单分子荧光共振能量转移用于生物大分子构象动态变化过程研究进展

生物大分子参与生命活动的各个过程,在单分子水平上实时观测和分析生物大分子自身的结构动态以及生物大分子相互作用的动态过程,对于深入理解生物大分子的作用机制具有重要意义。自提出以来,单分子荧光共振能量转移技术逐渐展现出其在研究生物大分子构象变化和相互作用过程等方面的巨大潜力,一系列新的作用机理陆续被提出。本文对单分子荧光共振能量转移技术在蛋白质与核酸分子构象动态变化、蛋白质-蛋白质相互作用以及蛋白质-核酸相互作用等方面取得的研究进展进行了综述。     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

生物分子的单分子检测研究

本文评述了生物单分子检测的方法及其在生物大分子结构与功能之间的关系、酶的活性、反应动力学、分子构象、DNA和RNA的转录、蛋白质折叠等生物学重要问题研究上的应用。对生物单分子检测技术这一研究领域的发展趋势作了展望。     

Vitamin D3对儿童桥本甲状腺炎IL-10、IL-12的调节作用

目的:分别以IL-12、IL-10作为Th1、Th2的代表,在1,α25(OH)2D3的干预下,在单核细胞水平分析人外周血单核细胞(PBMC)中IL-10、IL-12在儿童桥本甲状腺炎(HT)中的细微变化,并探讨1α,25(OH)2D3的免疫调节作用。方法:以27例HT患儿为研究对象,17名健康儿童作对照,采集外周静脉血,一部分用于分离并培养单核细胞,设1,α25(OH)2D3干预组和对照组,收集培养上清液,ELISA检测上清液中IL-10、IL-12水平。另一部分静脉血分离出血清,再提取25(OH)D3,用放射免疫法(RIA)测定25(OH)D3水平。结果:HT组25(OH)D3显著低于健康对照,分别为(21.85±5.73)ng/mL和(27.56±7.46)ng/mL(P<0.01),且HT组PBMC产生IL-12水平显著高于健康对照,分别为(119.18±28.65)pg/mL和(102.84±23.86)pg/mL(P<0.01),而IL-10的表达显著低于健康对照,分别为(132.99±12.04)pg/mL和(171.41±35.72)pg/mL(P<0.01)。1α,25(OH)2D3干预后HT患儿IL-12表达显著下调为(98.57±11.98)pg/mL(P<0.01),IL-10表达在HT组和健康对照组均上调,分别为(184.15±35.34)pg/mL(P<0.01)、(223.77±53.36)pg/mL(P<0.01),HT组ρ(IL-10)/ρ(IL-12)比值显著升高。结论:HT患儿的25(OH)D3水平不足。1α,25(OH)2D3参与单核细胞分泌细胞因子的调节过程,能抑制HT增强的Th1型细胞因子IL-12分泌,增加IL-10表达,纠正n(Th1)/n(Th2)细胞因子失衡。     

配体活性构象搜寻方法及其应用研究——Ⅰ.搜寻方法及凝血酶抑制剂活性构象的搜寻

在综合系统构象搜寻和配体-生物大分子对接(Dock)方法的基础上,发展了根据受体活性部位三维结构搜寻配体活性构象的搜寻方法BCSPL.用此方法搜寻了凝血酶抑制剂PPACK的活性构象,结果与晶体结构非常吻合,又用此方法搜寻了膦酰肽类和二肽、三肽类凝血酶抑制剂与人体α凝血酶结合时的活性构象,并在此基础上用分子力学计算了抑制剂与凝血酶的结合能,结果表明结合能与活性有很好的相关性,计算结果能合理地解释抑制剂与凝血酶的相互作用方式及结构与活性的关系.     

手性药物与生物大分子相互作用中对映选择性的表征方法研究进展

手性药物在临床上有着重要的影响,已成为人们关注的一个重点问题。本文对手性药物小分子与生物大分子间相互作用中对映选择性分析方法的研究进展作了概述,重点介绍了分子光谱分析法、等温滴定量热法、手性传感器、原子力显微镜、分子对接模拟等方法在手性药物小分子与生物大分子间对映选择性相互作用表征方法中的应用与进展。     

离子液与蛋白质和核酸相互作用的研究

离子液因其具有良好的生物兼容性和独特的理化性质,近年来在生物催化和生物大分子蛋白质与核酸的分离分析领域得到广泛应用。离子液与生物大分子相互作用的研究是离子液相关理论与应用研究的基础,有关离子液与蛋白质和核酸相互作用的机理研究受到关注。本文简要介绍了常用离子液的分类,离子液与蛋白质分子作用的机理,离子液与核酸分子作用的机理,以及离子液在酶催化反应、生物分子分离、生物分子电化学分析和毛细管电泳分析中的应用,并主要综述了近年的相关研究和应用进展。     

共振光散射技术的原理及其在生化研究和分析中的应用

共振光散射技术是一项在普通荧光分光光度法计上进行测量的光散射分析技术。本文在简要介绍该技术的基础上，作了可能的原理的探索和定量基础讨论，就共振光散射技术在有机染料分子的聚集、在生物大分子上的堆积以及对生物大分子构象所产生的影响方面作了综述，简要评价该技术在生物大分子分析中的应用，并对其在生化研究和分析中可能的发展进行了预测。     

