蛋白质组学在干细胞研究中的应用

蛋白质组学技术通过整合多项技术来分析生物体的全部蛋白质成分,通过考察不同状态下细胞或组织蛋白质组的变化情况来了解细胞活动的分子机理。干细胞分化过程中受外界条件的影响其蛋白表达模式也表现出一定的差异,对干细胞分化过程中进行蛋白质组学研究将有利于从蛋白质分子水平上阐明干细胞的分化机理。本文对蛋白质组学及其在干细胞研究中的应用加以评述。     

分子与细胞事件的光学可视化——解读2008年诺贝尔化学奖

绿色荧光蛋白（green fluorescent protein,GFP）及其突变体作为报告基因,已被广泛应用于基因表达调控、蛋白质空间定位、生物分子之间相互作用、转基因动物以及药物药效评价和作用机理研究等方面,极大地推动了现代生物学的发展．随着光电信息技术的不断进步,基于荧光报告基因的光学分子成像技术将在细胞、细胞网络、组织、器官和个体等不同层次实现分子与细胞事件的实时可视化,从而在重大疾病的早期诊断和药物研发中发挥重要作用．     

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。     

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。     

羟基喜树碱诱导肝癌细胞凋亡前后细胞核蛋白质组变化的定量分析

对羟基喜树碱诱导肝癌细胞凋亡前后的细胞核蛋白质组进行定量研究,获得凋亡前后细胞核蛋白质组中差异蛋白表达量相对变化的信息,为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供实验依据。分离提取羟基喜树碱诱导肝癌细胞凋亡前后的细胞核并鉴定其纯度,用含稳定同位素的化学试剂c-ICAT标记细胞凋亡前后的细胞核蛋白,对细胞核标记蛋白进行消化和纯化,利用基于多维色谱-线性离子阱/静电场轨道阱质谱联用技术的鸟枪（Shotgun）法策略及c-ICAT定量策略分析鉴定蛋白质,获得同一种肝癌细胞核蛋白质在凋亡前后细胞中的表达量变化比值。分析鉴定了在细胞凋亡前后的表达量差异有显著统计学意义（P〈0.05）的肝癌细胞核蛋白42个,其中,12个蛋白的表达量在羟基喜树碱诱导细胞凋亡后下调,30个蛋白的表达量在凋亡细胞中上调。这些差异蛋白的分子功能主要与细胞增殖、凋亡和分化、能量代谢、核酸代谢以及细胞骨架相关。     

植物应答非生物胁迫的蛋白质组学研究进展

逆境对植物的生长发育产生不利影响,植物在长期适应环境中演化出了相应的机制。蛋白质是基因表达的最终产物,是细胞生命活动的基础,在逆境条件下,许多与逆境相关的蛋白质表达量发生变化。蛋白质组学技术的发展为鉴定和研究逆境响应相关蛋白提供了手段,为阐述逆境应答分子机理提供了重要的依据。本综述根据近年来采用蛋白质组学研究植物应答非生物胁迫(干旱,高盐和低温)的结果,总结并讨论了不同逆境下蛋白表达的组织特异性特点,旨在为理解植物的逆境胁迫响应机制提供更多信息。     

酸胁迫下短乳杆菌NCL912蛋白质的差异表达及其作用

【目的】本文从蛋白质组水平,对本实验室分离的一株高产γ-氨基丁酸的短乳杆菌NCL912(Lactobacillus brevis)在酸胁迫下蛋白质的差异表达及其应激机理进行探讨。【方法】利用双向凝胶电泳技术对pH 5.0和pH 4.0条件下,不含L-谷氨酸钠的培养物的蛋白质组电泳图谱进行了分析,并对酸胁迫下差异表达的蛋白进行了比较。利用质谱检测技术和生物信息学技术对这些差异表达的蛋白进行了鉴定、功能分类和代谢途径分析等。【结果】通过双向凝胶电泳技术,可以得到均匀、背景清晰、分辨率高、重复性好的Lb.brevis NCL912的双向凝胶电泳图谱。对pH 5.0和pH 4.0条件下培养的该菌总蛋白质电泳图谱进行比较,发现有25个差异表达的蛋白点。对这25个差异表达的蛋白进行了质谱鉴定。由于缺乏短乳杆菌NCL912的全基因组,所以其中只有8个蛋白点被质谱鉴定和分析得到。它们分别参与了蛋白质的合成、核苷酸的合成、糖酵解代谢、细胞能量水平的调节等。【结论】酸应激下这些表达蛋白质可通过其相应的功能来保护细胞耐受酸胁迫,从而使菌能够在酸性环境下生存增值。这可能就是Lb.brevis NCL912的酸胁迫应激机理之一。     

