工程蛋白用作特异性结合试剂

随着蛋白质工程的发展和蛋白质文库选择技术的出现,我们开始了对大量人工改造的作为结合分子的蛋白支架的探索,并且已经获得了有价值的结合蛋白支架,这就预示着用于生物技术和临床的抗体有了新的替代物。主要介绍了不同类型的结合蛋白支架以及它们的结构特征。     

天然可降解生物材料在组织工程中的应用研究进展

细胞培养支架材料是组织工程学的重要研究内容之一 ,是实现产业化的关键。天然可降解生物材料是细胞培养支架材料中的重要组成部分 ,目前用于细胞培养支架的天然可降解生物材料主要有多糖类和蛋白质类。多糖类主要包括壳多糖、透明质酸 ;蛋白质类主要包括胶原纤维蛋白和血纤维蛋白。     

HIV感染导致MT4细胞蛋白表达差异的初步研究

为了比较MT4细胞株感染HIV-1的ⅢB株前后的蛋白质表达差异,我们分别提取MT4细胞及感染了人类免疫缺陷病毒(HIV)的MT4细胞的总蛋白质,通过双向电泳分离,使用Image Master 2D Elite 3.10图像分析软件分析获得的凝胶图谱,寻找差异点,使用质谱仪鉴定获得的差异点蛋白质.结果表明感染HIV和未感染HIV的MT4细胞有40个蛋白质点差异,HIV感染后减少的蛋白质点有12个,增多的有28个,通过质谱分析,29个蛋白质得到鉴定.其中HIV感染后下调的蛋白质有能量代谢相关蛋白、肌动蛋白相关蛋白及假想蛋白等;上调的蛋白有肌动蛋白、酶类蛋白、免疫蛋白及假想蛋白等.通过研究我们可以看出宿主细胞感染HIV病毒后有多个蛋白发生变化,可能和HIV与宿主细胞的相互作用有关.为了研究HIV感染的机制必须去除高丰度蛋白,针对特定功能的蛋白质进行具体研究.     

大豆种子萌发过程中的差异蛋白质组研究

运用蛋白质组学技术对大豆(Glycinemax)N2899种子萌发0h、8h、36h、60h4个时期蛋白质的差异表达情况进行了研究.结果发现,在考马斯亮蓝染色的双向电泳pH3～10胶上,PDQuest图像分析软件可识别的点约350个,其中表达量变化2.5倍以上的蛋白质点有24个,而绝大部分大豆种子贮藏蛋白在萌发期尚未降解.在萌发的第一阶段,24个差异表达蛋白中有10个蛋白质的丰度发生变化.第二阶段,差异表达蛋白的种类和量增加,其中15个蛋白质是动态变化的,14个蛋白质在胚根突破种皮时表达量达到峰值,表明吸胀后种子内的生命活动越来越强.对这24个蛋白质点进行胶内酶解,用基质辅助激光解析电离飞行时间质谱测定均获得肽质量指纹图谱.搜索大豆的UniGene库初步鉴定出6个蛋白质,分别是核苷二磷酸激酶、热激蛋白、硫氧还蛋白、35ku种子成熟蛋白及种子成熟蛋白PM36.对这些蛋白质在种子萌发过程中可能的作用进行了讨论.     

幽门螺杆菌毒素蛋白CagA诱导的蛋白的差异表达分析及其基因在胃癌组织中的表达

为了研究胃癌细胞中幽门螺杆菌(Hp)毒素蛋白CagA诱导的蛋白差异表达及其基因在人胃癌组织中的表达,用Hp感染胃癌细胞系SGC 7901和AGS及用含CagA基因的表达载体稳定转染SGC 7901细胞, 构建3组实验模型.提取各组细胞的总蛋白进行双向凝胶电泳,筛选3组重叠的差异表达蛋白质斑点进行质谱鉴定.共获得135个差异表达的蛋白质,其中上调蛋白质73个,下调蛋白质62个. 鉴定出10个差异表达蛋白质, 其中有6个差异表达蛋白是首次发现,它们主要参与细胞的能量代谢和信号转导等.最后定量检测了这10个差异表达蛋白基因在人胃癌组织中的表达, 发现有4个基因高表达和1个基因低表达. 本结果将为研究幽门螺杆菌感染引起胃癌的分子机制提供新的线索.     

