Protein-protein Interactions In Mammalian Cells Research Articles

Split T7 RNA polymerase biosensors to study multiprotein interaction dynamics.

Protein-protein interactions (PPIs) are involved in nearly all cellular processes. PPIs are particularly crucial for mediating selectivity along signaling pathways. Thus, measuring the competitive interplay between PPIs in a cell is important for both understanding fundamental cellular regulation and developing therapeutics targeting those whose dysregulation is associated with disease. A variety of split protein reporter-based tools are available to measure if two proteins interact within a cell and thereby characterize the general determinants of their interactions. PPIs, however, occur within complex networks facilitated by dynamic biophysical nuances that determine activity and selectivity. Evolved, proximity-dependent split T7 RNA polymerase (RNAP) biosensors have recently been used to perform deep mutational scanning of PPI interfaces, and to create synthetic gene circuits. In this chapter, we present the application of proximity-dependent split RNAP biosensors as a method to measure multidimensional PPIs in live cells. Orthogonal split RNAP "tags" encode each interaction in a unique RNA signal, thereby enabling the study of multiple competitive PPIs in live cells. Each unique RNA signal can be quantified via established RNA analysis methods. Herein, we provide advice and protocols to aid other researchers in using the split RNAP biosensor, focusing primarily on how to detect multiple PPIs in mammalian cells, including their dynamic interplay in the presence of small molecule inhibitors.

Time-gated detection of protein-protein interactions with transcriptional readout.

Transcriptional assays, such as yeast two-hybrid and TANGO, that convert transient protein-protein interactions (PPIs) into stable expression of transgenes are powerful tools for PPI discovery, screens, and analysis of cell populations. However, such assays often have high background and lose information about PPI dynamics. We have developed SPARK (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics), in which proteolytic release of a membrane-tethered transcription factor (TF) requires both a PPI to deliver a protease proximal to its cleavage peptide and blue light to uncage the cleavage site. SPARK was used to detect 12 different PPIs in mammalian cells, with 5 min temporal resolution and signal ratios up to 37. By shifting the light window, we could reconstruct PPI time-courses. Combined with FACS, SPARK enabled 51 fold enrichment of PPI-positive over PPI-negative cells. Due to its high specificity and sensitivity, SPARK has the potential to advance PPI analysis and discovery.

Fatty acid synthase cooperates with protrudin to facilitate membrane outgrowth of cellular protrusions

Cellular protrusion formation capacity is a key feature of developing neurons and many eukaryotic cells. However, the mechanisms underlying membrane growth in protrusion formation are largely unclear. In this study, photo-reactive unnatural amino acid 3-(3-methyl-3H-diazirin-3-yl)-propamino-carbonyl-Nε-l-lysine was incorporated by a genetic code expansion strategy into protrudin, a protein localized in acidic endosomes and in the endoplasmic reticulum, that induces cellular protrusion and neurite formation. The modified protrudin was used for covalent trapping of protrudin-interacting proteins in living cells. Fatty acid synthase (FASN), which synthesizes free fatty acids, was identified to transiently interact with protrudin. Further characterization revealed a unique cooperation mechanism in which protrudin cooperates with FASN to facilitate cellular protrusion formation. This work reveals a novel mechanism involved in protrusion formation that is dependent on transient interaction between FASN and protrudin, and establishes a creative strategy to investigate transient protein-protein interactions in mammalian cells.

LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks.

Protein-protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein-protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.

Current Approaches Toward Quantitative Mapping of the Interactome.

Protein–protein interactions (PPIs) play a key role in many, if not all, cellular processes. Disease is often caused by perturbation of PPIs, as recently indicated by studies of missense mutations. To understand the associations of proteins and to unravel the global picture of PPIs in the cell, different experimental detection techniques for PPIs have been established. Genetic and biochemical methods such as the yeast two-hybrid system or affinity purification-based approaches are well suited to high-throughput, proteome-wide screening and are mainly used to obtain qualitative results. However, they have been criticized for not reflecting the cellular situation or the dynamic nature of PPIs. In this review, we provide an overview of various genetic methods that go beyond qualitative detection and allow quantitative measuring of PPIs in mammalian cells, such as dual luminescence-based co-immunoprecipitation, Förster resonance energy transfer or luminescence-based mammalian interactome mapping with bait control. We discuss the strengths and weaknesses of different techniques and their potential applications in biomedical research.

An ALuc-Based Molecular Tension Probe for Sensing Intramolecular Protein-Protein Interactions.

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. The present protocol demonstrates an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. A unique design of single-chain probes was fabricated, in which a full-length artificial luciferase (ALuc(®)) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. A molecular tension probe comprising ALuc23 greatly enhances the bioluminescence in response to varying concentrations of rapamycin, and named "tension probe (TP)." The basic probe design can be further modified towards eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "combinational probe." TPs may become an important addition to the tool box of bioassays in the determination of protein dynamics of interest in mammalian cells.

Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

MCLIP Detection of Novel Protein-Protein Interactions at the Nuclear Envelope.

The organization and function of the nuclear envelope (NE) involves hundreds of nuclear membrane proteins and myriad protein-protein interactions, most of which are still uncharacterized. Many NE proteins interact stably or dynamically with the nuclear lamina or chromosomes. This can make them difficult to extract under nondenaturing conditions, and greatly limits our ability to explore and identify functional protein interactions at the NE. This knowledge is needed to understand nuclear envelope structure and the mechanisms of human laminopathy diseases. This chapter provides detailed protocols for MCLIP (membrane cross-linking immunoprecipitation) identification of novel protein-protein interactions in mammalian cells.

DULIP: A Dual Luminescence-Based Co-Immunoprecipitation Assay for Interactome Mapping in Mammalian Cells

Mapping of protein–protein interactions (PPIs) is critical for understanding protein function and complex biological processes. Here, we present DULIP, a dual luminescence-based co-immunoprecipitation assay, for systematic PPI mapping in mammalian cells. DULIP is a second-generation luminescence-based PPI screening method for the systematic and quantitative analysis of co-immunoprecipitations using two different luciferase tags. Benchmarking studies with positive and negative PPI reference sets revealed that DULIP allows the detection of interactions with high sensitivity and specificity. Furthermore, the analysis of a PPI reference set with known binding affinities demonstrated that both low- and high-affinity interactions can be detected with DULIP assays. Finally, using the well-characterized interaction between Syntaxin-1 and Munc18, we found that DULIP is capable of detecting the effects of point mutations on interaction strength. Taken together, our studies demonstrate that DULIP is a sensitive and reliable method of great utility for systematic interactome research. It can be applied for interaction screening and validation of PPIs in mammalian cells. Moreover, DULIP permits the specific analysis of mutation-dependent binding patterns.

A New in Vivo Cross-linking Mass Spectrometry Platform to Define Protein–Protein Interactions in Living Cells

Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.

Identifying nuclear protein-protein interactions using GFP affinity purification and SILAC-based quantitative mass spectrometry.

Many cellular proteins assemble into macromolecular protein complexes. Therefore, identifying protein-protein interactions (PPIs) is essential to gain insight into the function of proteins. Recently established quantitative mass spectrometry-based techniques have significantly improved the unbiased search for PPIs. In this chapter, we describe a single-step GFP affinity purification method combined with SILAC-based quantitative mass spectrometry that can be used to identify nuclear PPIs in mammalian cells.

A molecularly engineered split reporter for imaging protein-protein interactions with positron emission tomography

Improved techniques to non-invasively image protein-protein interactions (PPIs) are essential. We molecularly engineered a positron emission tomography (PET)-based split reporter (herpes simplex virus type 1 thymidine kinase [TK]), split between Thr265 and Ala266, and used this in a protein-fragment complementation assay (PCA) to quantitatively measure PPIs in mammalian cells and to microPET image them in living mice. An introduced point mutation (V119C) significantly enhanced TK complementation in PCAs based on rapamycin modulation of FRB (FKBP12-rapamycin-binding domain) and FKBP12 (FK506 binding protein), on interaction of hypoxia-inducible factor-1α and the von Hippel-Lindau tumor suppressor, and in an estrogen receptor intramolecular protein folding assay. Applications of this novel split TK are potentially far-reaching, including for example considerably more accurate monitoring of immune and stem cell therapies, allowing unprecedented fully quantitative and tomographic PET localization of PPIs in pre-clinical small and large animal models of disease.

A Modified Tandem Affinity Purification Technique Identifies That 14-3-3 Proteins Interact with Tiam1, an Interaction Which Controls Tiam1 Stability

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.

Application of quantitative immunoprecipitation combined with knockdown and cross‐linking to Chlamydomonas reveals the presence of vesicle‐inducing protein in plastids 1 in a common complex with chloroplast HSP90C

Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein-protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein-protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle-inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross-linking step (-X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross-linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions.

High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays

BackgroundMost of the biological processes rely on the formation of protein complexes. Investigation of protein-protein interactions (PPI) is therefore essential for understanding of cellular functions. It is advantageous to perform mammalian PPI analysis in mammalian cells because the expressed proteins can then be subjected to essential post-translational modifications. Until now mammalian two-hybrid assays have been performed on individual gene scale. We here describe a new and cost-effective method for the high-throughput detection of protein-protein interactions in mammalian cells that combines the advantages of mammalian two-hybrid systems with those of DNA microarrays.ResultsIn this cell array protein-protein interaction assay (CAPPIA), mixtures of bait and prey expression plasmids together with an auto-fluorescent reporter are immobilized on glass slides in defined array formats. Adherent cells that grow on top of the micro-array will become fluorescent only if the expressed proteins interact and subsequently trans-activate the reporter. Using known interaction partners and by screening 160 different combinations of prey and bait proteins associated with the human androgen receptor we demonstrate that this assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. Moreover, different strategies in respect to bait-prey combinations are presented.ConclusionWe demonstrate that the CAPPIA assay allows the quantitative detection of specific protein interactions in different types of mammalian cells and under the influence of different compounds. The high number of preys that can be tested per slide together with the flexibility to interrogate any bait of interest and the small amounts of reagents that are required makes this assay currently one of the most economical high-throughput detection assays for protein-protein interactions in mammalian cells.

