肿瘤坏死因子与其受体相互作用的计算机模拟研究

利用同源模建方法,以ＴＮＦＲ５５受体胞外区的晶体结构为参考模板,预测了ＴＮＦＲ７５受体胞外区Ｃｙｓ１８～Ｐｈｅ１４７片段的三维结构．根据Ｒ５５受体胞外区与ＬＴ相结合的复合物的晶体结构,预测了ＴＮＦ与Ｒ５５及Ｒ７５胞外区的复合物的三维结构,模拟了ＴＮＦ与受体之间的相互作用．由于ＴＮＦ与受体的作用形式是三聚体对三聚体,因此在模拟ＴＮＦ与受体相互作用时选择了包括一个非对称的ＴＮＦ三聚体和一个受体（Ｒ５５或Ｒ７５）单体的模拟系统．结合已有的突变体实验结果,利用计算机模拟分析手段,发现了一些ＴＮＦ突变体之所以具有受体选择性的三维结构基础和发挥了关键作用的氨基酸残基以及这些残基之间的主要作用形式．研究深化了对已有的突变体实验结果的认识,建立了不同的实验结果之间的内在关联,为以后有目的的新型突变体设计和实验研究打下了基础．     

VLDL-受体的配体结合结构域结构分析

极低密度脂蛋白受体(VLDL-R)的配体结合域具有8个富含半胱氨酸的配体结合重复序列(ligand-binding repeats,LBR),被认为是与配体结合的部位。该受体与含7个类似重复序列的低密度脂蛋白受体(LDL-R)的配体结合特性明显不同。为了明确VLDL-R中8个LBR在配体结合中的作用并探讨结合位点的结构,本研究采用计算机辅助蛋白质结构预测方法,在二级结构分析的基础上,通过同源建模方法预测受体N-端328个氨基酸的配体结合域空间结构,结果显示该区域呈现弧形口袋样结构,其中前3个LBR结构紧凑,呈棒状,负电荷相对集中,推测这一特征结构是配体结合的重要结构基础,结构分析同时表明在LBR5与LBR6之间连接区的弹性结构可以赋予结合位点一定的伸缩性,利于其与不同配体的结合。本研究预测结果首次提出了VLDL-R配体结构域结构及结合位点的结构特征,并与已有的实验结果一致。     

t-PA K1区的同源模建及Kringle区与赖氨酸相互作用的研究

利用同源模建方法预测了t-PAK1区的三维结构。通过结构叠合确定了t-PAK1、K2区,纤溶酶原K1、K4区及UKK区的赖氨酸结合口袋。静电势计算及疏水性分析表明,在t-PAK2区以及纤溶酶原K1、K4区与纤维蛋白裸露的赖氨酸之间存在明显的静电势互补和疏水面契合。确定了影响Kringle区结合口袋与赖氨酸亲和的重要氨基酸,分析了t-PAK1区、UKK区不能结合赖氨酸的原因,由此设计了具有赖氨酸亲和力的新型t-PAK1区及UKK区突变体。利用模拟残基突变技术预测了突变体的结构变化,分析了突变后t-PAK1区及UKK区与赖氨酸亲和力的变化,初步在理论上肯定了设计方案的合理性。     

肿瘤坏死因子受体研究新进展

肿瘤坏死因子（ＴＮＦ）具有广泛学活性,其作用由其细胞表面受体介导。已知ＴＮＦ有两种不同类型的细胞表面受体,分子量分别５５ｋＤ和７５ｋＤ,目前均建立了其ｃＤＮＡ克隆,确定了其氨基酸顺序;两类可溶性ＴＮＦ受体也已被分离和鉴定,并生产出重组可溶性受体。     

肿瘤坏死因子结构研究进展

肿瘤坏死因子是α-肿瘤坏死因子和淋巴毒素的统称,它们具有相同的细胞受体.国内外学者在阐明人肿瘤坏死因子三级结构的基础上,结合使用化学修饰,抗 体结构域定位、定点诱变等方法对人肿瘤坏死因子结构与其功能的关系进行了研究,确定了人肿瘤坏死因子分子中对其活性至关重要的部位,同时也基本阐明了人α-肿瘤坏死因子的受体结合位点的氨基酸组成以及它们在人肿瘤坏死因子三、四级结构上的位置.     