水稻叶片对镉胁迫响应的蛋白质差异表达

为揭示水稻镉抗性的分子机理,以抗镉水稻品种P1312777和镉敏感水稻品种IR24为材料,在镉离子浓度为0(对照)、50和100 μmol·L-1条件下水培处理7 d,应用蛋白质组学方法分析了2种水稻叶片对镉胁迫响应的蛋白质差异表达.结果表明:镉胁迫下水稻PI312777叶片中共检测到差异表达蛋白质点31个,通过MALDI-TOF/MS分析,鉴定了其中的24个蛋白质(包括20个不同蛋白质,4个重复检出蛋白质);IR24叶片中共检测到差异表达蛋白质点19个,其中15个蛋白质得到鉴定.PI312777叶片鉴定出的20个蛋白质覆盖了IR24叶片鉴定的15个蛋白质,前者有4个与光合作用相关,11个与细胞防御代谢相关,3个与其他代谢相关,2个为功能未知蛋白.与对照相比,不同浓度镉胁迫下,抗镉水稻PI312777叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型、果糖1,6-二磷酸醛缩酶、硫氧还蛋白和DNA重组修复蛋白均上调表达;镉敏感水稻IR24叶片中热激蛋白、谷胱甘肽还原酶、蛋白酶体α亚基6型的表达无显著差异,果糖1,6-二磷酸醛缩酶和硫氧还蛋白则下调表达.此外,DNA重组修复蛋白仅在镉胁迫的PI312777叶片中表达.水稻PI312777比IR24具有更强的镉抗性与这些差异表达的蛋白质密切相关.     

植物RACK1蛋白研究进展

RACK1（蛋白激酶C受体）是一种色氨酸-天门冬氨酸域（WD40结构）重复蛋白。它是一种多功能支架蛋白, 结合来自不同转导通路的信号分子并在多种哺乳动物发育过程中起关键作用。在植物中也存在RACK1同源基因, 如拟南芥基因组有3个编码RACK1蛋白质的基因, 这3个蛋白质与哺乳动物RACK1在氨基酸水平的相似性都超过75%。此外, 植物RACK1蛋白质包含的WD40数量、位置和蛋白激酶C结合位点的结构域在很大程度上是保守的。该文对植物RACK1蛋白的发现、结构及其在信号转导方面的功能进行综述。     

突变前核纤层蛋白A对细胞衰老的作用

早老症(Hutchinson-Gilford progeria syndrome,HGPS)是一种极其罕见的遗传性疾病,它是由LMNA基因突变引起的,产生一个截短的lamin A 蛋白称为 progerin.核纤层蛋白异常A加工积累的 progerin 能够破坏核纤层的支架功能,替代正常蛋白质与其配体结合,导致细胞核畸形和早老表型.     

PRP8蛋白质反式剪接系统的建立

真菌病原体Cryptococcus neoformansAD血清型剪接体蛋白PRP8蛋白质内含子是目前 发现的第2个存在于真核生物体核基因组中的蛋白质内含子.它的宿主基因prp8编码的PRP 8蛋白作为剪接体的1个组分,是1个高度保守的mRNA剪接蛋白.将组氨酸标签插入克隆自真菌病原体Cryptococcus neoformans AD血清型的PRP8蛋白质内含子中,并将该蛋白质内含子进行人工断裂,获得断裂蛋白质内含子,在大肠杆菌中鉴定其剪接活性.研究结果表明：所获得的改造型蛋白质内含子均表现出高效的剪接活性.利用此Cryptococcus neoformansAD血清型PRP8 断裂蛋白质内含子,成功构建了蛋白质反式剪接系统.这一反式剪接系统可用于其他蛋白质的连接与合成,有望成为蛋白质工程中的一种有用工具.     

