蛋白质组学技术在感染研究中的应用

蛋白质组学的目标在于阐明特定生物体、组织、细胞或亚细胞结构中全部蛋白质的表达模式和功能模式,其技术平台由高通量的蛋白质分离技术、鉴定技术和生物信息学组成。在许多研究领域,蛋白质组学技术为阐明疾病过程和生命现象的分子机制提供了全面、网络和动态的蛋白质组信息。感染是重要的基本致病因素之一,蛋白质组学的研究策略和技术方法有利于快速分离鉴定病原体蛋白质组、宿主免疫细胞蛋白质组、感染相关蛋白、疫苗候：选抗原蛋白、生物标志物和药物靶标,从而明显加快病原体、宿主反应、感染发病机制以及感染预防、诊断和治疗等相关研究的进程。     

细胞质膜蛋白质组学研究技术进展

质膜蛋白在细胞中执行着非常重要的功能。随着蛋白质组学的发展,细胞质膜蛋白质组学成为蛋白质组学研究的重要组成部分,它为质膜蛋白的生物功能研究及药物靶标的发现提供了新的途径。然而,质膜蛋白丰度低、疏水性强,对现有蛋白质组学研究技术提出了挑战。简要综述了近年来质膜蛋白质组研究的相关技术进展,包括富集、提取分离鉴定方法及定量和生物信息学研究方法等。     

蛋白质组学在病毒致病机理研究中的应用

近年来,蛋白质组学技术成为医学研究的热点。蛋白质组学是高通量的分析正常及病理条件下机体、组织、细胞或亚细胞成分中的全部蛋白质。对不同空间、不同时间上动态变化的蛋白质组的整体进行比较,分析不同蛋白质组之间在表达数量、表达水平和修饰状态上的差异。蛋白质组学分析作为对生物代谢调控分析的技术手段,以病毒为研究的对象和工具,该技术的研究主要集中在新蛋白的发现、致病机理、疫苗的研制及耐药机制等方面。本文主要概述了蛋白质组学在一些动物传染病病毒致病方面研究和应用,分析了蛋白质组学技术对蛋白功能研究存在的问题和未来发展趋势,以便使研究者了解该技术使用的现状,提供理论参考。     

蛋白质组学研究相关技术及进展

蛋白质组学以蛋白质组为研究对象,应用相关研究技术,从整体水平上来认识蛋白的存在及活动方式。随着人类基因组计划的完成,蛋白质组学的研究也得到了快速发展,与蛋白质组学研究相关的一些技术也日益得到完善和提高。简要综述了近年来蛋白质组学研究中最为重要的样品制备、蛋白质分离、蛋白质鉴定等技术及研究进展。     

酵母双杂交技术及其在蛋白质组研究中的应用

蛋白质组学是后基因组时代出现的一个新兴的研究领域,它的主要任务是识别鉴定细胞,组织或机体的全部蛋白质,并分析蛋白质的功能及其模式。因此,揭示蛋白质组中蛋白质间的相互作用关系也是蛋白质组学的重要内容之一。酵母双杂交技术是用来检测蛋白质间是否相互作用的一个非常有效的手段,该技术在酵母蛋白质组研究中的初步成功应用,表明它有望在人类蛋白质且研究中发挥重要作用。     

生物质谱技术在蛋白质组学研究中的应用

随着技术的进步,蛋白质组学的研究重心由最初旨在鉴定细胞或组织内基因组所表达的全部蛋白质转移到从整个蛋白质组水平上阐述包括蛋白翻译后修饰、生物大分子相互作用等反映蛋白质功能的层次。多种质谱离子化技术的突破使质谱技术成为蛋白质组学研究必不可少的手段。质谱技术联合蛋白质组学多角度、深层次探索生命系统分子本质成为现阶段生命科学研究领域的主旋律之一。本文简要综述了肽和蛋白质等生物大分子质谱分析的原理、方式和应用,并对其发展前景做出展望。     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究,来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术,使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前,种子蛋白质组研究主要集中在两个方面：一方面是鉴定尽可能多的蛋白,以创建种子特定生命时期的蛋白质组参照图谱;另一方面主要集中在差异蛋白质组,通过比较分析不同蛋白质组,以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展,内容包括种子发育过程中蛋白质组的变化,与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响;并对种子蛋白质组研究的热点问题进行了展望。     

种子蛋白质组的研究进展

蛋白质组学是通过对全套蛋白质动态的研究, 来阐明生物体、组织、细胞和亚细胞全部蛋白质的表达模式及功能模式。大量可用的核苷酸序列信息和灵敏高速的质谱鉴定技术, 使得蛋白质组学方法为分析模式植物和农作物的复杂功能开辟了新的途径。目前, 种子蛋白质组研究主要集中在两个方面: 一方面是鉴定尽可能多的蛋白, 以创建种子特定生命时期的蛋白质组参照图谱; 另一方面主要集中在差异蛋白质组, 通过比较分析不同蛋白质组, 以探明关键功能蛋白。该文综述了近年来种子蛋白质组的研究进展, 内容包括种子发育过程中蛋白质组的变化, 与种子休眠/萌发相关的蛋白质组、翻译后修饰蛋白质组、细胞与亚细胞差异蛋白质组以及环境因子对种子蛋白质组的影响; 并对种子蛋白质组研究的热点问题进行了展望。     