变构受体分子的设计及其在分子动态识别中的应用

变构调节(allostericregulation)普遍存在于自然界中,是生物体系实行精妙调控和准确表达的有效途径之一 [1]。在生物体系中,蛋白质分子具有的特定空间构象是表达其生物学功能所必需的,某些含有亚基的蛋白质,其功能往往是通过构象的变化来调节的。20世纪 60年代,Monod[2]在研究血红蛋白与氧结合时提出了蛋白质的变构效应。血红蛋白由两条α 亚基和两条β 亚基相互折叠彼此缠绕而成,亚基与亚基之间由于次级键的存在,处于T态 (tensestate),对氧的亲和力低于单独的α 亚基或β 亚基对氧的亲和力。随着氧浓度提高,氧首先与一个α 亚基中的亚…     

小分子与生物大分子间非共价相互作用分析方法研究进展

对小分子与生物大分子间非共价相互作用分析方法的研究进展作了较详细的评述。重点介绍了光谱、电化学、核磁共振、质谱等方法在小分子与生物大分子间相互作用研究中的应用及进展,总结了这些分析方法的优缺点,引用文献56篇。     

Inhibiting the NLRP3 Inflammasome

Inflammasomes are protein complexes which are important in several inflammatory diseases. Inflammasomes form part of the innate immune system that triggers the activation of inflammatory cytokines interleukin (IL)-1β and IL-18. The inflammasome most studied in sterile inflammation and non-communicable disease is the NLRP3 inflammasome. Upon activation by diverse pathogen or disease associated signals, NLRP3 nucleates the oligomerization of an adaptor protein ASC forming a platform (the inflammasome) for the recruitment and activation of the protease caspase-1. Active caspase-1 catalyzes the processing and release of IL-1β and IL-18, and via cleavage of the pore forming protein gasdermin D can drive pyroptotic cell death. This review focuses on the structural basis and mechanism for NLRP3 inflammasome signaling in the context of drug design, providing chemical structures, activities, and clinical potential of direct inflammasome inhibitors. A cryo-EM structure of NLRP3 bound to NEK7 protein provides structural insight and aids in the discovery of novel NLRP3 inhibitors utilizing ligand-based or structure-based approaches.     

NLRP3 Inflammasome Inhibitors in Cardiovascular Diseases

Virtually all types of cardiovascular diseases are associated with pathological activation of the innate immune system. The NACHT, leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is a protein complex that functions as a platform for rapid induction of the inflammatory response to infection or sterile injury. NLRP3 is an intracellular sensor that is sensitive to danger signals, such as ischemia and extracellular or intracellular alarmins during tissue injury. The NLRP3 inflammasome is regulated by the presence of damage-associated molecular patterns and initiates or amplifies inflammatory response through the production of interleukin-1β (IL-1β) and/or IL-18. NLRP3 activation regulates cell survival through the activity of caspase-1 and gasdermin-D. The development of NLRP3 inflammasome inhibitors has opened the possibility to targeting the deleterious effects of NLRP3. Here, we examine the scientific evidence supporting a role for NLRP3 and the effects of inhibitors in cardiovascular diseases.     

The Novel Role of MIF in the Secretion of IL-25, IL-31, and IL-33 from PBMC of Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is an autoimmune inflammatory joint disease with complex pathogenesis associated with cytokine dysregulation. Macrophage migration inhibitory factor (MIF) plays a role in systemic inflammation and joint destruction in RA and could be associated with the secretion of other immune-modulatory cytokines such as IL-25, IL-31, and IL-33. For the above, our main aim was to evaluate the IL-25, IL-31, and IL-33 secretion from recombinant human MIF (rhMIF)-stimulated peripheral blood mononuclear cells (PBMC) of RA patients. The rhMIF and lipopolysaccharide (LPS) plus rhMIF stimuli promote the secretion of IL-25, IL-31, and IL-33 (p < 0.05) from PBMC of RA patients. The study groups, the different stimuli, and the interaction between both showed a statistically significant effect on the secretion of IL-25 (p < 0.05) and IL-31 (p < 0.01). The study of the effect of the RA patient treatments and their interaction with the effect of stimuli did not show an interaction between them. In conclusion, our study generates new evidence for the role of MIF in the secretion of IL-25, IL-31, and IL-33 and its immunomodulatory effect on RA.     

Insight into the structures of Interleukin-18 systems

Structure-based molecular designs play a critical role in the context of next generation drug development. Besides their fundamental scientific aspects, the findings established in this approach have significant implications in the expansions of target-based therapies and vaccines. Interleukin-18 (IL-18), also known as interferon gamma (IFN-γ) inducing factor, is a pro-inflammatory cytokine. The IL-18 binds first to the IL-18α receptor and forms a lower affinity complex. Upon binding with IL-18β a hetero-trimeric complex with higher affinity is formed that initiates the signal transduction process. The present study, including structural and molecular dynamics simulations, takes a close look at the structural stabilities of IL-18 and IL-18 receptor-bound ligand structures as functions of time. The results help to identify the conformational changes of the ligand due to receptor binding, as well as the structural orders of the apo and holo IL-18 protein complexes.     