抗人肿瘤坏死因子-α单链抗体与其配体的相互作用

为了在大肠杆菌中表达纯化抗人 TNF- α单链抗体并检测其结合活性与中和活性 .利用GST融合蛋白系统在大肠杆菌中表达抗人 TNF- α单链抗体 E6Sc Fv;分离包含体后进行变性和复性 ,再用亲和层析法进行纯化 ;用 ELISA法和酵母双杂交系统检测 E6Sc Fv与配体的结合 ;用 L92 9细胞检测 E6Sc Fv对人 TNF- α细胞毒作用的中和活性 .经变性 ,复性与亲和层析 ,E6Sc Fv被纯化 ,在 SDS- PAGE上为单一蛋白带 ;体外结合与中和实验表明 ,表达纯化的 E6Sc Fv可与人 TNF-α结合并中和其细胞毒活性 ;进一步用酵母双杂交系统证明当表达于细胞内时 ,E6Sc Fv仍保持了与TNF-α相结合的能力 .     

应用进化踪迹及分子动力学模拟研究beta2肾上腺素受体突变活性

β2肾上腺素受体(β2adrenergic receptor,β2AR)是G蛋白耦联受体(G protein coupled receptors,GPCRs)超家族中的一员,也是研究治疗哮喘的关键药物受体靶标.采用进化踪迹(evolutionary trace,ET)方法分析肾上腺素受体家族跨膜区片段序列,识别出了44个保守的残基,然后将β2肾上腺素受体以及受体D130N活性突变体、D79N失活突变体进行分子动力学模拟,试图找出与受体不同功能状态相关的结构动力学特征.发现受体DRY motif中的D130远离R131而转向K149残基这一结构特征与受体活性高度关联,此外,从残基相互作用的变化推断出了受体helix 2,4 and 6伴随着受体活化而发生的运动.这些研究结果对进一步探索β2肾上腺素受体突变体的激活机制以及所诱发疾病的分子机理提供了依据.     

肿瘤坏死因子受体超家族成员在免疫系统和疾病中的研究

肿瘤坏死因子受体超家族 (tumor necrosis factor receptor superfamily, TNFRSF) 是细胞因子受体的一个蛋白质超家族,其显著特征是通过细胞外富含半胱氨酸结构域结合肿瘤坏死因子(tumor necrosis factor,TNF)。肿瘤坏死因子受体(tumor necrosis factor receptors,TNFRs)是古老的细胞因子,TNFRs同源基因最早可追溯到节肢动物果蝇中。TNFRs在炎症反应、细胞凋亡、淋巴细胞稳态和组织发育中发挥重要的作用,TNFRs最主要的功能是与免疫系统相关。鉴于其在免疫系统中发挥重要的作用,肿瘤坏死因子受体家族成员已成为治疗糖尿病、动脉粥样硬化、骨质疏松、自身免疫性疾病、移植排斥反应和癌症等人类疾病的靶点。随着科学技术发展,关于TNFRs的功能有了新的进展,在无脊椎动物和低等脊椎动物中已经有大量报道。在本篇综述中,主要总结了在高等哺乳动物中发现的29种TNFR成员的相关报道,包括8种死亡受体和21种非死亡受体,主要涉及在免疫系统以及与疾病相关领域的研究。大多数研究处于基础实验阶段,少数走向临床研究的案例取得的临床效果并不理想,靶向设计针对自身免疫性疾病、炎症和肿瘤疾病的治疗方案需要更深入的理解TNFRs功能。本文旨在对TNFRs成员发挥的功能有进一步的认识。     

肿瘤坏死因子受体超家族成员在免疫系统和疾病中的研究

肿瘤坏死因子受体超家族 (tumor necrosis factor receptor superfamily, TNFRSF) 是细胞因子受体的一个蛋白质超家族,其显著特征是通过细胞外富含半胱氨酸结构域结合肿瘤坏死因子(tumor necrosis factor,TNF)。肿瘤坏死因子受体(tumor necrosis factor receptors,TNFRs)是古老的细胞因子,TNFRs同源基因最早可追溯到节肢动物果蝇中。TNFRs在炎症反应、细胞凋亡、淋巴细胞稳态和组织发育中发挥重要的作用,TNFRs最主要的功能是与免疫系统相关。鉴于其在免疫系统中发挥重要的作用,肿瘤坏死因子受体家族成员已成为治疗糖尿病、动脉粥样硬化、骨质疏松、自身免疫性疾病、移植排斥反应和癌症等人类疾病的靶点。随着科学技术发展,关于TNFRs的功能有了新的进展,在无脊椎动物和低等脊椎动物中已经有大量报道。在本篇综述中,主要总结了在高等哺乳动物中发现的29种TNFR成员的相关报道,包括8种死亡受体和21种非死亡受体,主要涉及在免疫系统以及与疾病相关领域的研究。大多数研究处于基础实验阶段,少数走向临床研究的案例取得的临床效果并不理想,靶向设计针对自身免疫性疾病、炎症和肿瘤疾病的治疗方案需要更深入的理解TNFRs功能。本文旨在对TNFRs成员发挥的功能有进一步的认识。     