应用差异蛋白质组学方法分析作物化感作用的分子机理

试验旨在分析运用分子标记技术(QTL)和差异蛋白组学技术研究作物化感作用分子机理的差异性。首先运用差异蛋白组学技术探讨在生物胁迫(稗草)下水稻化感作用潜力变化的内在分子机理。分别用稗草和水稻的根系分泌物培养切自一株5叶龄化感水稻P I312777植株并经恢复的2个分蘖。7d后,提取处理和对照相同叶位叶片的全蛋白质并进行双向电泳,每张电泳胶片上获得800多个电泳胶点,其中差异表达的蛋白质点有4个。采用M ALD I-TOF-M S对各差异蛋白质点进行肽质量指纹图谱分析,经过SW ISS-PROT数据库查询,结果表明化感水稻P I312777在稗草胁迫下的特异蛋白分别与苯丙氨酸氨解酶(PAL)、硫还原型蛋白(T rx-m)、3-羟基-3-甲基戊二酰辅酶A还原酶(HM GR)和过氧化物酶(POD)相匹配。根据编码以上4个差异蛋白质的DNA序列,发现编码以上4个差异蛋白的基因分别位于水稻染色体4、7、8和12上的特定克隆位点,这就是与化感作用相关基因。前人也运用QTL方法开展作物化感作用的分子机理研究,但由于所采用的供体材料、受体植物及对表型性状的评价方法等的不同,定位结果存在较大的差异。综合比较两种方法后认为,运用差异蛋白组学技术分析水稻化感作用的分子机理,比QTL技术更加直接和深入。因为比较胁迫处理和对照植物组织的2-DE图谱将能鉴定出由表达候选基因编码的胁迫蛋白质,氨基酸残基序列的测定将揭示那些功能与胁迫性状密切相关的蛋白质,这种编码的基因就是兼具功能与表达的候选基因。     

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.     

Low doses of niclosamide and quinacrine combination yields synergistic effect in melanoma via activating autophagy-mediated p53-dependent apoptosis

Malignant melanoma is a highly aggressive, malignant, and drug-resistant tumor. It lacks an efficient treatment approach. In this study, we developed a novel anti-melanoma strategy by using anti-tapeworm drug niclosamide and anti-malarial drug quinacrine, and investigated the molecular mechanism by in vitro and in vivo assays. Meanwhile, other types of tumor cells, immortalized epithelial cells and bone marrow mesenchymal stem cells were used to evaluate the universal role of anti-cancer and safety of the strategy. The results showed, briefly, an exposure to niclosamide and quinacrine led to an increased apoptosis-related protein p53, cleaved caspase-3 and cleaved PARP and autophagy-related protein LC3B expression, and a decreased expression of autophagy-related protein p62, finally leading to cell apoptosis and autophage. After inhibiting autophagy by Baf-A1, flow cytometry and western blot showed that the expression of apoptosis-related proteins was down-regulated and the number of apoptotic cells decreased. Subsequently, in the siRNA-mediated p53 knockdown cells, the expression of apoptosis-related proteins and the number of apoptotic cells were also reduced, while the expression of autophagy-related proteins including LC3B, p62 did not change significantly. To sum up, we developed a new, safe strategy for melanoma treatment by using low doses of niclosamide and quinacrine to treat melanoma; and found a novel mechanism by which the combination application of low doses of niclosamide and quinacrine exerts an efficient anti-melanoma effect through activation of autophagy-mediated p53-dependent apoptosis. The novel strategy was verified to exert a universal anti-cancer role in other types of cancer.     