Selective inhibition of the K+ efflux sensitive NLRP3 pathway by Cl− channel modulation

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, and contributes to inflammation exacerbating disease. Fenamate non-steroidal anti-inflammatory drugs (NSAIDs) were recently described as NLRP3 inflammasome inhibitors via chloride channel inhibition. Fenamate NSAIDs inhibit cyclooxygenase (COX) enzymes, limiting their potential as therapeutics for NLRP3-associated diseases due to established side effects. The aim here was to develop properties of the fenamates that inhibit NLRP3, and at the same time to reduce COX inhibition. We synthesised a library of analogues, with feedback from in silico COX docking potential, and IL-1β release inhibitory activity. Through iterative screening and rational chemical design, we established a collection of chloride channel inhibiting active lead molecules with potent activity at the canonical NLRP3 inflammasome and no activity at COX enzymes, but only in response to stimuli that activated NLRP3 by a K⁺ efflux-dependent mechanism. This study identifies a model for the isolation and removal of unwanted off-target effects, with the enhancement of desired activity, and establishes a new chemical motif for the further development of NLRP3 inflammasome inhibitors.

The NLRP3 inflammasome regulates production of the pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18, and contributes to inflammation exacerbating disease.     

Chemical Modulation of the 1-(Piperidin-4-yl)-1,3-dihydro-2H-benzo[d]imidazole-2-one Scaffold as a Novel NLRP3 Inhibitor

In the search for new chemical scaffolds able to afford NLRP3 inflammasome inhibitors, we used a pharmacophore-hybridization strategy by combining the structure of the acrylic acid derivative INF39 with the 1-(piperidin-4-yl)1,3-dihydro-2H-benzo[d]imidazole-2-one substructure present in {"type":"entrez-nucleotide","attrs":{"text":"HS203873","term_id":"313347069"}}HS203873, a recently identified NLRP3 binder. A series of differently modulated benzo[d]imidazole-2-one derivatives were designed and synthesised. The obtained compounds were screened in vitro to test their ability to inhibit NLRP3-dependent pyroptosis and IL-1β release in PMA-differentiated THP-1 cells stimulated with LPS/ATP. The selected compounds were evaluated for their ability to reduce the ATPase activity of human recombinant NLRP3 using a newly developed assay. From this screening, compounds 9, 13 and 18, able to concentration-dependently inhibit IL-1β release in LPS/ATP-stimulated human macrophages, emerged as the most promising NLRP3 inhibitors of the series. Computational simulations were applied for building the first complete model of the NLRP3 inactive state and for identifying possible binding sites available to the tested compounds. The analyses led us to suggest a mechanism of protein–ligand binding that might explain the activity of the compounds.     

定点突变提高IL-4免疫毒素对淋巴瘤的选择性和杀伤活性

采用软件分析选择与IL-4分子结合与活性相关的重要位点13T,121R,通过定点突变得到IL-4突变基因cpIL4(13D121E),将其与绿脓杆菌外毒素突变基因PE38KDEL融合,成功地构建了编码免疫毒素cpIL4(13D121E)-PE38KDEL的融合基因.该基因在原核表达系统中得到了高效表达,表达量占细胞全蛋白的30%以上.表达产物经亲和色谱和阴离子交换色谱纯化后,进行细胞毒性实验,证明其对表达型IL-4受体的淋巴瘤细胞Daudi具有良好的细胞毒作用,活性是同类型IL-4免疫毒素的2倍,而对表达型IL-4受体的内皮细胞活性较低.     

Synthesis of DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] Derivatives and Their Regulatory Effects on Pro-Inflammatory Cytokine Production in IL-1β-Stimulated Primary Human Oral Cells

Interleukin-1 beta (IL-1β) has diverse physiological functions and plays important roles in health and disease. In this report, we focus on its function in the production of pro-inflammatory cytokines, including IL-6 and IL-8, which are implicated in several autoimmune diseases and host defense against infection. IL-1β activity is markedly dependent on the binding affinity toward IL-1 receptors (IL-1Rs). Several studies have been conducted to identify suitable small molecules that can modulate the interactions between 1L-1β and 1L-1R1. Based on our previous report, where DPIE [2-(1,2-Diphenyl-1H-indol-3-yl)ethanamine] exhibited such modulatory activity, three types of DPIE derivatives were synthesized by introducing various substituents at the 1, 2, and 3 positions of the indole group in DPIE. To predict a possible binding pose in complex with IL-1R1, a docking simulation was performed. The effect of the chemicals was determined in human gingival fibroblasts (GFs) following IL-1β induction. The DPIE derivatives affected different aspects of cytokine production. Further, a group of the derivatives enabled synergistic pro-inflammatory cytokine production, while another group caused diminished cytokine production compared to DPIE stimulation. Some groups displayed no significant difference after stimulation. These findings indicate that the modification of the indole site could modulate IL-1β:IL1R1 binding affinity to reduce or enhance pro-inflammatory cytokine production.