要途径,参与机体多个病理生理过程.色氨酸2,3-双加氧酶(tryptophan 2,3-dioxygenase,TDO2)是KP的初始限速酶之一,能够催化色氨酸(tryptophan,Trp)使其转化为犬尿氨酸(kynurenine,Kyn)...     

克木毒蛋白三种酶活性与色氨酸残基的关系

对一种新的核糖体失活蛋白──克木毒蛋白的研究表明,该分子中仅含一个色氨酸.此色氨酸与克木毒蛋白具有的三种酶活性有明显不同的关系.     

嗜热链球菌dlt操纵子终端亚基stDltD的晶体结构

细胞表面多聚物的酰基跨膜修饰对增强细菌的致病性至关重要. DltA/B/C/D操纵子介导的脂磷壁酸（LTA）D-丙酰化修饰是革兰氏阳性菌中重要的一类后修饰,其调节膜内外的电荷平衡. DltA/B/C/D操纵子主要由DltA、DltB、DltC 和DltD四种蛋白质亚基组成,其催化机制与结构在生物进化中高度保守. DltA/DltC介导的胞内D-丙酰胺的转移机理已有深入的研究,而跨膜O-酰基转移酶DltB和dlt操纵子末端DltD介导的跨膜催化过程并不清楚. 本文解析了来源于嗜热链球菌 （S. thermophilus）中stDltD膜外结构域2.94 ?分辨率的晶体三维结构. 结构比对分析表明,stDltD是dlt操纵子终端的酰基转移酶,属于SGNH-like家族,stDltD的活性中心,包括4个blocks和催化三联体,都保守存在于多种革兰氏阳性病原菌中. 此外,结构叠合分析表明,stDltD催化中心形成的正电荷窝沟正好可以结合一个脂磷壁酸骨架的单体即甘油磷酸分子. 在前人的研究基础上,我们提出了一个由Dlt/A/B/C/D操纵子介导跨膜D-丙酰化修饰的工作模型. 本研究对进一步阐明DltD的生物学功能以及Dlt/A/B/C/D操纵子酰基跨膜修饰的分子机制具有重要意义.     

MiRNA调控IGFBPs参与肿瘤发生的研究进展

作为一类广泛存在于真核细胞中的RNA分子,微小RNA(microRNA,miRNA)在生物体内的基因表达调控、生长发育等基本生命过程中发挥着不可替代的重要作用,与人类疾病密切相关。胰岛素样生长因子结合蛋白(insulin-like growth factor binding proteins,IGFBPs)是一类结合和调节胰岛素样生长因子(insulin-like growth factor,IGF)生物活性的蛋白质,在肿瘤中通过IGF依赖或非依赖的机制扮演着至关重要的角色。在肿瘤中,IGFBPs和mi RNA的异常表达与肿瘤细胞的增殖、侵袭和转移等生物学行为密切相关。IGFBPs能够作为mi RNA的直接靶因或者下游信号分子,参与多种疾病的发生与发展。该文总结了mi RNA在脑、胃肠道、肝胆、妇科等多种肿瘤性疾病中对IGFBPs的调控作用,以期为相关疾病的临床诊断、预后生物标志物或治疗靶点等的探索提供全面、专业的参考。     

基于rhaSR嵌合操纵子的穿梭表达载体及其在鱼腥藻细胞中表达的初步研究

通过PCR等基因重组技术,以pKT-215（起源于RSF1010）为载体,构建了带有PR序列（含rhaSR操纵子表达调控元件、rhaR基因、报告基因gst的嵌合操纵子）和PT序列（含rhaSR操纵子表达调控元件及rhaT基因）的二种穿梭表达载体pKT～PTR、pKT—PR。其中,在RhaR基因上游设计和插入了增强的SD序列。首先将pKT—PTR、pKT—PR分别转化入大肠杆菌BL21（DE3）中,在不同培养基条件下,即舍鼠李糖与不含鼠李糖的培养基中进行表达,紫外分光光度计测定GST蛋白（谷胱甘肽-S-转移酶）的活性。结果表明,含嵌合操纵子的穿梭表达载体能在L-鼠李糖的诱导下表达,gst基因的表达量诱导备件下比非诱导条件下高3倍左右,且表达载体上的thaT基因并不能提高gst的表达;其次,利用三亲结合的方法将2种表迭质粒转入鱼腥藻Annbaena sp．PCC 7120中,抗生素筛选后得到能稳定遗传的转基因藻,并以转基因藻的质粒DNA为模板进行PCR检测。最后,转基因藻GST酶活力分析结果表明,嵌合操纵子PR在蓝藻细胞中的诱导表达效率极低,表达条件还需进一步优化。尝试将诱导型的操纵子转入蓝藻,具有潜在的应用价值。     

Resolution of the fluorescence equilibrium unfolding profile of trp aporepressor using single tryptophan mutants.

