Applications of Protein Fragment Complementation Assays for

Reviews Cite This: J. Proteome Res. XXXX, XXX, XXX−XXX

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Applications of Protein Fragment Complementation Assays for Analyzing Biomolecular Interactions and Biochemical Networks in Living Cells Peipei Li,† Li Wang,†,‡ and Li-jun Di*,† †

Cancer Center, Faculty of Health Sciences, University of Macau, Macau, SAR of China Metabolomics Core, Faculty of Health Sciences, University of Macau, Macau, SAR of China

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‡

ABSTRACT: Protein−protein interactions (PPIs) are indispensable for the dynamic assembly of multiprotein complexes that are central players of nearly all of the intracellular biological processes, such as signaling pathways, metabolic pathways, formation of intracellular organelles, establishment of cytoplasmic skeletons, etc. Numerous approaches have been invented to study PPIs both in vivo and in vitro, including the protein-fragment complementation assay (PCA), which is a widely applied technology to study PPIs and biomolecular interactions. PCA is a technology based on the expression of the bait and prey proteins in fusion with two complementary reporter protein fragments, respectively, that will reassemble when in close proximity. The reporter protein can be the enzymes or fluorescent proteins. Recovery of the enzymatic activity or fluorescent signal can be the indicator of PPI between the bait and prey proteins. Significant effort has been invested in developing many derivatives of PCA, along with various applications, in order to address specific questions. Therefore, a prompt review of these applications is important. In this review, we will categorize these applications according to the scenarios that the PCAs were applied and expect to provide a reference guideline for the future selection of PCA methods in solving a specific problem. KEYWORDS: protein-fragment complementation assays, biomolecular interactions, biochemical network, split enzymes, BiFC

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excitation of the acceptor fluorophore and autofluorescence, while BRET signals are very weak and dim.12 In addition, this assay is only suitable for validating the suspected PPIs because overexpression of both proteins in fusion with distinct fluorophore is required. Yeast two-hybrid (Y2H) is another widely applied method for studying protein−protein interaction in vivo. This technology identifies PPIs based on fusing a “bait” protein to a DNA binding domain (DBD) and “prey” proteins to the transcription activation domain (TAD). Interaction of the suspected pair of proteins brings DBD and TAD together, which induces the expression of a reporter gene. This method is more appropriate to discover the novel PPIs.13 For transmembrane bound proteins, this method might not be useful since the hydrophobic membrane-binding domain of these proteins might prohibit the formation of a complex in the nucleus. Proximity-dependent labeling (PDL) of interacting proteins has recently been proposed by taking advantage of several enzymes such as promiscuous biotin ligase (BirA*) and ascorbate peroxidase (APEX), which synthesizes highly reactive molecules such as “5′-AMP-Biotin”, and these reactive molecules diffuse freely in the live cells within a limited distance, and the nearby proteins are highly susceptible to be modified covalently. 14−16 The limitation of the wide application of PDL technologies is the high background,

INTRODUCTION PPI is the main regulator of biological processes such as signal transduction, enzymatic reaction, protein complex assembly, etc. Usually, proteins form either stable or transient PPIs in participating in the relevant biological activities.1,2 Undoubtedly, the breakdown of the PPIs or formation of novel PPIs leads to disordered biological process and may result in diseases. Therefore, the PPIs are also attractive targets for drug design because of their independent role in mediating important biological processes.3 A variety of methods in investigating and identifying PPIs in vitro and in vivo have been developed, such as affinity tags mediated pull down,4 coimmunoprecipitation,5 biomolecular fluorescence resonance energy transfer (FRET),6 bioluminescence resonance energy transfer (BRET),7,8 and yeast/ mammalian cell two-hybrid approaches,9 etc. A systematic study of several of these technologies in identifying reliable PPIs indicated that none of them are perfect when being applied in a high throughput scenario.10 Affinity tags mediated pull-down and coimmunoprecipitation require the lysis of cells and the purification of proteins, and PPIs may be lost or abolished after a series of purification steps. Moreover, the identification of low-abundance PPIs and transient PPIs through this method is not easy.11 The advantage of FRET and BRET assay is the capability of detecting dynamic interactions of macromolecules instantaneously, but FRET has some disadvantages such as photobleaching, direct © XXXX American Chemical Society

Received: March 6, 2019 Published: June 24, 2019 A

DOI: 10.1021/acs.jproteome.9b00154 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research

Figure 1. The mechanism of PCAs. The diagram to illustrate the working mechanism of PCAs. PCA techniques are based on the fact that some fluorescent proteins or enzymes including DHFR, TEV, ubiquitin, β-lactamase, β-galactosidase, luciferase, BirA*, and APEX can be separated into two fragments, and these two fragments still recover the activity of the intact proteins or enzymes if the two fragments reassemble in the cells. These fluorescent proteins or enzymes, thus, can be applied as PCA reporters. By fusing two proteins to these two fragments respectively, the complex formation by these two proteins will promote the reassembly of the PCA reporters that can be detected by the measurement of signals such as fluorescence, cell survival, bioluminescence, absorbance, biotinylation and the active transcription of the reporter gene of choice mediated by transcriptional factor (TF), etc.

Principles of PCAs

probably owing to the free diffusion of the reactive molecules.17 Proximity ligation assay (PLA), another proximity-dependent approach, has been widely used to detect PPIs in situ.18 Similar to FRET-based assay, two antibodies are required to recognize the target proteins, and the reporter DNA probe with fluorescent dyes produce the detectable signals; thus, the quality and specificity of two antibodies are crucial, and it is useful in fixed samples. PCA is also a powerful technology to study PPIs in a living cell. This technique is based on the fact that some fluorescent proteins, such as GFP, can be expressed as two separated fragments, C-GFP and N-GFP, and these two fragments still give rise to the fluorescence signal if these two fragments reassemble in the live cells. By fusing two potentially interacting proteins to the two fragments of GFP respectively, a measurable and visible signal can be obtained through the recovered GFP and can be used to determine the location and function of the protein complex formed by the pair of target proteins.19 Besides the GFP fluorescent proteins, PCAs can also be established through various reporters, such as the ubiquitin, intein, various fluorescent proteins, split enzymes, dihydrofolate reductase (DHFR), TEV protease (Tobacco Etch Virus), β-lactamase, β-galactosidase, and luciferase, etc.20−25 (Figure 1). PCAs have been used not only for verification of the PPIs,26 but also for investigating subcellular localizations of PPIs, tracking protein complexes,27 identifying novel molecules of PPI networks,28 observing the protein oligomerization,29 and discovering drugs, etc. PCAs have been widely applied in numerous research areas from bacteria to plants, as well as mammalian cells.30−32 In addition, PCAs are relatively easy and straightforward without requiring any special equipment and complicated data interpretation. However, appropriate and sufficient controls should be included for each experiment to avoid the false calling of positive signals. In this review, we will introduce these PCAs based on various reporter systems and give a detailed explanation of the pros and cons of each application on different platforms.