单显性细胞核雄性不育油菜花雷表达蛋白比较研究

以受1对显性基因控制的单显性细胞核雄性不育油菜为材料,运用蛋白双向电泳技术对初花期不育花蕾和可育花蕾中蛋白质表达差异进行分析.结果表明,可育花蕾中表达的蛋白质总点数高于不育系.不育花蕾和可育花蕾中共有的蛋白点为223个,不育花蕾特有的蛋白质点数为103个,而可育花蕾中特有的蛋白质点数为160个.可育花蕾中表达的特有蛋白质分子量主要分布在60kD以下的小分子量区域,30kD尤为丰富;不育花蕾中表达的特有蛋白按分子量分布相对均匀.两者表达特有蛋白都相对集中在pI6.0～7.5区域,多为中性蛋白.     

Effect of binding in cyclic phosphorylation-dephosphorylation process and in energy transformation

The effects of binding on the phosphorylation-dephosphorylation cycle (PDPC) - one of the key components of the signal transduction processes - is analyzed based on a mathematical model. The model shows that binding of proteins, forming a complex, diminishes the ultrasensitivity of the PDPC to the differences in activity between kinase and phosphatase in the cycle. It is also found that signal amplification depends upon the strength of the binding affinity of the protein (phosphorylated or dephosphorylated) to other proteins . It is also observed that the amplification of signal is not only dependent on phosphorylation potential but also on binding properties and resulting adjustments in binding energies.     

Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction.

Cyclosporin A, the major immunosuppressive drug in transplantation, and the more potent therapeutic drug candidate, FK506, have led to the discovery of two superfamilies of immunosuppressant binding proteins, the cyclophilins and the FK binding proteins. These proteins, enzymes with high kcat values for isomerization of X-Pro bonds in peptides and protein substrates, are distributed in all cell compartments where protein folding normally occurs. It is likely that they play major roles in the protein folding and protein trafficking in the cell. It is also likely that they have been suborned in T cells by the immunosuppressant drugs that are potent pseudosubstrate ligands that selectively block the signal transduction cascade. The discovery of the inhibition of protein phosphatase 2B (calcineurin) by the drug-immunophilin complex (CsA-CyP or FK506-FKBP) provides evidence for a specific downstream target of the drug-immunophilin complexes and may prompt a search for endogenous ligands of cyclophilin and FKBP that may effect signal transduction regulation. The molecular insights gained over a short time in this area have been remarkable; they promise to elucidate the steps in T cell activation and delineate new targets for immunosuppressive therapy.     

Protein Clusters in Phosphotyrosine Signal Transduction

Signal transduction systems based on tyrosine phosphorylation are central to cell–cell communication in multicellular organisms. Typically, in such a system, the signal is initiated by activating tyrosine kinases associated with transmembrane receptors, which induces tyrosine phosphorylation of the receptor and/or associated proteins. The phosphorylated tyrosines then serve as docking sites for the binding of various downstream effector proteins. It has long been observed that the cooperative association of the receptors and effectors produces higher-order protein assemblies (clusters) following signal activation in virtually all phosphotyrosine signal transduction systems. However, mechanistic studies on how such clustering processes affect signal transduction outcomes have only emerged recently. Here we review current progress in decoding the biophysical consequences of clustering on the behavior of the system, and how clustering affects how these receptors process information.     

Population shift of binding pocket size and dynamic correlation analysis shed new light on the anticooperative mechanism of PII protein

P_II protein is one of the largest families of signal transduction proteins in archaea, bacteria, and plants, controlling key processes of nitrogen assimilation. An intriguing characteristic for many P_II proteins is that the three ligand binding sites exhibit anticooperative allosteric regulation. In this work, P_II protein from Synechococcus elongatus, a model for cyanobacteria and plant P_II proteins, is utilized to reveal the anticooperative mechanism upon binding of 2‐oxoglutarate (2‐OG). To this end, a method is proposed to define the binding pocket size by identifying residues that contribute greatly to the binding of 2‐OG. It is found that the anticooperativity is realized through population shift of the binding pocket size in an asymmetric manner. Furthermore, a new algorithm based on the dynamic correlation analysis is developed and utilized to discover residues that mediate the anticooperative process with high probability. It is surprising to find that the T‐loop, which is believed to be responsible for mediating the binding of P_II with its target proteins, also takes part in the intersubunit signal transduction process. Experimental results of P_II variants further confirmed the influence of T‐loop on the anticooperative regulation, especially on binding of the third 2‐OG. These discoveries extend our understanding of the P_II T‐loop from being essential in versatile binding of target protein to signal‐mediating in the anticooperative allosteric regulation. Proteins 2014; 82:1048–1059. © 2013 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Protein-protein interactions in signaling cascades