干旱胁迫条件下马铃薯耐旱品种宁蒗182叶片蛋白质组学分析

开展马铃薯抗旱分子机理的研究对培育马铃薯抗旱品种, 减少干旱造成的损失至关重要。文章利用双向电泳技术对云南地方耐旱马铃薯品种宁蒗182在干旱与正常处理条件下叶片表达差异蛋白质组进行对比研究。经电泳图谱分析和MALDI-TOF-TOF/MS质谱鉴定获得12个表达差异蛋白点, 并进行了功能分类。结果发现, 在差异蛋白中具有保护马铃薯光和系统以及线粒体正常运转的酶类; 调节该植株对环境胁迫响应的信号传导以及调控其组织内N、C运输系统的功能蛋白, 这些蛋白在受到干旱胁迫时表达量均升高。这一结果揭示出该类蛋白是马铃薯在干旱条件下产生的耐受相关蛋白。文章为阐释马铃薯抗旱品种通过多种路径和水平的调控提高其抗性的分子机理提供了理论依据。     

Punicalagin promotes the apoptosis in human cervical cancer (ME-180) cells through mitochondrial pathway and by inhibiting the NF-kB signaling pathway

Increasing attention of plant derived therapeutic agents against cancer, investigating the anti-proliferative efficiency of plant derived chemicals have achieved increasing momentum for the design of anticancer drug. Punicalagin, dietary phytochemical altered the various cell signal transduction pathways associated with cell apoptosis and proliferation. This investigation was intended to examine the efficiency of punicalagin lying on cell viability so as to examine the molecular based punicalagin mechanism stimulated apoptosis via exploring the expression of Bcl-2 family proteins, and caspases also the cell cycle regulatory proteins p53 and NF-κB signaling in human cervical cancer cells. We also analyzed the morphological characteristic changes through mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, TUNEL assay, AO/EtBr analysis in cervical cancer cells. Our findings demonstrated that punicalagin repressed the viability of cervical cancer cells in a dosereliant mode via stimulating mitochondrial mediated apoptosis. Moreover, our this study demonstrated that punicalagin blocked cervical cancer cell proliferation and stimulated cell apoptosis by suppressing NF-kappa B activity. Hence our study suggested that punicalagin exhibits opposing actions on NF-kappa B signaling networks to block cancer cell progression acts as a classical candidate for anticancer drug designing.     

Plant cell organelle proteomics in response to abiotic stress

Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.     

抗白血病药物三尖杉酯碱对L1210细胞着丝粒蛋白含量及CenpB基因表达的影响

以小鼠白血病细胞系L1210为对象,探讨了抗白血病药物三尖杉酯碱(Harringtonine,HT或Har)对细胞内着丝粒蛋白含量及着丝粒蛋白CenP基因表达的影响。间接免疫荧光(IIF)检测结果显示,随HT作用时间延长,L1210细胞着丝粒荧光斑点减弱;免疫印迹(Western blot)检测结果显示,所用的抗着丝粒抗血清(ACA 血清)能够识别8种不同分子量的着丝粒蛋白：140、80、70、56、37、34、32和17kD。受HT作用,细胞中这些着丝粒蛋白的含量不同程度地降低。在L1210细胞中识别17、80 and l40kD蛋白质的ACA抗体也分别与分子量相当的已知为CenpA、CenpB和CenpC的3种蛋白发生交叉反应。Northern和Dot blot显示,HT的抑制作用使细胞中CenpB mRNA表达水平较之对照细胞下降。结果表明,HT可(通过抑制基因mRNA的表达)降低细胞中某些着丝粒蛋白的含量;HT对细胞的杀伤及诱导凋亡作用可能与CenpB等着丝粒蛋白基因的表达抑制有关。     

Comparative proteomics analysis of human stem cells from dental gingival and periodontal ligament

