Protein complementation as tool for studying protein-protein interactions in living cells

The association and dissociation of protein-protein complexes play an important role in various processes in living cells. The disruption of protein-protein interactions is observed in various pathologies. The study of the nature of these interactions will contribute to a better understanding of the molecular basis of the pathogenesis of the disease and the development of new approaches to therapy. Now there is a set of methods that allow one to reveal and analyze the interaction of proteins in vitro. However, more accurate data can be obtained by studying protein-protein interactions in vivo. One of a few prospective methods is based on the effect of the complementation of fragments of reporter proteins. These reporter systems are based on the change in the fluorescent properties or enzymatic activity of the proteins that can be measured using colorimetric, fluorescent, or other substrates. The principle of the complementation is widely used to analyze protein interactions, to determine of order of interaction of protein partners in different signaling pathways, as well as in high-performance screening studies for detecting and mapping previously unknown protein-protein interactions. The possibilities of existing complementation reporter systems allow one to solve problems that are far beyond the simple registration of the interactions of two or more proteins.     

Protein-protein interaction through beta-strand addition

Protein-protein interactions have essential roles at almost every level of organization and communication in living cells. During complex formation, proteins can interact via covalent, surface-surface or peptide-surface contacts. Many protein complexes are now known to involve the binding of linear motifs in one of the binding partners. An emerging mechanism of such non-covalent peptide-surface interaction involves the donation or addition of a beta strand in the ligand to a beta sheet or a beta strand in the receptor. Such 'beta-strand addition' contacts can dictate or modulate binding specificity and affinity, or can be used in more promiscuous protein-protein contacts. Three main classes of beta-strand addition can be distinguished: beta-sheet augmentation; beta-strand insertion and fold complementation; and beta-strand zippering. A survey of protein-protein complexes in the protein data bank identifies beta-strand additions in many important metabolic pathways. Targeting these interactions might, thus, provide novel routes for rational drug design.     

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.     

In vivo quantification of protein-protein interactions in Saccharomyces cerevisiae using bimolecular fluorescence complementation assay

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.     

Application of protein-fragment complementation assays in cell biology

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein-protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. Here we discuss the application of PCA to different aspects of cell biology.     

Light resonance energy transfer-based methods in the study of G protein-coupled receptor oligomerization

Since most of the functions in cells are mediated by multimeric protein complexes, the determination of protein-protein interactions is an important step in the study of cellular mechanisms. Traditionally, after screening for possible target interactors by means of a yeast two-hybrid screen, several methods are used to validate the initial result before carrying out functional experiments. Nowadays, non-invasive fluorescence-based methods like Bioluminescence Resonance Energy Transfer (BRET) and Fluorescence Resonance Energy Transfer (FRET) are widely used in the study of protein-protein interactions in living cells. In the present review, we address the individual strengths and weaknesses of both RET approaches, providing information on their possible future use in the study of G protein-coupled receptor oligomerization.     

Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells

Bimolecular fluorescence complementation (BiFC) assay makes it possible to visualize protein-protein interactions in living cells. In this assay, Venus, a bright-yellow variant of green fluorescent protein, is known to produce fluorescent backgrounds due to non-specific interactions. In this study we found that the V150A mutation increased by 8.6-fold the signal-to-noise ratio in the BiFC assay of bFos-bJun interaction.     

Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions

Protein-protein interactions play a pivotal role in coordinating many cellular processes. Determination of subcellular localization of interacting proteins and visualization of dynamic interactions in living cells are crucial to elucidate cellular functions of proteins. Using fluorescent proteins, we previously developed a bimolecular fluorescence complementation (BiFC) assay and a multicolor BiFC assay to visualize protein-protein interactions in living cells. However, the sensitivity of chromophore maturation of enhanced yellow fluorescent protein (YFP) to higher temperatures requires preincubation at lower temperatures prior to visualizing the BiFC signal. This could potentially limit their applications for the study of many signaling molecules. Here we report the identification of new fluorescent protein fragments derived from Venus and Cerulean for BiFC and multicolor BiFC assays under physiological culture conditions. More importantly, the newly identified combinations exhibit a 13-fold higher BiFC efficiency than originally identified fragments derived from YFP. Furthermore, the use of new combinations reduces the amount of plasmid required for transfection and shortens the incubation time, leading to a 2-fold increase in specific BiFC signals. These newly identified fluorescent protein fragments will facilitate the study of protein-protein interactions in living cells and whole animals under physiological conditions.     

Visualization of ternary complexes in living cells by using a BiFC-based FRET assay

Studies of protein interactions have increased our understanding and knowledge of biological processes. Assays that utilize fluorescent proteins, such as fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC), have enabled direct visualization of protein interactions in living cells. However, these assays are primarily suitable for a pair of interacting proteins, and methods to visualize and identify multiple protein complexes in vivo are very limited. This protocol describes the recently developed BiFC-FRET assay, which allows visualization of ternary complexes in living cells. We discuss how to design the BiFC-FRET assay on the basis of the validation of BiFC and FRET assays and how to perform transfection experiments for acquisition of fluorescent images for net FRET calculation. We also provide three methods for normalization of the FRET efficiency. The assay employs a two-chromophore and three-filter FRET setup and is applicable to epifluorescence microscopes. The entire protocol takes about 2-3 weeks to complete.     