PCA relies on the ability of the reporter proteins to recover their signals or enzymatic activities when the two fragments are brought into proximity by the PPI. Thus, not quite many of the proteins are suitable for being the PCA reporter. The first PCA was established by splitting ubiquitin, a 76-residue, singledomain protein in 1994 by Johnson et al.,33 who validated that the split-ubiquitin fragments conditionally restore the ability to recruit the ubiquitination machinery when fused to interacting protein pairs. After that, more enzymes or fluorescent proteins were found to recover the enzyme activity or fluorescence emission when fused to interacting proteins as two separate fragments. In addition to the reporters that emit the measurable signal directly, some other reporters without emitting measurable signals were also designed. These unmeasurable signals must be relayed to the secondary reporter that generates a measurable signal. For instance, some proteases have been selected as PCA reporter and recovery of the protease activity results in the release of another reporter factor such as transcriptional factor, to drive expression of a luciferase gene or another measurable gene. Full or partial recovery of the function of the reporter protein requires the two fragments to be in proximity or even bind to each other. Ideally, the β sheet of reporter protein seems to have the advantage of mediating the interaction between the two fragments,34,35 although there are relatively limited studies to prove that. Extremely low signal, if not none, when both fragments of the reporter present in the same cell in any type of PCA is preferred and this all-or-none feature might because of the nonspontaneous assembly of the two fragments and could be the critical reason that only limited types of the reporter were created.26 Apparently, the folding of each fragment separately is distinct from that of the whole protein. A study suggested that most of the single fragment may tend to be unfolded or even misfolded.36 Or these fragments fail to maintain a static conformation and will rapidly convert the conformations. Thus, it is almost impossible for the two fragments to form a functional reporter without fusing each of the fragments to the partner protein pairs. The fused interaction proteins possibly promote the assembly of the B


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Figure 2. Mechanism of the split-ubiquitin yeast two-hybrid (SU-Y2H) assay. The membrane-bound bait protein is fused with C-terminal half of ubiquitin (Cub) and LexA-VP16 (PLV), while the prey protein is fused to a mutated version of N-terminal half of ubiquitin (NubG). When the bait-prey forms PPI, the intact ubiquitin is formed. The ubiquitin-specific protease recognizes the reconstituted ubiquitin and releases PLV, which enters into the nucleus and activates reporter genes including LacZ, HIS3, or ADE2. If the bait and prey proteins do not form PPI, reporter genes will not be activated and the cells will die.

negative controls should have very low/no signal. Moreover, introducing mutations to the pair of candidate proteins should be designed to observe the disruption of PCA signal. For positive controls, the coexpression of the known interacting protein, including the protein pairs forming a constitutive complex or the protein pairs forming a conditional complex, should be performed separately. In addition to these controls, coimmunoprecipitation and fusion tag mediated pull down are recommended to validate the PPIs independently.

fragments due to an increased rate of fragment interactions, the thermodynamic stability of the macromolecules complex, and the local concentration of the fragments in a limited space.35,37,38 Sometimes, the pair of candidate proteins form a complex without direct interaction but mediated by other proteins, which still recovers the signal of the reporter possibly owing to the increased local concentration of the fragments.39 The PCA approaches also share several common features making this technology an ideal tool to investigate the PPIs in vivo. For example, the fusion proteins are expressed in the relevant intracellular context, and the PPIs are based on the native state and correct modification of these proteins in living cells.20 In addition, since the PPIs can be determined directly by identifying fluorescence signals or enzymatic activities, not depending on secondary events and complicated data processing, PCA can be performed with common equipment. Moreover, PCA is comprised of a collection of different assays using distinct reporters, which provide the flexibility to choose a proper assay according to the object or specific platform. PCA can be easily modified to fit into the high-throughput screening platform and provide the convenience to screen for compounds, inhibitors, or growth factors, etc.40−42 However, some concerns were also raised about the PCA. For instance, when the reporter fragments are fused to the protein pairs, steric constraints may impede the association of the fragments. This can be partially eliminated by designing a flexible linker (GGGGS) to separate the two fusion parts.43 (GGGGS)2 linker is usually chosen because it is more flexible and long enough to allow for the two fragments to interact and fold.44 In fact, there was a report to demonstrate that extended linkers in PCAs may improve the detection of protein−protein interactions.45 Another concern is that PCAs also include transient overexpression of the fusion proteins. Thus, the overexpression of both proteins fragments in the cells needs to be verified before any PCA analysis.46 The negative controls for PCA assays include the observation of signals from free fragments of a reporter, the target protein pair without fusion to the fragments of a reporter, and noninteraction pair of protein with fusion to the fragments of reporter, etc. All these

PCAs in High Throughput Screening for PPIs

PCA is a robust tool to probe protein−protein interactions in most cell lines.47 Besides being applied to analyze individual PPI event, PCA can be developed as a high throughput screening tool to discover the novel PPIs. The advantage of PCA-dependent PPI screening is that variable reporters can be chosen with the high signal-to-noise ratio. For example, the PCA reporter ubiquitin (Ub) was split into an N-terminal peptide of 35 residues (Nub) and a C-terminal peptide of 41 residues (Cub) and applied for screening of PPIs.48 In this assay, the occurrence of PPI and the recovery of intact Ub leads to the release of LexA-VP16 that is a gene specific activator and expressed in fusion with one of the Ub fragment. LexA-VP16 will bind to the promoters of genes such as his3 or ade2 that are essential for the cell survival by breaking down the nutrient substrates such as histidine or adenine presenting in the auxotroph medium.49,50 Another similar approach is known as the split-ubiquitin yeast two-hybrid (SU-Y2H) assay, which breaks the limitation of traditional Y2H that is not suitable for identifying PPIs occurring outside of the nucleus. In this application, a bait-CubPLV protein was generated by fusing the bait protein to Cub-LexA-VP16 (CubPLV), and a prey construct was created by fusing the prey protein to a mutated version of Nub (NubG). When the prey and the bait proteins interact, a functional ubiquitin protein forms and could be recognized by a ubiquitin-specific protease of the host cells.51 PLV could be cleaved and released. Then PLV transfers into the nucleus and activates the genes with LexA binding motifs at their promoters such as LacZ, HIS3, and ADE2, etc. C


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Figure 3. Characterization of dihydrofolate based PCA system. Methotrexate (MTX) is a chemical that can inhibit DHFR and kill the cells. The mutant DHFR (mDHFR) is insensitive to MTX. A pair of candidate proteins (Protein A and Protein B) can be fused to the fragments of mDHFR, respectively. If the two proteins do not form PPI, the mDHFR will not form and the cells treated with MTX will die due to the suppression of endogenous DHFR. Otherwise, the reassembled mDHFR by PPI between Protein C and Protein D will rescue the cells.