叶绿体的蛋白质组学研究进展

蛋白质组学是以基因组编码的所有蛋白为研究对象,高通量地从细胞及整体水平上研究蛋白质的组成及其功能的新兴学科。在后基因组时代的今天,蛋白质组学的研究正逐渐深入到生命科学的各个领域,21世纪蛋白质组学将成为生命科学中最热门的学科。蛋白质组分析已成为鉴定植物功能的有力工具之一,叶绿体作为比较重要的细胞器,在植物蛋白质组学中已有较多的研究,,随着双向电泳技术的改进和质谱法的出现,并与不断增多的拟南芥、水稻、玉米等植物的序列数据相结合,叶绿体蛋白质组可以被快速鉴定。本文主要介绍了植物蛋白质组学、叶绿体及其蛋白质组学研究技术和研究进展,并对蛋白质组学的研究趋势进行了展望。     

病毒蛋白质组学: 病毒学研究前沿

病毒寄生于宿主细胞中, 需要不断地适应和改变宿主的环境. 它们能够编码多种多功能蛋白质, 这些蛋白能与宿主蛋白发生一系列的相互作用以完成病毒的各种功能. 迄今, 尽管许多病毒的基因组已测序完成, 但由于受到病毒影响而发生相应改变的宿主蛋白组、宿主蛋白翻译后修饰, 以及蛋白酶剪接过程还未被完全阐明. 近年来新兴的高通量技术, 如基于质谱技术的定量或半定量蛋白组方法, 已被广泛应用于病毒宿主相互作用的研究中, 且有望在上述领域取得突破性进展. 本综述主要探讨蛋白质组学研究中的病毒颗粒蛋白质组学, 病毒结构蛋白质组学和病毒影响的宿主蛋白质组学等病毒蛋白质组学中的前沿领域.     

病毒感染蛋白质组学研究进展

病毒的侵入会导致宿主细胞蛋白表达模式的改变,这种改变将影响宿主细胞的正常生理功能并决定病毒的致病进程和结果.因此,病毒感染蛋白质组学研究有助于揭示病毒与宿主的相互作用机制和病毒的分子致病机制,以及寻找病毒早期感染的分子标记、建立早期诊断方法、评价治疗效果和预后.本文介绍了病毒感染蛋白质组学研究技术、病毒诱导宿主细胞蛋白质组改变和病毒感染宿主血清差异蛋白质组等方面的研究进展.     

综述: 病毒与宿主细胞的糖基化修饰及相关功能

病毒的复制和对宿主的入侵与自身结构蛋白的糖基化修饰密切相关．对于宿主而言,在病毒感染宿主和宿主抗病毒的过程中,宿主的糖基化过程一方面可抑制病毒的复制和入侵,另一方面可促进病毒对宿主的感染,抑制宿主糖苷酶可抑制病毒的复制．从病毒方面来看,由于病毒自身缺乏糖基化修饰系统,病毒的糖基化过程是借宿主细胞内的合成系统对自身进行糖基化修饰．病毒的糖基化过程对病毒蛋白的折叠与稳定、病毒的感染和入侵、参与识别宿主细胞受体和参与病毒的免疫逃逸等过程起着重要的作用．随着糖基化研究技术的发展,以糖基化为基础的功能应用也越来越深入：如新型病毒疫苗和新型抗病毒药物的研制,以糖蛋白质组学研究为基础的质谱技术和生物信息学方法的发展,以及利用糖基化对病毒性疾病的诊断和治疗等,这些均为糖基化深入研究发展奠定了基础．本文就病毒与宿主细胞糖基化过程、相关功能以及研究应用等进展作一综述．     

White spot syndrome virus proteins and differentially expressed host proteins identified in shrimp epithelium by shotgun proteomics and cleavable isotope-coded affinity tag