Reporter gene imaging of protein-protein interactions in living subjects

In the past few years there has been a veritable explosion in the field of reporter gene imaging, with the aim of determining the location, duration and extent of gene expression within living subjects. An important application of this approach is the molecular imaging of interacting protein partners, which could pave the way to functional proteomics in living animals and might provide a tool for the whole-body evaluation of new pharmaceuticals targeted to modulate protein-protein interactions. Three general methods are currently available for imaging protein-protein interactions in living subjects using reporter genes: a modified mammalian two-hybrid system, a bioluminescence resonance energy transfer (BRET) system, and split reporter protein complementation and reconstitution strategies. In the future, these innovative approaches are likely to enhance our appreciation of entire biological pathway systems and their pharmacological regulation.     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

Protein complementation assays: Approaches for the in vivo analysis of protein interactions

The in vivo identification and characterization of protein-protein interactions (PPIs) are essential to understand cellular events in living organisms. In this review, we focus on protein complementation assays (PCAs) that have been developed to detect in vivo protein interactions as well as their modulation or spatial and temporal changes. The uses of PCAs are increasing, spanning different areas such as the study of biochemical networks, screening for protein inhibitors and determination of drug effects. Emphasis is given to approaches that rely on signals of spectroscopic nature (i.e. fluorescence or luminescence), the ones that are more directly related to bioimaging.     

Construction of intramolecular luciferase complementation probe for detecting specific RNA

Intermolecular enzyme complementation assay is a useful method for detecting protein-protein interactions. Specifically, bioluminescent signals produced from reconstructed split luciferase fragments are powerful tools for in vivo analysis because the bioluminescent signals have been visualized both in cultured cells and living animals. However, they are limited for detection and evaluation of biological events relevant to intermolecular protein-protein interactions. In this study, we constructed an intramolecular luciferase complementation probe for detecting target biomolecules other than protein-protein interactions. It consists of peptide-inserted firefly luciferase (PI-FLuc) containing a short peptide between internally divided firefly luciferase. The inserted short peptide triggers FLuc complementation or discomplementation and subsequent reactivation or inactivation of FLuc activity through its induced fit conformational changes. We chose RNA binding arginine rich motif (ARM) peptides, Rev and/or Tat, for model peptide insertion, and expressed constructed PI-FLuc probe variants using a wheat germ cell-free protein synthesis system. They showed FLuc activity changes, reactivation, or inactivation after binding to their specific RNA targets. Furthermore, to expand the versatility of the PI-FLuc RNA detection system, we designed split-RNA probes built to reform the ARM peptide binding site in the presence of arbitrarily selected target-RNA. As a result, the target RNA was homogeneously detected by FLuc luminescent signals mediated by a cooperative function of the PI-FLuc and split-RNA probe sets.     

Visualizing Interactions along the Escherichia coli Twin-Arginine Translocation Pathway Using Protein Fragment Complementation

The twin-arginine translocation (Tat) pathway is well known for its ability to export fully folded substrate proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Studies of this mechanism in Escherichia coli have identified numerous transient protein-protein interactions that guide export-competent proteins through the Tat pathway. To visualize these interactions, we have adapted bimolecular fluorescence complementation (BiFC) to detect protein-protein interactions along the Tat pathway of living cells. Fragments of the yellow fluorescent protein (YFP) were fused to soluble and transmembrane factors that participate in the translocation process including Tat substrates, Tat-specific proofreading chaperones and the integral membrane proteins TatABC that form the translocase. Fluorescence analysis of these YFP chimeras revealed a wide range of interactions such as the one between the Tat substrate dimethyl sulfoxide reductase (DmsA) and its dedicated proofreading chaperone DmsD. In addition, BiFC analysis illuminated homo- and hetero-oligomeric complexes of the TatA, TatB and TatC integral membrane proteins that were consistent with the current model of translocase assembly. In the case of TatBC assemblies, we provide the first evidence that these complexes are co-localized at the cell poles. Finally, we used this BiFC approach to capture interactions between the putative Tat receptor complex formed by TatBC and the DmsA substrate or its dedicated chaperone DmsD. Our results demonstrate that BiFC is a powerful approach for studying cytoplasmic and inner membrane interactions underlying bacterial secretory pathways.     

Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.     

Methods to map protein interactions in mammalian cells: different tools to address different questions

In the post-genome era, functional annotation of the predicted gene-sets will be one of the most important upcoming challenges. So-called interactome analysis positions a protein in its subcellular environment by mapping its interaction partners. Such interaction maps are essential for an accurate insight into protein function since many cellular processes are organised to operate in protein complexes. These assemblies have dynamic structures and can interact with each other, two properties which are often controlled by regulated protein expression and modification. Various methods exist to unravel protein interaction circuitries, which can be roughly divided into biochemical and genetic strategies. In this review we focus on the different strategies to study protein-protein interactions in living mammalian cells. Recently developed analytical and screening methods are also addressed.     

Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein-protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein-protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein-protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein-protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein-protein interactions in a dynamic context- and phosphorylation site-specific manner.     

基于TurboID的植物蛋白邻近标记实验方法

邻近标记作为近些年发展起来的一项检测活细胞内蛋白互作关系和亚细胞结构蛋白组的新型技术,已成功应用于多种动植物体系的研究。该技术通过给诱饵蛋白融合一个具有特定催化连接活性的酶,在酶的催化作用下将小分子底物(如生物素)共价连接到酶邻近的内源蛋白,通过富集和分析被标记的蛋白可获得与诱饵互作的蛋白组。经定向进化产生的生物素连接...