A model of the complex between interleukin-4 and its receptors

A three-dimensional model of interleukin-4 (IL-4) bound to one molecule each of the high- and low-affinity receptors (IL-4R and IL-2Rγ) was built, using the crystal structure of the complex of human growth hormone (HGH) with its receptor (HGHR) as a starting model. The modeling of IL-4 with its receptors was based on the conservation of the sequences and on the predicted structural organization for cytokine receptors, and assuming that the binding mode of the ligands would be similar. Analysis of the interface between IL-4 and both receptor molecules was carried out to reveal which residues are important for complex formation. The modeling procedures showed that there were no major problems in maintaining a reasonable fit of IL-4 with the two receptor molecules, in a manner analogous to the complex of HGH–HGHR. Many of the residues that appear by modeling to be important for binding between IL-4 and the receptors have been previously implicated in that role by different methods. A striking motif of aromatic and positively charged residues on the surface of the C-terminal domains of the receptors is highly conserved in the structure of HGH–HGHR and in the models of IL-4 complexed with its receptors. © 1995 Wiley-Liss, Inc.     

Three dimensional modeling of N-terminal region of galanin and its interaction with the galanin receptor

The neuropeptide galanin comes under the powerful and versatile modulators of classical neurotransmitters and is present in brain tissues, which are intimately involved in epileptogenesis. It acts as appealing targets for studying basic mechanisms of seizure initiation and arrest, and for the development of novel approaches for various neurodegenerative diseases. Galanin is widely distributed in the mammalian brain which controls various processes such as sensation of pain, learning, feeding, sexual behaviour, carcinogenesis, pathophysiology of neuroendocrine tumors and others. The function of galanin can be exploited through its interaction with three G-protein coupled receptors subtypes such as GalR1, GalR2 and GalR3. The N-terminal region of galanin comprises about highly conserved 15 amino acid residues, which act as the crucial region for agonist-receptor binding. We have constructed a theoretical structural model for the N-terminal region of galanin from Homo sapiens by homology modeling. The stereochemistry of the model was checked using PROCHECK. The functionally conserved regions were identified by surface mapping of phylogenetic information generated by online web algorithm ConSurf. The docking studies on the pharmacologically important galanin receptors with the theoretical model of N-terminal region of galanin predicted crucial residues for binding which would be useful in the development of novel leads for neurodegenerative disorders.     

1,3-丙二醇氧化还原酶结构与功能关系的探讨

用生物信息学方法对来源于klebsiella pneumonide的1,3-丙二醇氧化还原酶(即1,3-丙二醇氧化还原酶,PDOR)进行高级结构模建,并搜索其功能位点,以所得三维结构为对象,定位其铁信号结合位点、辅酶NADP大致位置以及可能的底物结合部位;在此基础上模拟PDOR活性部位,探讨该酶的构效关系。     

白细胞介素6及其受体结构与功能关系研究进展

白细胞介素6(IL-6)的多种生物学功能是通过其特异性受体白细胞介素6受体(IL-6R)和公共受体gp130共同传递的。近年来,对IL-6、IL-6R及gp130的结构与功能进行了大量的研究工作,为进一步揭示它们之间作用的分子机制以及该系统拮抗剂设计提供了思路。     

轮状病毒与细胞表面受体相互作用的分子机制研究

目的轮状病毒是导致婴幼儿重症腹泻的重要病因。轮状病毒感染宿主细胞是一个多因素参与的复杂过程,包括病毒表面两种外壳蛋白与细胞表面唾液酸、整合素、热应激同源蛋白70等多种受体分子的相互作用。就轮状病毒与细胞受体相互作用的分子机制作了简要论述。     

Characterization of the interaction between anthrax toxin and its cellular receptors

