玉米酵母双杂交cDNA文库的构建及ZmCEN互作蛋白的筛选

为阐明玉米中心蛋白(ZmCEN)的生物学功能,采用酵母双杂交技术对其互作蛋白进行研究。提取玉米(Zea mays L.)自交系‘郑58’幼苗的总RNA,利用SMART技术反转录合成ds cDNA,构建以pGBKT7为载体的酵母双杂交cDNA文库;依据ZmCEN基因的CDS序列设计引物,构建重组诱饵载体(pGBKT7-ZmCEN)转化酵母菌株Y2HGold,检测诱饵载体的毒性与自激活能力后,筛选与玉米中心蛋白(ZmCEN)互作的猎物蛋白。将筛选的互作蛋白NAC67和TONNEAU1b(TON1b)再次验证相互作用,并选取互作蛋白TON1b,采用BiFC实验分别构建ZmCEN-pSPYNE和TON1b-pSPYCE BiFC半分子重组载体,转化拟南芥原生质体,进一步验证它们在细胞内的互作;并利用Uniprot和KEGG在线网站对互作蛋白进行gene ontology(GO)注释分析。结果表明:玉米全株幼苗的cDNA文库库容量达到2.56×107 CFU,文库滴度5.36×108 CFU/mL,符合建库要求。经检测诱饵载体无毒性也无自激活功能,所筛选的cDNA文库经测序和Blast比对分析以及共转验证,最终得到28个与诱饵蛋白ZmCEN互作的蛋白质。GO注释显示互作蛋白参与的生物过程有21种。BiFC结果显示,蛋白TON1b与ZmCEN在拟南芥原生质体细胞内互作而形成互补,从而产生黄色荧光,进一步证实了两者存在互作关系。酵母双杂交系统cDNA文库的成功构建与筛选,为进一步研究玉米ZmCEN及其与互作蛋白的作用机制奠定了基础。     

运用BioID技术筛选S100A7互作蛋白*

目的: 通过邻近生物素鉴定(BioID)技术筛选S100A7互作蛋白并进行验证。方法: 采用邻近生物素BioID和质谱相结合的方法,筛选S100A7互作蛋白。通过免疫荧光和免疫共沉淀方法,对相互作蛋白进行验证。结果: 与对照组比对,通过BioID技术共获得94个可能与 S100A7 相互作用的候选蛋白质。选取 Annexin A2(AnxA2)蛋白在 HEK293 细胞中进行了验证,结果表明S100A7 与 AnxA2 存在直接的相互作用,且二者共定位于细胞质和细胞膜。结论: 可将BioID技术作为互作蛋白筛选的一项新技术,通过该技术发现S100A7和AnxA2存在相互作用。     

活细胞内亚细胞结构蛋白质组学研究新技术——几种邻近标记策略的应用及比较

真核细胞内多种无膜及有膜细胞器为各种生物学过程的发生提供场所.被膜细胞器通过它们之间的膜接触位点所进行的信息交流和物质交换是维持生命活动所必需的.绘制活细胞中细胞器或膜接触位点等处的蛋白质组图谱,将有助于解析这些部位的生物学功能及作用机制,并为研究细胞器相互作用提供基础.但由于无膜细胞器或膜接触位点很难分离纯化,传统的生化方法难以系统解析其中的蛋白质组.最近报道的几种基于酶类的蛋白质邻近标记技术,则为系统分析上述空间受限的蛋白质组这一难题提供了有效的解决方案.通过将能催化产生活性自由基(最常见的是生物素及其衍生物的自由基)的酶连接到目标蛋白上,可对其邻近的蛋白质组进行共价标记,从而使后者的分离和鉴定成为可能,并可以运用于活细胞中的动态标记.我们在此综述了几种最新的邻近标记策略的原理及应用,并对它们的优势与局限性进行了比较,以期为细胞器互作的蛋白质组学研究提供参考.     

利用酵母双杂交技术筛选鉴定与番茄环纹斑点病毒NSs蛋白互作的西花蓟马蛋白

【目的】筛选鉴定西花蓟马Franiklinella ocicdentalis体内与番茄环纹斑点病毒(tomato zonate spot virus, TZSV)NSs蛋白互作的介体因子。【方法】利用酵母双杂交技术筛选与TZSV NSs互作的西花蓟马蛋白;进行序列分析鉴定后,将捕获的蛋白与NSs基因回转到酵母细胞,利用营养缺陷型培养基鉴定蛋白互作情况。再利用GST Pull-down技术验证TZSV NSs与鉴定出的西花蓟马蛋白的体外互作关系。【结果】构建了TZSV NSs的酵母双杂交诱饵质粒pGBKT7-NSs,确定了诱饵质粒对酵母AH109细胞无毒性,并且无自激活活性。序列分析发现与TZSV NSs互作的西花蓟马蛋白为类电压依赖性阴离子通道(voltage-dependent anion-selective channel-like, VDAC)。酵母回转实验显示TZSV NSs与西花蓟马VDAC在酵母细胞内存在特异性互作。GST Pull-down结果表明TZSV NSs与西花蓟马VDAC在体外存在相互作用。【结论】通过酵母双杂交和GST Pull-down技术,分别在酵母细胞内和体外证实了TZSV NSs和西花蓟马VDAC存在特异性互作。这些结果有助于揭示西花蓟马VDAC蛋白调控西花蓟马持久传毒的机制,为虫传病毒病的防治提供理论基础。     