The process of signal transduction is dependent on specific protein-protein interactions. In many cases these interactions are mediated by modular protein domains that confer specific binding activity to the proteins in which they are found. Rapid progress has been made in the biochemical characterization of binding interactions, the identification of binding partners, and determination of the three-dimensional structures of binding modules and their ligands. The resulting information establishes the logical framework for our current understanding of the signal transduction machinery. In this overview a variety of protein interaction modules are discussed, and issues relating to binding specificity and the significance of a particular interaction are considered.     

The role of the CGRP-receptor component protein (RCP) in adrenomedullin receptor signal transduction.

G protein-coupled receptors are usually thought to act as monomer receptors that bind ligand and then interact with G proteins to initiate signal transduction. In this study we report an intracellular peripheral membrane protein named the calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) required for signal transduction at the G protein-coupled receptor for adrenomedullin. Cell lines were made that expressed an antisense construct of the RCP cDNA, and in these cells diminished RCP expression correlated with loss of adrenomedullin signal transduction. In contrast, loss of RCP did not diminish receptor density or affinity, therefore RCP does not appear to act as a chaperone protein. Instead, RCP represents a novel class of protein required to couple the adrenomedullin receptor to the cellular signal transduction pathway. A candidate adrenomedullin receptor named the calcitonin receptor-like receptor (CRLR) has been described, which forms high affinity adrenomedullin receptors when co-expressed with the accessory protein receptor-activity modifying protein 2 (RAMP2). RCP co-immunoprecipitated with CRLR and RAMP2, indicating that a functional adrenomedullin receptor is composed of at least three proteins: the ligand binding protein (CRLR), an accessory protein (RAMP2), and a coupling protein for signal transduction (RCP).     

Heterotrimeric G protein activation by G-protein-coupled receptors

Heterotrimeric G proteins have a crucial role as molecular switches in signal transduction pathways mediated by G-protein-coupled receptors. Extracellular stimuli activate these receptors, which then catalyse GTP-GDP exchange on the G protein alpha-subunit. The complex series of interactions and conformational changes that connect agonist binding to G protein activation raise various interesting questions about the structure, biomechanics, kinetics and specificity of signal transduction across the plasma membrane.     

Why cytoplasmic signalling proteins should be recruited to cell membranes

It has been suggested that localization of signal-transduction proteins close to the cell membrane causes an increase in their rate of encounter after activation. We maintain that such an increase in the first-encounter rate is too small to be responsible for truly enhanced signal transduction. Instead, the function of membrane localization is to increase the number (or average lifetime) of complexes between cognate signal transduction proteins and hence increase the extent of activation of downstream processes. This is achieved by concentrating the proteins in the small volume of the area just below the plasma membrane. The signal-transduction chain is viewed simply as operating at low default intensity because one of its components is present at a low concentration. The steady signalling level of the chain is enhanced 1000-fold by increasing the concentration of that component. This occurs upon 'piggyback' binding to a membrane protein, such as the activated receptor, initiating the signal-transduction chain. For the effect to occur, the protein translocated to the membrane cannot be free but has to remain organized by being piggyback bound to a receptor, membrane lipid(s) or scaffold. We discuss an important structural constraint imposed by this mechanism on signal transduction proteins that might also account for the presence of adaptor proteins.     

14-3-3蛋白与植物细胞信号转导

14-3-3蛋白通过直接蛋白质-蛋白质相互作用对植物代谢关键酶、质膜H^＋ -ATP酶等发挥广泛调节作用。越来越多证据显示14-3-3蛋白通过与转录因子和其他信号分子结合参与调控植物细胞信号转导。对植物细胞中14-3-3蛋白调控信号转导途径,尤其是植物细胞对胁迫响应的调控机制进行了综述。