Protein-protein interaction detection in plants: from classical approaches to future perspectives.

Split-protein sensors have become an important tool for the analysis of protein-protein interactions in living cells. In general, two interacting proteins are expressed as fusion proteins with a pair of inactive fragments of a reporter enzyme. Interaction-induced reassembly of the two fragments then results in a functional enzyme and a detectable phenotypic readout. Despite the constantly expanding repertoire of methods, new split-protein sensors that could detect and screen for protein-protein interactions both in the cytosol and in the membrane would be very useful. In a first attempt to create new split-protein sensors, cytochrome c peroxidase (CCP) from the yeast Saccharomyces cerevisiae was rationally dissected into two fragments, which were fused to two interacting proteins. Activity of reassembled peroxidase might be visually detected using a simple colony screen [1]. However, this approach failed due to the insolubility of the chosen fragments. A random approach based on circular permutation originally developed by Graf and Schachmann was therefore adapted to isolate suitable fragmentation sites [2]. Unfortunately, only split-proteins expressing quasi wild-type protein were isolated, resulting from cuts close to the N or the C terminus of CCP. Two reasons can account for this: (i) the fragile active site environment of CCP could considerably hamper the fragmentation of the enzyme; (ii) the method itself favors the isolation of quasi wild-type proteins. The combinatorial method for the generation of new split-protein sensors was therefore further modified to circumvent the isolation of quasi wild-type proteins and successfully applied to the (β/α)8-barrel enzyme N-(5'-phosphoribosyl)-anthranilate isomerase Trp1p from Saccharomyces cerevisiae. The generated split-Trp protein sensors allow for the detection of protein-protein interactions in the cytosol as well as the membrane by enabling trp1 cells to grow on medium lacking tryptophan. In addition, split-Trp can be used as reporter for the detection of small molecule-protein interactions. This powerful selection thus complements the repertoire of the currently used split-protein sensors and provides a new tool for high-throughput interaction screening. Furthermore, the combinatorial approach should be able to generate split-protein sensors of almost any protein, thereby yielding tailor-made sensors for different applications.

The detection of protein-protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein-protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein-protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.

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.

A novel protein molecular targeting system was created using a cytoplasmic face of a plasma membrane-targeting system in Saccharomyces cerevisiae. The technique involves a molecular display for the creation of a novel reaction site and interaction sites in the field of biotechnology. In a model system, a fluorescent protein was targeted as a reporter to the cytoplasmic face of the plasma membrane. The C-terminal transmembrane domain (CTM) of Ras2p and Snc2p was examined as the portions with anchoring ability to the cytoplasmic face of the plasma membrane. We found that the CTM of Snc2p targeted the enhanced cyan fluorescent protein (ECFP)-protein A fusion protein on the cytoplasmic face of the plasma membrane more strongly than that of Ras2p. To develop it for use as a detection system for protein-protein interactions, the Fc fragment of IgG (Fc) was genetically fused with the enhanced yellow fluorescent protein (EYFP) and expressed in the cytoplasm of the ECFP-protein A-anchored cell. A microscopic analysis showed that fluorescence resonance energy transfer (FRET) between ECFP-protein A and EYFP-Fc occurred, and the change in fluorescence was observed on the cytoplasmic face of the plasma membrane. The detection of protein-protein interactions at the cytoplasmic face of a plasma membrane using FRET combined with a cytoplasmic face-targeting system for proteins provides a novel method for examining the molecular interactions of cytoplasmic proteins, in addition to conventional methods, such as the two-hybrid method in the nuclei.

The occurrence, development and prediction of various biological processes and diseases are inseparable from the protein-protein interaction (PPI), so it is extremely meaningful to perfect PPI networks. However, shortcomings of traditional detection methods, such as protein degradation, long detection time, complex operation, poor automation and high cost, restrict the rapid development of PPI networks. Here, a low-temperature digital microfluidic (LTDMF) system-based PPI detection box (LTDMF-PPI-Box) was developed to achieve rapid, lossless and efficient PPI detection. It consists of a PMMA shell, LTDMF-PPI and an integrated temperature control system. LTDMF reduces the PPI detection time from tens of hours to 1.5 hours by programmatically controlling the movement of droplets. Moreover, an integrated thermoelectric cooler (TEC) ensures an operating temperature of 4 °C, resulting in a protein protection up to 90%. The interaction between RILP protein and Rab26 protein which has a close connection to insulin secretion was demonstrated as a prototype to illustrate the feasibility of the LTDMF-PPI-Box. LTDMF with automation characteristics is capable of meeting the requirement of high-throughput screening of interacting proteins; therefore, the LTDMF-PPI-Box is expected to accelerate the establishment of the PPI network in the future.

Chapter 45 - Detection of Protein-Protein Interactions in vivo Using Cyan and Yellow Fluorescent Proteins

We report a new method for detection of protein-protein interactions in vitro and in cells. One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA's "acceptor peptide" (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3epsilon phosphoserine/threonine binding protein. Protein-protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.