运用BioID技术筛选水稻GS3互作蛋白

水稻异源三聚体G蛋白系统中的非典型γ亚基GS3,是一个控制籽粒大小的主效数量效应基因座,在调节籽粒大小中发挥负调控因子的功能。BioID(proximity-dependent biotin identification)为邻近蛋白标记技术,其工作原理是生物素连接酶能使其周围的蛋白带上生物素,同时生物素又能和链霉亲和素紧密结合,所以能够利用链霉亲和素偶联的磁珠富集目标蛋白。该技术具有灵敏、高效和周期短等特点,为筛选互作蛋白提供了新方法。为了解析GS3的蛋白调控网络,该研究以水稻原生质体为材料,采用BioID技术对GS3在水稻中的互作蛋白进行了筛选。Western-blot结果表明:融合蛋白Bir AG-GS3在原生质体中成功表达并生物素化GS3邻近蛋白。使用链霉亲和素磁珠富集生物素化后的蛋白,并进行蛋白质谱测序,获得了与GS3邻近的可能存在直接或间接互作的蛋白。将获得的蛋白进行功能富集与注释,并构建蛋白-蛋白互作网络。对部分蛋白进行了BiFC验证,发现GS3可能与ICL、PPDK、RPN7和RH15发生相互作用,涉及能量代谢的调节、种子淀粉物质的储存、泛素-蛋白酶体系统以及凋亡途径等生物过程。     

利用酵母双杂交筛选与苹果褪绿叶斑病毒MP互作的寄主因子

为了找到与苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)运动蛋白(Movement protein,MP)互作的寄主因子,首先构建了ACLSV MP酵母双杂交诱饵载体,然后通过顺序转化的方法自前期已构建好的苏俄苹果(Malus sylvestris cv.R12740-7A)cDNA文库中筛选互作基因;在GenBank中对互作寄主基因进行BLAST分析,根据基因注释推测其在病毒与寄主互作过程中可能发挥的作用。结果表明,构建的酵母双杂交诱饵载体pGBKT7-MP无自激活性、无毒性。筛选到69个与ACLSV MP互作的苏俄苹果寄主因子,包括水解酶活性、病程相关蛋白、氧化还原酶活性、磷酸酶活性、催化活性、连接酶活性、苯丙氨酸解氨酶活性、过氧化物酶活性、DNA结合功能和未知蛋白,共10类不同功能的蛋白。通过生物信息学分析,推断蛋白磷酸酶、病程相关蛋白及3-磷酸甘油醛脱氢酶可能在互作过程中起重要作用。该研究为深入了解ACLSV致病特征及病原与寄主互作机制奠定基础。     

基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化、纯化和检测方法

目的:建立并优化基于Avi-tag标签技术的人胚肾细胞增强型绿色荧光蛋白(eGFP)的定点生物素化标记、纯化和检测方法。方法:分别构建具有Avi-tag标签的eGFP真核表达载体plenti-Avi-eGFP和BirA酶真核过表达载体pQCXIH-BirA,将plenti-Avi-eGFP和pQCXIH-BirA共转染人胚肾293T细胞,12 h后观察Avi-tag标签对eGFP蛋白在细胞内定位的影响;48 h后裂解细胞,用链霉亲和素珠子纯化生物素标记的eGFP,SDS-PAGE观察eGFP纯化和富集情况,并优化基于Western印迹的生物素化eGFP检测方法。结果:Avi-tag标签对eGFP在细胞内的定位无影响,同时BirA酶在293T细胞内可将带Avi-tag标签的eGFP标记上生物素;生物素化的eGFP可特异性地被链霉亲和素珠子纯化和富集,纯度可达95%;Western印迹检测生物素化蛋白的最终条件为5%的BSA作为封闭液和终浓度为100 ng/mL的链霉亲和素-HRP。结论:建立了基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化标记、纯化与检测方法,为该方法的广泛应用奠定了前期技术基础。     

生物素化FOXM1真核表达体系的构建及鉴定

建立基于生物素AP-tag标签的FOXM1真核表达载体,利用生物素与链霉亲和素的高特异性、高亲和力的结合性质对FOXM1蛋白进行纯化,为后续鉴定其互作分子的研究奠定基础。分别构建真核表达载体pc DNA3.1-AP-FOXM1和大肠杆菌生物素连接酶Bir A真核表达载体pcDNA3.1-Bir A,将pcDNA3.1-AP-FOXM1和pcDNA3.1-BirA共转染人胚肾293T(HEK293T)细胞,用银染及Western印迹检测细胞裂解液,确定相关蛋白表达;用链霉亲和素琼脂糖珠纯化生物素标记的FOXM1,并考察FOXM1的互作蛋白是否可以同时被洗脱下来。研究发现构建的生物素标签真核表达体系能有效表达生物素化的目标蛋白FOXM1;该蛋白在Western印迹、pull-down等实验中可实现无需抗体的一步法应用;该体系可用于FOXM1互作蛋白的鉴定和功能分析。结果表明建立了基于生物素AP-tag标签的FOXM1真核表达体系,为FOXM1互作蛋白与功能的深入研究奠定了技术基础。     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