Dental stem cells isolated from oral tissues have been shown to provide with high proliferation ability and multilineage differentiation potential. Gingival mesenchymal stem cells (GMSCs) and periodontal ligament stem cells (PDLSCs), kinds of dental stem cells, can be used as substitutes for tissue repair materials because of their similar regenerative functions. In this study, we aim to explore the similarities and differences between the protein profiles of GMSCs and PDLSCs through quantitative proteomics. A total of 2821 proteins were identified and retrieved, of which 271 were upregulated and 57 were downregulated in GMSCs compared to PDLSCs. Gene Ontology (GO) analysis demonstrated that the 328 differentially abundant proteins (DAPs) were involved in the regulation of gene expression, metabolism, and signal transduction in biological process, mainly distributed in organelles related to vesicle transport, and involved in the molecular function of binding protein. And Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the DAPs were committed to regulating the synthesis of proteasome and spliceosome. Real-time quantitative polymerase chain reaction (RT-qPCR) results showed that ARPC1B, PDAP1, and SEC61B can be used as special markers to distinguish GMSCs from PDLSCs. This research contributes to explaining the molecular mechanism and promoting the clinical application of tissue regeneration of GMSCs and PDLSCs.     

Foreign protein production using plant cell and organ cultures: Advantages and limitations

Plants and plant tissue cultures are used as host systems for expression of foreign proteins including antibodies, vaccines and other therapeutic agents. Recombinant or stably transformed plants and plant cell cultures have been applied for foreign protein production for about 20 years. Because the product concentration achieved exerts a major influence on process economics, considerable efforts have been made by commercial and academic research groups to improve foreign protein expression levels. However, post-synthesis product losses due to protease activity within plant tissues and/or extracellular protein adsorption in plant cell cultures can negate the benefits of molecular or genetic enhancement of protein expression. Transient expression of foreign proteins using plant viral vectors is also a practical approach for producing foreign proteins in plants. Adaptation of this technology is required to allow infection and propagation of engineered viruses in plant tissue cultures for transient protein expression in vitro.     

Building Disease-Specific Drug-Protein Connectivity Maps from Molecular Interaction Networks and PubMed Abstracts

The recently proposed concept of molecular connectivity maps enables researchers to integrate experimental measurements of genes, proteins, metabolites, and drug compounds under similar biological conditions. The study of these maps provides opportunities for future toxicogenomics and drug discovery applications. We developed a computational framework to build disease-specific drug-protein connectivity maps. We integrated gene/protein and drug connectivity information based on protein interaction networks and literature mining, without requiring gene expression profile information derived from drug perturbation experiments on disease samples. We described the development and application of this computational framework using Alzheimer''s Disease (AD) as a primary example in three steps. First, molecular interaction networks were incorporated to reduce bias and improve relevance of AD seed proteins. Second, PubMed abstracts were used to retrieve enriched drug terms that are indirectly associated with AD through molecular mechanistic studies. Third and lastly, a comprehensive AD connectivity map was created by relating enriched drugs and related proteins in literature. We showed that this molecular connectivity map development approach outperformed both curated drug target databases and conventional information retrieval systems. Our initial explorations of the AD connectivity map yielded a new hypothesis that diltiazem and quinidine may be investigated as candidate drugs for AD treatment. Molecular connectivity maps derived computationally can help study molecular signature differences between different classes of drugs in specific disease contexts. To achieve overall good data coverage and quality, a series of statistical methods have been developed to overcome high levels of data noise in biological networks and literature mining results. Further development of computational molecular connectivity maps to cover major disease areas will likely set up a new model for drug development, in which therapeutic/toxicological profiles of candidate drugs can be checked computationally before costly clinical trials begin.     

Coated vesicles in plant cells

Coated vesicles represent vital transport intermediates in all eukaryotic cells. While the basic mechanisms of membrane exchange are conserved through the kingdoms, the unique topology of the plant endomembrane system is mirrored by several differences in the genesis, function and regulation of coated vesicles. Efforts to unravel the complex network of proteins underlying the behaviour of these vesicles have recently benefited from the application in planta of several molecular tools used in mammalian systems, as well as from advances in imaging technology and the ongoing analysis of the Arabidopsis genome. In this review, we provide an overview of the roles of coated vesicles in plant cells and highlight salient new developments in the field.