fluorescent protein will not split anymore. This irreversibility may benefit the detection of weak and transient interactions since the signal may accumulate. For example, a library of colonies in yeast was created with 5911 open reading frames (ORFs), which nearly covers 95% of genome-wide ORFs, with their 3′ end fusing with the N-terminal fragment of Venus (VN) fluorescent protein. Simultaneously, another colony was generated with the expression of fusion protein formed by small ubiquitin-related modifier (SUMO) and C-terminal fragment of Venus (VC). Mating of the VC strain and each of the VN strain enabled the high content screening of fluorescent signal of recovered Venus, revealing the proteins that interact with SUMO.56 Similar methods were performed to map the interactome network of Pex11, which regulates the number of peroxisomes in yeast.57 However, the high throughput screening based on the BiFC in the mammalian system is difficult owing to the large genome size and the difficulties in culturing the cells, not even mentioning the difficulties in maintaining a mammalian library of colonies. Thus, Lepur et al. attempted BiFC by including 94 colonies of cells with each of them expressing one human gene in the screening of the interacting proteins of S-adenosyl homocysteine hydrolase (AHCY) in human cells.58 The results were promising with the identification of Galectin-3 as AHCY interacting protein. Similarly, most of the eukaryotic cells do not have the luciferase gene, and thus the bimolecular luminescence complementation (BiLC) is suitable to identify low abundance PPIs. In one study, split Gaussia luciferase protein fragment complementation assay (GLuc PCA) was developed to monitor programmed cell death network. In this study, a library of 63 apoptotic and autophagic proteins was fused to luciferase-based PCA reporter fragments. Through analyzing 3600 candidate PPIs between any two of the 63 proteins, a detailed network of the apoptotic and autophagic pathway was constructed.32 In another recent study, Kolkhof et al. developed the BiLC based on the same luciferase to detect proteins that interact with lipid droplets. Forty-seven LD-

(Figure 2). The expression of these reporter genes enables the survival of the host cell growing on the selective medium and allows the further identification of the growing clones.52 Thus, this assay almost resembles the traditional Y2H assay but can be used to analyze PPIs outside of the nucleus. Dihydrofolate reductase has also been used as a reporter protein in many PCAs, especially the ones for the screening of the novel protein−protein interactions. For example, two leucine zipper libraries were designed to combine either Nterminus of mDHFR (N-mDHFR) fragment or C-terminus of mDHFR (C-mDHFR) fragment of the murine dihydrofolate reductase (mDHFR), and the libraries were used to transform Escherichia coli. Analysis of the survived colonies indicated that some zipper coding sequences were enriched, suggesting that these leucine zipper pairs have a higher affinity to form dimers.53 In another example, Tarassov et al. conducted a genome-wide binary screen for PPIs in S. cerevisiae by a PCA based on mDHFR. In total, 2770 PPIs were detected among 1124 endogenous proteins, and most of the interactions were unknown before.54 Since the recovery rate of mDHFR correlates with the growth rate of colonies, in a recent high throughput study of PPIs, the PPI efficiency was quantitated based on the mDHFR recovery rate, or the colony growth rate in live Saccharomyces cerevisiae (Figure 3). In fact, the sensitivity of this approach is high enough to distinguish the PPIs formed by paralog proteins.55 Fluorescent and luminescent proteins are another group of widely used PCA reporters but the signals of these reporters need to be measured by imaging instruments. For example, bimolecular fluorescence complementation (BiFC) is based on the recovery of a functional fluorescent protein in the live cells. A unique advantage of fluorescence or luminescence based PCAs is that these signals also indicate the intracellular localization of PPIs, providing more details of the biological activities. BiFC provides the required sensitivity for many applications because of the minimum background. Of noting is that once the fluorescent signal recovers in BiFC assay, the signal becomes stabilized because the two halves of the D


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Journal of Proteome Research associated proteins were selected as bait proteins from Drosophila and mouse based on previous reports. In total, this study screened 1561 pairs of PPIs and 487 protein pairs were identified.59 Of noting is that the formation of the complemented luminescence signal is reversible in this BiLC assay. Thus, this reporter has the potential to be developed as a real-time reporter to reflect the dynamic changes associated with cell status upon different treatments.

agonists, Ronan et al. applied Gluc based PCA that had the sensitivity to distinguish the effects caused by the different EGF agonists. Interestingly, the results indicated that all the tested kinases were rapidly recruited and dose-dependent; however, the speed and efficiency of response to different ligands were quite different, and thus different biological effects were induced in the intracellular context.61 In fact, many studies applied luciferase-based PCA for compounds screening such as potent synthetic opioids,62 NF-κB inhibitors,63 drug targeting α345(IV) trimers to treat Alport syndrome,64 etc. Combination of PCAs and high throughput reporter assay has also been applied to monitor the downstream gene transcription activity of membrane-bound receptors. For example, the expressed oligonucleotide tags (EXT) assay was combined with TEV as the PCA reporter. In this application, the downstream effects of PPIs between different ERBB family proteins can be monitored by high-throughput microarray measuring of barcoded reporters, which are transcripts promoted by either constitutive GAL4-VP16 (GV) or specific TF.65 Galinski et al. further applied NGS (next-generation sequencing), in combination with the EXT and TEV based PCA, to decide the identity of the transcripts in their analysis of PPIs of GPCR in living cells.66

PCAs in High Throughput Screening for PPI Inhibitors and Compounds

PCAs are important tools for screening of PPI inhibitors and compounds.3 For example, BiFC was recently used in highthroughput screening for novel molecular inhibitors of PPIs in mammalian cells. Yue et al. applied the BiFC approach to screen for inhibitors of the interaction between Survivin and Borealin. Survivin and its interacting partner Borealin form the chromosomal passenger complex (CPC) that contributes to the development of hepatocellular carcinoma. Surprisingly, the traditional chemotherapeutic drug Etoposide was finally found as one of the effective chemicals out of 10 400 chemicals.41 Since the exact working mechanism of Etoposide is not very clear, this study provided an interesting novel target of Etoposide and might bring new opportunities to improve the therapeutic effect of this drug. In another application, estrogen receptor alpha (ERα) was created as a biosensor for monitoring compounds by using PCAs, a series of estrogen sensors were designed through flanking the ligand-binding domain of the ERα (estrogen receptor alpha) by fragments of a split mVenus fluorescent protein fused at several different Nterminal and C-terminal positions. Since each sensor had a unique combination of N-terminal and C-terminal sequence flanking the ligand binding domain of ERα, these sensors showed very different responses to estrogen agonist or antagonist and thus provided the flexibility to identify or determine the quality of estrogenic molecules.42 The oligomerization of β-amyloid (Aβ) is a well-recognized symptom associated with the genesis and development of Alzheimer’s disease. By using mDHFR as the PCA reporter, Acerra et al. screened a library of the peptide in E. coli and identified some of the candidate peptides that reduced the accumulation of amyloid in E. coli.60 Schlecht et al. established hundreds of barcoded PCA colonies with candidate interacting proteins fused with the split fragments of mDHFR. They also applied an mDHFR mutant that enables the growth of cells even with the presence of mDHFR inhibitor MTX. Thus, the hundreds of PCA colonies can be pooled together for culture with the presence of MTX. After the pooled colonies were treated by 80 compounds respectively, the growth of each colony in the pool was evaluated on the basis of the quantitation of barcode DNA. They successfully discovered at least 12 PPI-chemical pairs in their initial study.40 Interestingly, both the inhibiting and promoting PPI-chemical pairs were reported in this study, highlighting the potential application of this technology in the drug discovery field. Comparably, Gluc based PCA is more sensitive and can be applied to disclose some trivial change of PPIs upon treatment by compounds or peptides. For example, several EGF analogues were developed as EGF agonist over the past years. Previous studies barely found a significant difference in the recruitment of the downstream kinases by EGFR upon treatment by these EGF agonists. To observe if there is any difference in inducing the downstream effects by these EGF