Single tryptophan mutants of the trp aporepressor, tryptophan 19-->phenylalanine (W19F) and tryptophan 99-->phenylalanine (W99F), were used in this study to resolve the individual steady-state and time-resolved fluorescence urea unfolding profiles of the two tryptophan residues in this highly intertwined, dimeric protein. The wild-type protein exhibits a large increase in fluorescence intensity and lifetime, as well as a large red shift in the steady-state fluorescence emission spectrum, upon unfolding by urea (Lane, A.N. & Jardetsky, O., 1987, Eur. J. Biochem. 164, 389-396; Gittelman, M.S. & Matthews, C.R., 1990, Biochemistry 29, 7011-7020; Fernando, T. & Royer, C.A., 1992, Biochemistry 31, 6683-6691). Unfolding of the W19F mutant demonstrated that Trp 99 undergoes a large increase in intensity and a red shift upon exposure to solvent. Lifetime studies revealed that the contribution of the dominant 0.5-ns component of this tryptophan tends toward zero with increasing urea, whereas the longer lifetime components increase in importance. This lifting of the quenching of Trp 99 may be due to disruption of the interaction between the two subunits upon denaturation, which abolishes the interaction of Trp 99 on one subunit with the amide quenching group of Asn 32 on the other subunit (Royer, C.A., 1992, Biophys. J. 63, 741-750). On the other hand, Trp 19 is quenched in response to unfolding in the W99F mutant. Exposure to solvent of Trp 19, which is buried at the hydrophobic dimer interface in the native protein, results in a large red shift of the average steady-state emission.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Tryptophan to Tryptophan Energy Transfer in Proteins

Förster resonance energy transfer (FRET) studies usually involve observation of intensity or life-time changes in the donor or acceptor molecule and usually these donor and acceptor molecules differ (heterotransfer). The use of polarization to monitor FRET is far less common, although it was one of the first methods utilized. In 1960, Weber demonstrated that homotransfer between tryptophan molecules contributes to depolarization. He also discovered that the efficiency of homotransfer becomes much less effective upon excitation near the red-edge of the absorption. This “red-edge effect” was shown to be a general phenomenon of homotransfer. We have utilized Weber's red-edge effect to study tryptophan homotransfer in proteins. Specifically, we determined the polarization of the tryptophan fluorescence upon excitation at 295 nm and 310 nm (near the red-edge). Rotational diffusion leads to depolarization of the emission excited at either 295 nm or 310 nm, but homotransfer only contributes to depolarization upon excitation at 295 nm. Hence, the 310/295 polarization ratio gives an indication of tryptophan to tryptophan energy transfer. In single tryptophan systems, the 310/295 ratios are generally below 2 whereas in multi-tryptophan systems, the 310/295 ratios can be greater than 3.     

The groESL Operon of the Halophilic Lactic Acid Bacterium Tetragenococcus halophila

The groESL operon of the halophilic lactic acid bacterium Tetragenococcus halophila was cloned by a PCR-based method. The molecular masses of GroES and GroEL proteins were calculated to be 10,153 and 56,893 Da, respectively. The amount of groESL mRNA was increased 3.8-fold by heat shock (45°C), and 4-fold by high NaCl (3-4 M). The Bacillus subtilis σ^A-like constitutive promoter existed in front of groES, and was used under both normal and stress (heat shock and high salinity) conditions.     

Differential expression of triplicate phosphoribosylanthranilate isomerase isogenes in the tryptophan biosynthetic pathway of Arabidopsis thaliana (L.) Heynh.

Phosphoribosylanthranilate isomerases (PAI) in the tryptophan biosynthetic pathway of Arabidopsis thaliana are encoded by a gene family. Expression patterns of each individual PAI isogene were investigated by analyzing expression of translation-fusions of promoter-β-glucuronidase (GUS) chimeras in transgenic plants. Quantification and histochemical staining of GUS activities expressed in PAI transgenic plants demonstrated that, first, expression of the three PAI isogenes was differentially regulated under normal growth conditions. Both PAI1 and PAI3 showed approximately 10-fold stronger expression than PAI2. Second, PAI isogenes differentially responded to environmental stresses such as ultraviolet irradiation and the abiotic elicitor silver nitrate. PAI2 displayed a stronger response to stresses than the other two PAI isogenes. Third, each individual PAI isogene was differentially expressed in a tissue- and cell-type-specific manner. Fourth, expression of PAI isogenes was coordinated to meet the requirement for normal growth and development of A. thaliana. Deletion of PAI1 is partially responsible for abnormal growth and development in the PAI deletion mutant trp6 as well as strong blue fluorescence in young leaves under ultraviolet irradiation. Received: 15 June 2000 / Accepted: 16 August 2000     

内皮联蛋白在血管生成中的调控机制及作用

机体新生血管的形成和稳态的维持是保证组织细胞正常生命活动的重要基础。参与血管生成这一生理过程的因子众多,血管生成机制复杂,该过程的异常与血管疾病、肿瘤和癌症的发生密切相关。内皮联蛋白(endoglin,ENG)是一种主要在内皮细胞上表达的I型跨膜糖蛋白,其作为转化生长因子β家族的辅助受体在调控血管生成与稳态中发挥着重要作用。随着基质金属蛋白酶14(matrix metalloproteinase 14,MMP14)、整合素、血管生成性糖蛋白LRG1(leucine-rich alpha-2-glycoprotein-1,LRG1)、G蛋白信号调节体-G alpha相互作用蛋白C端(GAIP interacting protein C-terminus,GIPC)等越来越多与ENG具有相互作用的蛋白质被鉴定出来,关于ENG调控血管生成的分子机制的研究已有了一定的进展,但各个蛋白质之间错综复杂的调控网络仍有待探究和梳理。阐明ENG及其互作的蛋白质的特征、对信号传导的影响和对血管生成的贡献,对于理解机体在生理、病理条件下如何精细、有序地调控血管生成至关重要,且对于相关疾病的临床治疗手段的研究具有重要意义。本文系统总结了ENG与TGF-β及非TGF-β家族相关蛋白质的互作及在调控血管生成中的作用,并对未来ENG相关研究方向做出展望,以期为研究ENG在相关血管疾病中的机制提供分子层面的指导。