Mutations in capillary morphogenesis gene 2 (CMG2), one of the two closely related proteins that act as anthrax toxin receptors, cause two rare human autosomal recessive conditions, juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). Here we demonstrate that CMG2 proteins with certain JHF- and ISH-associated single amino acid substitutions in their von Willebrand factor A domain or transmembrane region do not function as anthrax toxin receptors. However, an ISH-associated CMG2 variant having a truncated cytosolic domain does still function as an anthrax receptor, and in fact makes cells hyper-sensitive to toxin, distinguishing the roles of CMG2 in physiology and anthrax pathology. Site-specific mutagenesis was used to characterize the role that domain 2 of the anthrax toxin protective antigen (PA) plays in interaction with CMG2, focusing on the interaction between the PA 2beta(3)-2beta(4) loop and a pocket (Glu-122 pocket) adjacent to the metal ion-dependent adhesion site in CMG2. Substitutions that disrupted the salt bridge between PA Arg-344 and CMG2 Glu-122 decreased the affinity of PA to CMG2 three- to fourfold. Furthermore, mutation of CMG2 Tyr-119 (within the Glu-122 pocket) to His lowered the pH threshold for PA prepore-to-pore conversion in the endocytic pathway.     

Homology modeling of human serum paraoxonase1 and its molecular interaction studies with aspirin and cefazolin

Human serum paraoxonase1 (HuPON1) belongs to the family of A-esterases (EC.3.1.8.1). It is associated with HDL particle and prevents atherosclerosis by cleaving lipid hydroperoxides and other proatherogenic molecules of oxidized low density lipoproteins (LDL). Since the precise structure of HuPON1 is not yet available, the structure-function relationship between HuPON1 and activators/inhibitors is still unknown. Therefore, a theoretical model of HuPON1 was generated using homology modelling and precise molecular interactions of an activator aspirin and an inhibitor cefazolin with PON1 were studied using Autodock software. The ligand binding residues were found to be similar to the predicted active site residues. Both cefazolin and aspirin were found to dock in the vicinity of the predicted active sites of PON1; cefazolin bound at residues N166, S193 and Y71, while aspirin at residues N309, I310 and L311. Binding region in the PON1 by prediction (3D2GO server) and docking studies provide useful insight into mechanism of substrate and inhibitor binding to the enzyme active site.     

The Relay/Converter Interface Influences Hydrolysis of ATP by Skeletal Muscle Myosin II

The interface between relay and converter domain of muscle myosin is critical for optimal myosin performance. Using Drosophila melanogaster indirect flight muscle S1, we performed a kinetic analysis of the effect of mutations in the converter and relay domain. Introduction of a mutation (R759E) in the converter domain inhibits the steady-state ATPase of myosin S1, whereas an additional mutation in the relay domain (N509K) is able to restore the ATPase toward wild-type values. The R759E S1 construct showed little effect on most steps of the actomyosin ATPase cycle. The exception was a 25–30% reduction in the rate constant of the hydrolysis step, the step coupled to the cross-bridge recovery stroke that involves a change in conformation at the relay/converter domain interface. Significantly, the double mutant restored the hydrolysis step to values similar to the wild-type myosin. Modeling the relay/converter interface suggests a possible interaction between converter residue 759 and relay residue 509 in the actin-detached conformation, which is lost in R759E but is restored in N509K/R759E. This detailed kinetic analysis of Drosophila myosin carrying the R759E mutation shows that the interface between the relay loop and converter domain is important for fine-tuning myosin kinetics, in particular ATP binding and hydrolysis.     

Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes

We have overexpressed in Escherichia coli the thymidylate kinase of Mycobacterium tuberculosis (TMPKmt). Biochemical and physico-chemical characterization of TMPKmt revealed distinct structural and catalytic features when compared to its counterpart from yeast (TMPKy) or E. coli (TMPKec). Denaturation of the dimeric TMPKmt by urea under equilibrium conditions was studied by intrinsic fluorescence and circular dichroism (CD) spectroscopy. It suggested a three-state unfolding mechanism with a monomeric intermediate. On the other hand, 3'-azido-3'-deoxythymidine monophosphate (AZT-MP), which is substrate for TMPKy and TMPKec acts as a potent competitive inhibitor for TMPKMT: We propose a structural model of TMPKmt in which the overall fold described in TMPKy and TMPKec is conserved and slight differences at the level of primary and 3D-structure explain strong variations in the phosphorylation rate of substrate analogs. According to the model, we synthesized dTMP analogs acting either as substrates or specific inhibitors of TMPKMT: This approach based on slight structural differences among similar proteins could be applied to other essential enzymes for the design of new species-specific antimicrobials.