Investigation of Biochemical Networks by PCAs

The most useful application of PCA is identifying the PPIs that have important roles in the metabolic pathways, signal transduction pathways, and transcriptional regulatory network. Some of the PPIs are transient and hardly captured by traditional immunoprecipitation technology. As we introduced above, since the recovery of the activities of some of the PCA reporters accumulates, the weak signals owing to the transient PPIs may be obtained. In addition, PCA reporters are expressed with the target protein pairs in fusion. Since many target pair of proteins are likely to maintain their native conformation and cellular localization, the detection of PCA signal may represent the reproduction of PPIs of these proteins under the natural conditions. This is an obvious advantage for PCA technology and making PCA suitable for many difficult scenarios especially those PPIs occurring between the membrane-bound proteins. Capturing Cytoplasm Membrane-Bound Protein Complexes. PCA is a suitable technology to study a very large family of proteins known as G protein-coupled receptors (GPCRs). GPCRs belong to seven-transmembrane domain receptors, and their main function is sensing the extracellular stimulus and transducing the signal to intracellular proteins, leading to the activation of the signaling pathway. Thus, GPCRs often mediate various effects such as cell proliferation, cell differentiation, and cell apoptosis in response to the distinct extracellular signals. The proper function of GPCRs relies on the formation of a protein complex between GPCRs and the cofactors such as the G-proteins, β-arrestins, etc. For instance, the interactions of GPCRs with different subtypes of β-arrestins were studied by fusing the C-terminus of luciferase to GPCRs and fusing the different subtypes of β-arrestin to Nterminus of luciferase. The luminance of restored luciferase was used as the indicator of the interaction between GPCRs and different subtypes of β-arrestin.67 In another example, Split-TEV PCA using TEV as the reporter was applied to investigate the interacting partners of GPCR. Since TEV is a protease, recovery of TEV upon the interacting between GPCR and β-arrestin2, the two target proteins that were fused E


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mutants of the CBR luciferase (Click Beetle in Red) from Caribbean Pyrophorus plagiophthalamus especially the ones with mutations at the C terminal fragment, which is relevant to the spectrum of luciferase emission. One mutant of C-CBR was identified to recover the luciferase activity by combining with the N-Fluc (N-terminal fragments of firefly luciferases from firefly, Photinus pyralis), N-CBG (Click Beetle in Green from Brazilian Cratomorphus distinctus), and N-CBR. Importantly, the recovered luciferase signal showed significant differences according to the different pairing fragment in the spectrum of the signal, which can be distinguished by the luminometer.76 In another study, the different emission wavelengths of luciferases can be observed between the NCBR/C-CBG and N-CBG/C-CBG pairs and were applied to demonstrate the dual PPI events in the same mammalian cells (Figure 4).77 Multiplexed PCAs were also developed based on

to two halves of TEV protease, induced the cleavage of an NTEV linked transcriptional factor. Then the transcription factor entered into the nucleus and activated the transcription of firefly luciferase.68 Neuregulin-induced the heterodimerization of ErbB2/ErbB4 receptor tyrosine kinase was confirmed using split TEV.21 Thus, application of this technology solved the common difficulties in isolating the membrane proteins and investigating the membrane-based PPIs. A similar approach was also applied to the cytokine receptors, another family of membrane-bound proteins.69 Upon binding by the cytokines such as EPO or other cytokines, the receptors dimerize and induce the autophosphorylation of JAK kinase. STAT is a family of transcriptional factors that enter the nucleus to regulate gene expression upon phosphorylation by JAK. A study applied mDHFR PCA assay to reveal that EPO binding to its receptors alters the conformation of the dimerized receptors, which is required for the phosphorylation of JAK.70 Capturing the Transient PPIs in Signal Transduction by PCAs. A relatively challenging task in biochemistry is to capture the transient PPIs. For example, the traditional biochemical characterization of PPIs via immunoprecipitation relies on the presence of a large number of candidate proteins and the formation of stable PPI between the candidate proteins. Thus, these technologies can hardly be applied to analyze those transient, low abundance PPIs such as the interaction between a kinase and its substrate. On the contrary, PCAs are techniques “recording” the PPIs, including “keeping the records of past interactions over a period of time”. Thus, PCAs are an ideal tool to investigate transient PPIs. Split TEV was first executed in Drosophila cell culture combined with RNAi screening, the constitutive (Hpo dimerization) and regulated (Yki/14.3.3) interactions were successfully validated, and split TEV is well proven invaluable in identifying transient PPIs.71 Pelletier et al. tried mDHFR based PCA assay in analyzing the transient PPIs.53,72 By monitoring the cell survival, which depends on the recovery of mDHFR, they validated the interaction of p21 ras-GTPase and Ser/Thrrafras-binding domain. Another example was from the study of cMyc that is known as a critical transcription factor promoting tumorigenesis and tumor metastasis. Mo et al. established Nanoluc (luciferase of a luminous deep-sea shrimp, Oplophorus gracilirostris) based PCA to investigate the MYC interacting partners among 83 cancer-associated proteins in 8 different human cancer cell lines from various tissues. In addition to some previously reported MYC-interacting partners, some novel ones were surprisingly identified such as LKB1 and AKT1.73 Since LKB1 and AKT1 have kinase activities, this hinted that MYC might be a target of these two kinases. Because LKB1 and AKT1 are all important players in intracellular signaling pathways, the interaction between MYC and these two kinases may point to a possibility that these signaling pathways work through the MYC mediated transcriptional program. BiFC assays are also suitable to study transient PPIs involved in the signaling transduction pathways such as the MAPK and NFκB signaling pathway in mammalian cells.74,75 Multiplexed PCAs in Analyzing PPI Networks. Multiplexed PCAs have also been designed to observe multiple PPIs in the same host cells simultaneously. Specifically, reporters need to be designed because of the possible overlapping of reporter signals. Also, sophisticated software for signal deconvolution and capturing of signals with high sensitivity are essential. An earlier study systematically screened the

Figure 4. Schematic representations of the dual-color luciferase PCA system. Click beetle green luciferase (CBG) can be fragmented into C-terminal half (C-CBG) and N-terminal half (N-CBG). The C-CBG reassembles CBG and releases Green luciferase output by interacting with N-CBG. The C-CBG also has the ability to interact with N-CBR, the N-termini of click beetle red luciferase (N-CBR), and generates Red luciferase output. By fusion Protein B to C-CBG, and Protein A to N-CBG or Protein C to N-CBR, the interaction between Protein A and Protein B, and Protein B and Protein C can be detected simultaneously.