Protein-protein interactions (PPIs) are responsible for various biological processes and cellular functions of all living organisms. The detection of PPIs helps in understanding the roles of proteins and their complex structure. Proteins are commonly represented by amino acid sequences. The method of identifying PPIs is divided into two steps. Firstly, a feature vector from protein representation is extracted. Then, a model is trained on these extracted feature vectors to reveal novel interactions. These days, with the availability of multimodal biomedical data and the successful adoption of deep-learning algorithms in solving various problems of bioinformatics, we can obtain more relevant feature vectors, improving the model’s performance to predict PPIs. Current work utilizes multimodal data as tertiary structure information and sequence-based information. A deep learning-based model, ResNet50, is used to extract features from 3D voxel representation of proteins. To get a compact feature vector from amino acid sequences, stacked autoencoder and quasi-sequence-order (QSO) are utilized. QSO converts the symbolic representation (amino acid sequences) of proteins into their numerical representation. After extracting features from different modalities, these features are concatenated in pairs and then fed into the bi-directional GRU-based classifier to predict PPIs. Our proposed approach achieves an accuracy of 0.9829, which is the best accuracy of 3-fold cross-validation on the human PPI dataset. The results signify that the proposed approach’s performance is better than existing computational methods, such as state-of-the-art stacked autoencoder-based classifiers.

Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs invivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the β-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta.

Protein-protein interactions regulate essentially all cellular processes. Understanding these interactions, including the quantification of binding parameters, is crucial for unraveling the molecular mechanisms underlying cellular pathways and, ultimately, their roles in cellular physiology and pathology. Current methods for measuring protein-protein interactions in vitro generally require amino acid conjugation of fluorescent tags, complex instrumentation, large amounts of purified protein, or measurement at extended surfaces. Here, we present an elegant nanoparticle-based platform for the optical detection of protein-protein interactions in the solution phase. We synthesized gold-coated silver decahedral nanoparticles possessing high chemical stability and exceptional optical sensing properties. The nanoparticle surface is then tailored for specific binding to commonly used polyhistidine tags of recombinant proteins. Sequential addition of proteins to the nanoparticle suspension results in spectral shifts of the localized surface plasmon resonance that can be monitored by conventional UV-vis spectrophotometry. With this approach, we demonstrate both the qualitative detection of specific protein-protein interactions and the quantification of equilibrium and kinetic binding parameters between small globular proteins. Requiring minimal protein quantities and basic laboratory equipment, this technique offers a simple, economical, and modular approach to characterizing protein-protein interactions, holds promise for broad use in future studies, and may serve as a template for future biosensing technologies.

Protein–protein interactions (PPI) by homo-, hetero- or oligo-merization in the cellular environment regulate cellular processes. PPI can be inhibited by antibodies, small molecules or peptides, and this inhibition has therapeutic value. A recently developed method, the proximity ligation assay (PLA), provides detection of PPI in the cellular environment. However, most applications using this assay are for proteins expressed in the same cell. We employ PLA for the first time to study PPI of cell surface proteins on two different cells. Inhibition of PPI using a peptide inhibitor is also quantified using this assay; PLA is used to detect PPI of CD2 and CD58 between Jurkat cells (T cells) and human fibroblast-like synoviocyte-rheumatoid arthritis cells that are important in the immune response in the autoimmune disease rheumatoid arthritis. This assay provides direct evidence of inhibition of PPI of two proteins on different cell surfaces.

Split focal adhesion kinase for probing protein–protein interactions

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells.

The activities of many regulatory factors involve interactions with other proteins. We demonstrate here that the ERF-associated amphiphilic repression (EAR) motif repression domain (SRDX) can convert a transcriptional complex into a repressor via transrepression that is mediated by protein-protein interactions and show that transrepressive activity of SRDX can be used to detect such protein-protein interactions. When we fused a protein that interacts with a transcription factor with SRDX and co-expressed the product with the transcription factor in plant cells, the expression of genes that are targets of the transcription factor was suppressed by transrepression. We demonstrated the transrepressive activity of SRDX using FOS and JUN as a model system and used two MADS box plant proteins, PISTILLATA and APETALA3, which are known to form heterodimers. Furthermore, the transgenic plants that expressed TTG1, which is a WD40 protein and interacts with bHLH transcription factors, fused to SRDX exhibited a phenotype similar to ttg1 mutants by transrepression and the regions of TTG1 required for interaction to the bHLH protein were detected using our system. We also used this system to analyse a protein factor that might be incorporated into a transcriptional complex and identified an Arabidopsis WD40 protein PWP2 (AtPWP2) interacting with AtTBP1 through comparison of phenotypes induced by 35S:AtPWP2-SRDX with that induced by the chimeric repressor. Our results indicate that the transrepression mediated by SRDX can be used to detect and confirm protein-protein interactions in plants and should be useful in identifying factors that form transcriptional protein complexes.

The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.