Shrimp subcuticular epithelial cells are the initial and major targets of white spot syndrome virus (WSSV) infection. Proteomic studies of WSSV-infected subcuticular epithelium of Penaeus monodon were performed through two approaches, namely, subcellular fractionation coupled with shotgun proteomics to identify viral and host proteins and a quantitative time course proteomic analysis using cleavable isotope-coded affinity tags (cICATs) to identify differentially expressed cellular proteins. Peptides were analyzed by offline coupling of two-dimensional liquid chromatography with matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. We identified 27, 20, and 4 WSSV proteins from cytosolic, nuclear, and membrane fractions, respectively. Twenty-eight unique WSSV proteins with high confidence (total ion confidence interval percentage [CI%], >95%) were observed, 11 of which are reported here for the first time, and 3 of these novel proteins were shown to be viral nonstructural proteins by Western blotting analysis. A first shrimp protein data set containing 1,999 peptides (ion score, > or =20) and 429 proteins (total ion score CI%, >95%) was constructed via shotgun proteomics. We also identified 10 down-regulated proteins and 2 up-regulated proteins from the shrimp epithelial lysate via cICAT analysis. This is the first comprehensive study of WSSV-infected epithelia by proteomics. The 11 novel viral proteins represent the latest addition to our knowledge of the WSSV proteome. Three proteomic data sets consisting of WSSV proteins, epithelial cellular proteins, and differentially expressed cellular proteins generated in the course of WSSV infection provide a new resource for further study of WSSV-shrimp interactions.     

Poxvirus Proteomics and Virus-Host Protein Interactions

Summary: Studies of the functional proteins encoded by the poxvirus genome provide information about the composition of the virus as well as individual virus-virus protein and virus-host protein interactions, which provides insight into viral pathogenesis and drug discovery. Widely used proteomic techniques to identify and characterize specific protein-protein interactions include yeast two-hybrid studies and coimmunoprecipitations. Recently, various mass spectrometry techniques have been employed to identify viral protein components of larger complexes. These methods, combined with structural studies, can provide new information about the putative functions of viral proteins as well as insights into virus-host interaction dynamics. For viral proteins of unknown function, identification of either viral or host binding partners provides clues about their putative function. In this review, we discuss poxvirus proteomics, including the use of proteomic methodologies to identify viral components and virus-host protein interactions. High-throughput global protein expression studies using protein chip technology as well as new methods for validating putative protein-protein interactions are also discussed.     

New insights into viral structure and virus–cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus–cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV–cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.     

Molecular mimicry: a mechanism for autoimmune injury.

Many mechanisms may account for immune-mediated pathology after viral infections. Although several means have been hypothesized to play a role in disease, a widely accepted mechanism for viral-induced autoimmunity is molecular mimicry. It is thought that damage could result from an immune response to similar regions shared between virus and the host. Using computer-aided analysis, many sequence homologies have been identified between virus and host antigens. Using peptides corresponding to these regions, immunologic cross-reactivity has been found. In some cases, monoclonal antibodies to peptides of these regions have been shown to directly induce or augment disease in animal models. Using this approach to identify similar regions, it is possible to associate a known autoantigen with an infectious agent in autoimmune diseases in which there is no known etiologic agent. Conversely, it would also be possible to associate a known viral constituent with an unknown host antigen. Furthermore, identification of disease-inducing regions of autoantigens or viral proteins may lead to immunotherapeutic approaches to establish tolerance or anergy to such disease-inducing regions.     

Virological Surveillance of Aedes aegypti Vectors Identifies All Four Dengue Serotypes in a Hyperendemic Region

EcoHealth - Dengue virus (DENV) 1–4 is the etiological agent of dengue, the most important viral infection transmitted by Aedes spp mosquitoes to humans. Our goal was to identify the...     

Identification of a pyridopyrimidinone inhibitor of orthopoxviruses from a diversity-oriented synthesis library

Orthopoxviruses include the prototypical vaccinia virus, the emerging infectious agent monkeypox virus, and the potential biothreat variola virus (the causative agent of smallpox). There is currently no FDA-approved drug for humans infected with orthopoxviruses. We screened a diversity-oriented synthesis library for new scaffolds with activity against vaccinia virus. This screen identified a nonnucleoside analog that blocked postreplicative intermediate and late gene expression. Viral genome replication was unaffected, and inhibition could be elicited late in infection and persisted upon drug removal. Sequencing of drug-resistant viruses revealed mutations predicted to be on the periphery of the highly conserved viral RNA polymerase large subunit. Consistent with this, the compound had broad-spectrum activity against orthopoxviruses in vitro. These findings indicate that novel chemical synthesis approaches are a potential source for new infectious disease therapeutics and identify a potentially promising candidate for development to treat orthopoxvirus-infected individuals.     

New insights into viral structure and virus-cell interactions through proteomics

Although genomics techniques such as DNA microarrays have been widely used in virology, much more limited use has been made of proteomics. Although difficult, proteomics can greatly contribute to an understanding of virus-cell interactions, including the ternary structure of viral receptors at the cell surface, post-translational modifications and isoforms of critical viral and cellular proteins and even to the structure of viruses. Proteomics techniques also offer the potential for discovering markers for diagnostic and prognostic tests of viral infections in vivo. This review describes the use of several proteomic approaches for the analysis of HIV-cellular receptor interactions, the molecular mechanisms of transport of herpes simplex virus within neurons, and the structure of the tegument of herpes simplex virus.