Fluc and nanoLuc. In this application, the PCA fragments, in fusion with distinct pairs of interacting proteins, were put into the same cells and different substrates were provided. As expected, Fluc and nanoLuc recovered their luciferase activity with a different wavelength of emission that can be distinguished.78 Capturing of Ternary Complexation by PCAs

PCAs have also been modified to study ternary complexes including proteins, DNA, RNA, iron−sulfur clusters, and other macromolecules (Figure 5).79 For example, a dual-color TriFC (trimolecules fragment complementation) based on mCherry and mVenus was demonstrated in living plant cells. Fundamentally, C terminal fragments of mCherry and mVenus were expressed in fusion with one protein of interest by flanking this protein. The two candidate interacting proteins are fused to the respective N terminal fragments of mCherry and mVenus. Consequently, only if the protein of interest F


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Figure 5. Detection ternary complexation using PCA. Protein A and protein C are fused with fragments of PCA reporter, respectively. The interaction between protein A and protein C is mediated by protein B (the left), DNA (the middle), or RNA (the right). The formation of the ternary complex will recover PCA reporter activity.

GFP-Trap. GFP-Trap is an affinity-dependent pull down by recognizing the epitope, which only appears when the two fragments of Venus bind to each other. Thus, only the recombined Venus can be pulled down by GFP-trap along with the fusion partners, the EBRR2 dimer in this case, and the other associated proteins. The coprecipitated proteins can be identified by the follow-up LC−MS.90 This technology effectively improves the targeting preciseness when the interactome of the dimerized proteins need to be determined and should have much broader application in different proteomics fields.

interacts with both candidate interacting proteins, efficient red and yellow fluorescence complementation occurs simultaneously in the same complex.80 TriFC has also been applied to analyze the protein−DNA interaction. In this example, a known ZNF domain was fused to the N-terminal fragment of the fluorescent protein, and MDB domain (methylated DNA binding domain), which recognizes the methylated CpG dinucleotides, was fused to the C-terminal fragment of the fluorescent protein. If the ZNF domain binds to the target sequence while the DNA sequence also has a methylated CpG, the functional fluorescent protein will be reassembled.81 Similarly, a combination of the MDB domain and OGG1 (oxoguanine glycosylase 1, detecting 8-oxoguanine caused by exposure to reactive oxygen species) in the Fluc PCA enables the detection of DNA damages associated with specified DNA sequence.82 TriFC has also been reported to identify RNA binding proteins, monitor the localization and dynamics of RNA−protein interaction in different living cells.83−85

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CONCLUSIONS AND PERSPECTIVE As a powerful tool in investigating the PPIs, PCA has been rarely studied for its working principle. Although a wellrecognized working mechanism is that the reporter protein, after being split into two fragments, recovers the enzymatic activity based on the spatial juxtaposition of the two fragments brought by the candidate PPIs,91 how the fragments reestablish the enzymatic domain is still a mystery. When each fragment is fused with the respective protein, how each fragment folded and whether there is conformational adaptation during the formation of the intact reporter is largely unknown. The shortage of sufficient knowledge of the working mechanism actually prevents the further improvement of the efficiency of the reporters, although some studies attempted to solve this issue by sequentially mutating the critical amino acids at the interacting interface. But those studies are still too preliminary because there are no conclusive results being obtained that might provide some guidance to improve the PCA assays.92 Even though some studies indicated that every single fragment might be in an unfolded status or in an unstable folded status when it is expressed in fusion, the direct evidence is still missing. Instead, it is more reasonable to suspect that the “half” protein will be folded, no matter in the correct or incorrect conformation, with the assistance of its fusion partner.93 This will raise another question of whether a remodeling process will be involved when the two halves of fragment meet each other and start to build up a functional enzyme, so the expression of fragments should be optimized. The false positive signal associated with PCAs has not been systematically investigated so far, which becomes another uncertainty when this useful technology is applied. In the BiFC application, when both GFP fragments are transfected into the same cell without fusion to other partner proteins, there is a constitutive background signal, indicating the formation of reconstituted GFP.94 Thus, an ideal signal-to-noise ratio is desirable to get confident results. The inclusion of the control that both GFP fragments are transfected into the same cell without fusion to other partner proteins, however, still cannot

Other PCA Derivatives

The PCA assays based on splitting the enzymes, known as PDL enzymes including horseradish peroxidase (HRP), ascorbate peroxidase (APEX), and E. coli biotin ligase BirA, etc., represent a more complicated application of this technology. For example, APEX2 is derived from soybean ascorbate peroxidase with quadruple mutants and capable of biotinylating the proximal proteins within 20 nm.86 Xue et al. tested 12 N-terminal truncations and 6 C-terminal truncations of APEX2 by anchoring with the known interaction pairs FKBP and FRB domain in the presence of rapamycin; they got the optimal split APEX2 pairs. By using these optimized pairs of APEX2 fragments, the PCA was further performed by fusing both APEX2 fragments to the cytoplasmic domain of STIM1, an endoplasmic reticulum-localized Ca2+ sensor. Since the recovery of the biotinylation activity of APEX2 could be detected, this indicates that the cytoplasmic domain of STIM1 might be responsible for the oligomerization of STIM.87 The mutant of BirA, also known as BirA*, has a very similar working mechanism as APEX. In fact, the split-BirA* based PCA has also been recently demonstrated by detecting the PPI between the catalytic and regulatory subunits of the protein phosphatase 1.88 Schopp et al. designed the split-BirA* based PCA and applied the approach to discover the novel interacting proteins of the phosphorylation-dependent heterodimer Cdc25C/14-3-3ε and miRNA-mediated silencing heterodimers Ago/Dicer or Ago/TNRC6.89 Another novel approach derived from BiFC was also reported, and this method is known as bimolecular complementation affinity purification (BiCAP). As demonstrated by Croucher et al., this technology resembles BiFC, but the recovered Venus fluorescent protein can be pulled down by G


H

bioluminescence

bioluminescence

29

77

19

61

20

36

β-lactamase

β-galactosidase

nanoluciferase

Click beetle luciferase Gaussia luciferase Renilla luciferase firefly luciferase BiFC