邻近标记在蛋白质组学中的发展及应用

人体内各种复杂的生命活动离不开蛋白质之间的相互作用。这种相互作用具有瞬时性和结合力弱等特点,并受到多种动态调节,特别是蛋白质翻译后修饰（post-translation modifications, PTM）。传统的亲和质谱检测方法存在蛋白纯化的局限性,在高效检测到动态变化方面存在不足。邻近标记是一种能够给与靶蛋白质瞬时靠近,或者互作（邻近）的蛋白质加上生物素的技术,它与质谱检测技术的联合使用能检测细胞过程中弱的、瞬时的蛋白质相互作用,有效解决上述问题。本文综述了基于生物素的邻近标记方法的发展现状,从依赖于融合序列的生物素标记开始,依次介绍有关生物素连接酶、过氧化物酶及其进化后的2代标记方法等经典生物素标记的方法和原理,比较各个方法间的差异和优缺点;也列举了一些近年来新出现的标记方法,如将生物素连接酶进行拆分、鉴定蛋白质在不同复合物中功能的方法、抗体靶向的标记方法,以及其他来源的生物素连接酶突变体,例如枯草芽孢杆菌（Bacillus subtilis）的C端氨基酸突变的生物素连接酶,能够应用在苍蝇和蠕虫中的生物素连接酶突变体。本文对这些方法进行归纳总结,旨在为初步接触该领域的科研工作者提供参考,同时也希望能够提供一些新的思路,推动蛋白质相互作用组学的发展。     

Defining Proximity Proteome of Histone Modifications by Antibody-mediated Protein A-APEX2 Labeling

Proximity labeling catalyzed by promiscuous enzymes, such as APEX2, has emerged as a powerful approach to characterize multiprotein complexes and protein–protein interactions. However, current methods depend on the expression of exogenous fusion proteins and cannot be applied to identify proteins surrounding post-translationally modified proteins. To address this limitation, we developed a new method to label proximal proteins of interest by antibody-mediated protein A-ascorbate peroxidase 2 (pA-APEX2) labeling (AMAPEX). In this method, a modified protein is bound in situ by a specific antibody, which then tethers a pA-APEX2 fusion protein. Activation of APEX2 labels the nearby proteins with biotin; the biotinylated proteins are then purified using streptavidin beads and identified by mass spectrometry. We demonstrated the utility of this approach by profiling the proximal proteins of histone modifications including H3K27me3, H3K9me3, H3K4me3, H4K5ac, and H4K12ac, as well as verifying the co-localization of these identified proteins with bait proteins by published ChIP-seq analysis and nucleosome immunoprecipitation. Overall, AMAPEX is an efficient method to identify proteins that are proximal to modified histones.     

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.     

MicroID2: A Novel Biotin Ligase Enables Rapid Proximity-Dependent Proteomics

Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions.     

Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.     

A proteomics strategy to elucidate functional protein-protein interactions applied to EGF signaling

Mass spectrometry-based proteomics can reveal protein-protein interactions on a large scale, but it has been difficult to separate background binding from functionally important interactions and still preserve weak binders. To investigate the epidermal growth factor receptor (EGFR) pathway, we employ stable isotopic amino acids in cell culture (SILAC) to differentially label proteins in EGF-stimulated versus unstimulated cells. Combined cell lysates were affinity-purified over the SH2 domain of the adapter protein Grb2 (GST-SH2 fusion protein) that specifically binds phosphorylated EGFR and Src homologous and collagen (Shc) protein. We identified 228 proteins, of which 28 were selectively enriched upon stimulation. EGFR and Shc, which interact directly with the bait, had large differential ratios. Many signaling molecules specifically formed complexes with the activated EGFR-Shc, as did plectin, epiplakin, cytokeratin networks, histone H3, the glycosylphosphatidylinositol (GPI)-anchored molecule CD59, and two novel proteins. SILAC combined with modification-based affinity purification is a useful approach to detect specific and functional protein-protein interactions.     

Detection of protein-protein interactions using nonimmune IgG and BirA-mediated biotinylation

Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.     

Split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts

We developed a split luciferase complementation assay to study protein-protein interactions in Arabidopsis protoplasts. In this assay, the N- and C-terminal fragments of Renilla reniforms luciferase are translationally fused to bait and prey proteins, respectively. When the proteins interact, split luciferase becomes activated and emits luminescence that can be measured by a microplate luminometer. Split luciferase activity was measured by first transforming protoplasts with a DNA vector in a 96-well plate. DNA vector expressing both bait and prey genes was constructed through two independent in vitro DNA recombinant reactions, Gateway and Cre-loxP. As proof of concept, we detected the protein-protein interactions between the nuclear histones 2A and 2B, as well as between membrane proteins SYP (syntaxin of plant) 51 and SYP61, in Arabidopsis protoplasts.     

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.