Efficiency of protein-protein interactions in mammalian cells determined by a dual-light assay

Efficiency of protein-protein interactions in mammalian cells determined by a dual-light assay

A retrovirus-based protein complementation assay screen reveals functional AKT1-binding partners.

We developed a retrovirus-based protein-fragment complementation assay (RePCA) screen to identify protein-protein interactions in mammalian cells. In RePCA, bait protein is fused to one fragment of a rationally dissected fluorescent protein, such as GFP, intensely fluorescent protein, or red fluorescent protein. The second, complementary fragment of the fluorescent protein is fused to an endogenous protein by in-frame exon traps in the enhanced retroviral mutagen vector. An interaction between bait and host protein (prey) places the two parts of the fluorescent molecule in proximity, resulting in reconstitution of fluorescence. By using RePCA, we identified a series of 24 potential interaction partners or substrates of the serine/threonine protein kinase AKT1. We confirm that alpha-actinin 4 (ACTN4) interacts physically and functionally with AKT1. siRNA-mediated ACTN4 silencing down-regulates AKT phosphorylation, blocks AKT translocation to the membrane, increases p27(Kip1) levels, and inhibits cell proliferation. Thus, ACTN4 is a critical regulator of AKT1 localization and function.

Association of RNA Helicase A with Human Immunodeficiency Virus Type 1 Particles

RNA helicase A (RHA) belongs to the DEAH family of proteins that are capable of unwinding double-stranded RNA structure. In addition to its involvement in the metabolism of cellular RNA, RHA has been shown to stimulate RNA transcription from the long terminal repeat promoter of human immunodeficiency virus type 1 (HIV-1) as well as to enhance Rev/Rev response element-mediated gene expression. In this study, we provide evidence that RHA associates with HIV-1 Gag in an RNA-dependent manner. This interaction results in specific incorporation of RHA into HIV-1 particles. Knockdown of endogenous RHA in virus producer cells leads to generation of HIV-1 particles that are less infectious in part as a result of restricted reverse transcription. Therefore, RHA represents the first example of cellular RNA helicases that participate in HIV-1 particle production and promote viral reverse transcription.

A dual-light reporter system to determine the efficiency of protein-protein interactions in mammalian cells

Methods for determining protein–protein interactions in mammalian cells typically rely on single reporter functions and are susceptible to variations between samples particularly in regard to levels of transcription, processing and translation. A method has been developed for determining protein–protein interactions in mammalian cells, which bypasses these variables confounding single reporter assays. The approach utilizes two units of gene expression linked to reporter functions that are interposed by a deactivation–activation unit in such a way that the downstream expression unit is switched off. Hence upstream expression occurs regardless of protein–protein interaction, leading to the production of the upstream reporter. In the event of protein–protein interactions, the downstream expression unit is switched on leading to dual reporter read outs. Thus, the ratio of the two reporter activities provides a measure to determine the efficiency of protein–protein interactions. To access the system we screened a mutant of BMPR2 where the interaction between BMPR-II and LIMK is abrogated. BMPR-II is a type II receptor of the TGFβ superfamily and plays a key role in the pathogenesis of familial pulmonary arterial hypertension. This system has potential for high-throughput screening of libraries (peptide, chemical, cDNA, etc.) to isolate agents that are capable of interfering with highly selective protein–protein interaction.

Protein photo-cross-linking in mammalian cells by site-specific incorporation of a photoreactive amino acid

We report a method of photo-cross-linking proteins in mammalian cells, which is based on site-specific incorporation of a photoreactive amino acid, p-benzoyl-L-phenylalanine (pBpa), through the use of an expanded genetic code. To analyze the cell signaling interactions involving the adaptor protein Grb2, pBpa was incorporated in its Src homology 2 (SH2) domain. The human GRB2 gene with an amber codon was introduced into Chinese hamster ovary (CHO) cells, together with the genes for the Bacillus stearothermophilus suppressor tRNA(Tyr) and a pBpa-specific variant of Escherichia coli tyrosyl-tRNA synthetase (TyrRS). The Grb2 variant with pBpa in the amber position was synthesized when pBpa was included in the growth medium. Upon exposure of cells to 365-nm light, protein variants containing pBpa in the positions proximal to the ligand-binding pocket were cross-linked with the transiently expressed epidermal growth factor (EGF) receptor in the presence of an EGF stimulus. Cross-linked complexes with endogenous proteins were also detected. In vivo photo-cross-linking with pBpa incorporated in proteins will be useful for studying protein-protein interactions in mammalian cells.