BirA* or APEX

bioluminescence

9

ubiquitin

35 or 28

61 27 (GFP)

gene reporter

25

biotinylation

bioluminescence fluorescence

gene reporter, cell survival fluorescence, absorbance fluorescence, absorbance, bioluminescence bioluminescence

fluorescence, cell survival

21

readout

dihydrofolate reductase (DHFR) TEV protease

reporter

reporter size (KDa)

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

detection of PPIs in vitro

Table 1. Overview of PCAs in This Review

yes

yes yes

yes

yes

yes

yes

limited

limited

yes

yes

yes

detection of PPIs in vivo

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

throughput of PPIs detection

limited

limited yes

limited

limited

limited

limited

no

no

no

no

limited

location visualization

no

yes limited

yes

yes

yes

yes

no

no

no

no

no

reversibility

application

yes

no no

no

no

no

no

no

no

no

no

no

genetic modification

yes

yes no

yes

yes

yes

yes

yes

yes

yes

yes

yes

enzyme reaction

yes

yes yes

yes

yes

yes

yes

limited

limited

limited

limited

yes

screen unknown interaction

no

yes yes

yes

yes

yes

yes

yes

yes

no

no

yes

bacterial cells

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

plant cells

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

nonmammalian cells

organism

yes

yes yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

mammalian cells

yes

yes yes

yes

yes

yes

yes

no

no

yes

no

no

living animals

27, 77, 78 41, 42, 56−58, 74, 75, 80−85 86−89

31, 63

24, 32, 59

62, 64, 73, 78 76, 77

22

23

21, 65, 66, 68 48−51

40, 53−55, 63, 70, 72

ref

Journal of Proteome Research Reviews



■

exclude the possibility that the signal shows up owing to the random collisions of the two fusion proteins without the actual formation of PPIs. In that occasion, careful checking of the signal-to-noise ratio is necessary, and only a very dramatic increase in the signal-to-noise ratio might be considered as acceptable. However, the criteria to determine the extent that the positive signal overrides the noise is hardly defined. There is a possibility that some true PPIs may be ignored owing to the false negative PCA signal. For instance, the fusion of the target protein to the reporter may lead to structural constraints that prevent the correct reassembly of the PCA reporter. However, this possibility can be restrained by applying longer linkers or redesigning the fusion expression of the reporter fragments. In fact, to maximize the discovery of novel PPIs, reciprocal fusion designs, different linkers, as well as more than one type of PCA reporters should be considered. Increasing the expression level of the fusion reporter fragments might also promote the reassembly of the PCA reporter and, thus, may help to prevent the false negative PPIs. However, overexpression of the PCA fusion reporter will also increase the chance of the false positive PPI.91 In addition, overexpression of the PCA fusion reporter is incapable of compensating the absence or shortage of the proteins that might be essential to mediate some of the false negative PPIs. Therefore, while PCAs are an ideal research tool for explorative studies and validation experiments, some limitations do exist just like any other technologies. The application of PCA as the conclusive technology is still immature, and other complementary methods are necessary to validate the results obtained from PCA. PCA has been widely applied in many cell types including mammalian cells, plant cells, invertebrate cells, yeast, and bacteria as well as mice models. Various proteins and enzyme can be used as PCA reporters. Therefore, the readout of the reporters is quite diversified. Given that there is the constitutive formation of reconstituted signal or the noise by the random collision of the two fusion proteins as discussed above, the reporter has to be carefully selected to identify the one with the lowest noise. Since each type of PCA is based on a distinct reporter, some limitations may exist when being applied to some experimental conditions (Table 1). For example, the mDHRF based assays may be more suitable for the bacterial based experiments since the bacterial cells are more sensitive to the nutrients supply. The same rule may be applied to other enzymes dedicated to providing nutrients for bacterial cell growth. Nevertheless, with the increasing application of PCA and the emergence of novel applications of this technology, the selection of the right assay may become easier. Conclusively, PCA is a useful technology with potential applications in many research fields. By carefully choosing the appropriate reporter and the readout signal, PCA can be applied in solving some important questions with some flexibility. Gradually, those defects or disadvantages associated with some PCA assays will be overcome, which will warrant more sophisticated applications such as dissecting the PPI networks, investigating the regulatory modules, discovering novel drug targets, exploring new compounds, etc.

Reviews

AUTHOR INFORMATION

Corresponding Author

*Tel.: 853-88224497. Fax: 853-88222314. E-mail: lijundi@um. edu.mo. ORCID

Peipei Li: 0000-0001-8862-1503 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is supported by the Science and Technology Development Fund (FDCT) of Macao SAR to LD (FDCT/ 0014/2018/A1) from Macau, the Multi-Year Research Grant from the University of Macau to LD (MYRG2018-00158FHS), and the National Natural Science Foundation of China (NSFC 81772980) to LD from China. This work is also supported by the Multi-Year Research Grant from the University of Macau to LW (MYRG2016-00251-FHS).

■

REFERENCES

(1) Nooren, I. M.; Thornton, J. M. Diversity of protein-protein interactions. EMBO J. 2003, 22 (14), 3486−92. (2) Ngounou Wetie, A. G.; Sokolowska, I.; Woods, A. G.; Roy, U.; Loo, J. A.; Darie, C. C. Investigation of stable and transient proteinprotein interactions: Past, present, and future. Proteomics 2013, 13 (3−4), 538−57. (3) Wehr, M. C.; Rossner, M. J. Split protein biosensor assays in molecular pharmacological studies. Drug Discovery Today 2016, 21 (3), 415−29. (4) Rigaut, G.; Shevchenko, A.; Rutz, B.; Wilm, M.; Mann, M.; Seraphin, B. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 1999, 17 (10), 1030−2. (5) Lin, J. S.; Lai, E. M. Protein-Protein Interactions: CoImmunoprecipitation. Methods Mol. Biol. 2017, 1615, 211−219. (6) Kenworthy, A. K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 2001, 24 (3), 289−96. (7) Pfleger, K. D. G.; Eidne, K. A. Monitoring the formation of dynamic G-protein-coupled receptor−protein complexes in living cells. Biochem. J. 2005, 385 (3), 625−637. (8) Coriano, C.; Powell, E.; Xu, W. Monitoring Ligand-Activated Protein-Protein Interactions Using Bioluminescent Resonance Energy Transfer (BRET) Assay. Methods Mol. Biol. 2016, 1473, 3−15. (9) Mehla, J.; Caufield, J. H.; Uetz, P. The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harbor Protoc. 2015, 2015 (5), 425−430. (10) Braun, P.; Tasan, M.; Dreze, M.; Barrios-Rodiles, M.; Lemmens, I.; Yu, H.; Sahalie, J. M; Murray, R. R; Roncari, L.; de Smet, A.-S.; Venkatesan, K.; Rual, J.-F.; Vandenhaute, J.; Cusick, M. E; Pawson, T.; Hill, D. E; Tavernier, J.; Wrana, J. L; Roth, F. P; Vidal, M. An experimentally derived confidence score for binary proteinprotein interactions. Nat. Methods 2009, 6 (1), 91−97. (11) Struk, S.; Jacobs, A.; Sanchez Martin-Fontecha, E.; Gevaert, K.; Cubas, P.; Goormachtig, S. Exploring the protein-protein interaction landscape in plants. Plant, Cell Environ. 2019, 42 (2), 387−409. (12) Xie, Q.; Soutto, M.; Xu, X.; Zhang, Y.; Johnson, C. H. Bioluminescence resonance energy transfer (BRET) imaging in plant seedlings and mammalian cells. Methods Mol. Biol. 2011, 680, 3−28. (13) Vidal, M.; Fields, S. The yeast two-hybrid assay: still finding connections after 25 years. Nat. Methods 2014, 11 (12), 1203−1206. (14) Roux, K. J.; Kim, D. I.; Raida, M.; Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 2012, 196 (6), 801−10. I


Reviews

Journal of Proteome Research (15) Rhee, H. W.; Zou, P.; Udeshi, N. D.; Martell, J. D.; Mootha, V. K.; Carr, S. A.; Ting, A. Y. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 2013, 339 (6125), 1328−1331. (16) Li, P.; Li, J.; Wang, L.; Di, L. J. Proximity Labeling of Interacting Proteins: Application of BioID as a Discovery Tool. Proteomics 2017, 17 (20), 1700002. (17) Hung, V.; Zou, P.; Rhee, H. W.; Udeshi, N. D.; Cracan, V.; Svinkina, T.; Carr, S. A.; Mootha, V. K.; Ting, A. Y. Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging. Mol. Cell 2014, 55 (2), 332−41. (18) Soderberg, O.; Gullberg, M.; Jarvius, M.; Ridderstrale, K.; Leuchowius, K. J.; Jarvius, J.; Wester, K.; Hydbring, P.; Bahram, F.; Larsson, L. G.; Landegren, U. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 2006, 3 (12), 995−1000. (19) Ghosh, I.; Hamilton, A. D.; Regan, L. Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein. J. Am. Chem. Soc. 2000, 122 (23), 5658−5659. (20) Remy, I.; Michnick, S. W. Application of protein-fragment complementation assays in cell biology. BioTechniques 2007, 42 (2), 137. 139, 141 passim. (21) Wehr, M. C.; Laage, R.; Bolz, U.; Fischer, T. M.; Grunewald, S.; Scheek, S.; Bach, A.; Nave, K. A.; Rossner, M. J. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 2006, 3 (12), 985−93. (22) Burkard, C.; Bloyet, L. M.; Wicht, O.; van Kuppeveld, F. J.; Rottier, P. J.; de Haan, C. A.; Bosch, B. J. Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of beta-galactosidase. PLoS One 2014, 9 (7), No. e101762. (23) Galarneau, A.; Primeau, M.; Trudeau, L. E.; Michnick, S. W. Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat. Biotechnol. 2002, 20 (6), 619−22. (24) Remy, I.; Michnick, S. W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat. Methods 2006, 3 (12), 977−9. (25) Lake, M. C.; Aboagye, E. O. Luciferase fragment complementation imaging in preclinical cancer studies. Oncoscience 2014, 1 (5), 310−25. (26) Michnick, S. W. Protein fragment complementation strategies for biochemical network mapping. Curr. Opin. Biotechnol. 2003, 14 (6), 610−617. (27) Ilagan, M. X.; Lim, S.; Fulbright, M.; Piwnica-Worms, D.; Kopan, R. Real-time imaging of notch activation with a luciferase complementation-based reporter. Sci. Signaling 2011, 4 (181), No. rs7. (28) Michnick, S. W.; Ear, P. H.; Manderson, E. N.; Remy, I.; Stefan, E. Universal strategies in research and drug discovery based on protein-fragment complementation assays. Nat. Rev. Drug Discovery 2007, 6 (7), 569−82. (29) Zhang, X. E.; Cui, Z.; Wang, D. Sensing of biomolecular interactions using fluorescence complementing systems in living cells. Biosens. Bioelectron. 2016, 76, 243−50. (30) Lee, L. Y.; Gelvin, S. B. Bimolecular fluorescence complementation for imaging protein interactions in plant hosts of microbial pathogens. Methods Mol. Biol. 2014, 1197, 185−208. (31) Hatzios, S. K.; Ringgaard, S.; Davis, B. M.; Waldor, M. K. Studies of dynamic protein-protein interactions in bacteria using Renilla luciferase complementation are undermined by nonspecific enzyme inhibition. PLoS One 2012, 7 (8), No. e43175. (32) Gilad, Y.; Shiloh, R.; Ber, Y.; Bialik, S.; Kimchi, A. Discovering protein-protein interactions within the programmed cell death network using a protein-fragment complementation screen. Cell Rep. 2014, 8 (3), 909−21. (33) Johnsson, N.; Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (22), 10340−10344.

(34) Kerppola, T. K. Complementary methods for studies of protein interactions in living cells. Nat. Methods 2006, 3 (12), 969−71. (35) Kerppola, T. K. Visualization of molecular interactions using bimolecular fluorescence complementation analysis: characteristics of protein fragment complementation. Chem. Soc. Rev. 2009, 38 (10), 2876−86. (36) Gegg, C. V.; Bowers, K. E.; Matthews, C. R. Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. Protein Sci. 1997, 6 (9), 1885−92. (37) Hu, C.-D.; Chinenov, Y.; Kerppola, T. K. Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Mol. Cell 2002, 9 (4), 789−798. (38) Robida, A. M.; Kerppola, T. K. Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association. J. Mol. Biol. 2009, 394 (3), 391−409. (39) Walter, M.; Chaban, C.; Schutze, K.; Batistic, O.; Weckermann, K.; Nake, C.; Blazevic, D.; Grefen, C.; Schumacher, K.; Oecking, C.; Harter, K.; Kudla, J. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 2004, 40 (3), 428−38. (40) Schlecht, U.; Miranda, M.; Suresh, S.; Davis, R. W.; St Onge, R. P. Multiplex assay for condition-dependent changes in protein-protein interactions. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (23), 9213− 9218. (41) Yue, L.; Li, L.; Li, D.; Yang, Z.; Han, S.; Chen, M.; Lan, S.; Xu, X.; Hui, L. High-throughput screening for Survivin and Borealin interaction inhibitors in hepatocellular carcinoma. Biochem. Biophys. Res. Commun. 2017, 484 (3), 642−647. (42) McLachlan, M. J.; Katzenellenbogen, J. A.; Zhao, H. A new fluorescence complementation biosensor for detection of estrogenic compounds. Biotechnol. Bioeng. 2011, 108 (12), 2794−803. (43) Chen, X.; Zaro, J. L.; Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Delivery Rev. 2013, 65 (10), 1357−69. (44) Remy, I.; Michnick, S. W. Visualization of biochemical networks in living cells. Proc. Natl. Acad. Sci. U. S. A. 2001, 98 (14), 7678−83. (45) Chretien, A. E.; Gagnon-Arsenault, I.; Dube, A. K.; Barbeau, X.; Despres, P. C.; Lamothe, C.; Dion-Cote, A. M.; Lague, P.; Landry, C. R. Extended linkers improve the detection of protein-protein interactions (PPIs) by dihydrofolate reductase protein-fragment complementation assay (DHFR PCA) in living cells. Mol. Cell. Proteomics 2018, 17 (3), 549. (46) Smaczniak, C.; Immink, R. G.; Muino, J. M.; Blanvillain, R.; Busscher, M.; Busscher-Lange, J.; Dinh, Q. D.; Liu, S.; Westphal, A. H.; Boeren, S.; Parcy, F.; Xu, L.; Carles, C. C.; Angenent, G. C.; Kaufmann, K. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (5), 1560−5. (47) Chumakov, S. P.; Kravchenko, Y. E.; Chumakov, P. M. Protein complementation as tool for studying protein-protein interactions in living cells. Mol. Biol. 2012, 46 (5), 627−638. (48) Johnsson, N.; Varshavsky, A. Split Ubiquitin as a Sensor Of Protein Interactions In-Vivo. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (22), 10340−10344. (49) Moreno, D.; Neller, J.; Kestler, H. A.; Kraus, J.; Dunkler, A.; Johnsson, N. A fluorescent reporter for mapping cellular proteinprotein interactions in time and space. Mol. Syst. Biol. 2013, 9, 647. (50) Dunkler, A.; Muller, J.; Johnsson, N. Detecting protein-protein interactions with the Split-Ubiquitin sensor. Methods Mol. Biol. 2012, 786, 115−30. (51) Stagljar, I.; Korostensky, C.; Johnsson, N.; te Heesen, S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (9), 5187−5192. J


Reviews


context using protein-fragment complementation assays. Assay Drug Dev. Technol. 2003, 1 (6), 811−22. (70) Remy, I.; Wilson, I. A.; Michnick, S. W. Erythropoietin Receptor Activation by a Ligand-Induced Conformation Change. Science 1999, 283 (5404), 990−993. (71) Wehr, M. C.; Holder, M. V.; Gailite, I.; Saunders, R. E.; Maile, T. M.; Ciirdaeva, E.; Instrell, R.; Jiang, M.; Howell, M.; Rossner, M. J.; Tapon, N. Salt-inducible kinases regulate growth through the Hippo signalling pathway in Drosophila. Nat. Cell Biol. 2013, 15 (1), 61−71. (72) Pelletier, J. N.; Campbell-Valois, F. X.; Michnick, S. W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. U. S. A. 1998, 95 (21), 12141−12146. (73) Mo, X.; Qi, Q.; Ivanov, A. A.; Niu, Q.; Luo, Y.; Havel, J.; Goetze, R.; Bell, S.; Moreno, C. S.; Cooper, L. A.; Johns, M. A.; Khuri, F. R.; Du, Y.; Fu, H. AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay. Mol. Pharmacol. 2017, 91 (4), 339−347. (74) Yamada, K.; Yamaguchi, K.; Yoshimura, S.; Terauchi, A.; Kawasaki, T. Conservation of Chitin-Induced MAPK Signaling Pathways in Rice and Arabidopsis. Plant Cell Physiol. 2017, 58 (6), 993−1002. (75) Zhou, X. Y.; Hu, D. X.; Chen, R. Q.; Chen, X. Q.; Dong, W. L.; Yi, C. L. 14−3-3 Isoforms Differentially Regulate NFkappaB Signaling in the Brain After Ischemia-Reperfusion. Neurochem. Res. 2017, 42 (8), 2354−2362. (76) Hida, N.; Awais, M.; Takeuchi, M.; Ueno, N.; Tashiro, M.; Takagi, C.; Singh, T.; Hayashi, M.; Ohmiya, Y.; Ozawa, T. Highsensitivity real-time imaging of dual protein-protein interactions in living subjects using multicolor luciferases. PLoS One 2009, 4 (6), No. e5868. (77) Villalobos, V.; Naik, S.; Bruinsma, M.; Dothager, R. S.; Pan, M. H.; Samrakandi, M.; Moss, B.; Elhammali, A.; Piwnica-Worms, D. Dual-color click beetle luciferase heteroprotein fragment complementation assays. Chem. Biol. 2010, 17 (9), 1018−29. (78) Verhoef, L. G.; Mattioli, M.; Ricci, F.; Li, Y. C.; Wade, M. Multiplex detection of protein-protein interactions using a next generation luciferase reporter. Biochim. Biophys. Acta, Mol. Cell Res. 2016, 1863 (2), 284−92. (79) Shekhawat, S. S.; Ghosh, I. Split-protein systems: beyond binary protein-protein interactions. Curr. Opin. Chem. Biol. 2011, 15 (6), 789−97. (80) Offenborn, J. N.; Waadt, R.; Kudla, J. Visualization and translocation of ternary Calcineurin-A/Calcineurin-B/Calmodulin-2 protein complexes by dual-color trimolecular fluorescence complementation. New Phytol. 2015, 208 (1), 269−79. (81) Stains, C. I.; Furman, J. L.; Segal, D. J.; Ghosh, I. Site-specific detection of DNA methylation utilizing mCpG-SEER. J. Am. Chem. Soc. 2006, 128 (30), 9761−5. (82) Furman, J. L.; Mok, P. W.; Badran, A. H.; Ghosh, I. Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA. J. Am. Chem. Soc. 2011, 133 (32), 12518−27. (83) Gelderman, G.; Sivakumar, A.; Lipp, S.; Contreras, L. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria. Biotechnol. Bioeng. 2015, 112 (2), 365−75. (84) Milev, M. P.; Brown, C. M.; Mouland, A. J. Live cell visualization of the interactions between HIV-1 Gag and the cellular RNA-binding protein Staufen1. Retrovirology 2010, 7, 41. (85) Han, Y.; Wang, S.; Zhang, Z.; Ma, X.; Li, W.; Zhang, X.; Deng, J.; Wei, H.; Li, Z.; Zhang, X. E.; Cui, Z. In vivo imaging of proteinprotein and RNA-protein interactions using novel far-red fluorescence complementation systems. Nucleic Acids Res. 2014, 42 (13), No. e103. (86) Lam, S. S.; Martell, J. D.; Kamer, K. J.; Deerinck, T. J.; Ellisman, M. H.; Mootha, V. K.; Ting, A. Y. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 2015, 12 (1), 51−4.

(52) Bashline, L.; Gu, Y. Using the split-ubiquitin yeast two-hybrid system to test protein-protein interactions of transmembrane proteins. Methods Mol. Biol. 2015, 1242, 143−58. (53) Pelletier, J. N.; Arndt, K. M.; Pluckthun, A.; Michnick, S. W. An in vivo library-versus-library selection of optimized protein-protein interactions. Nat. Biotechnol. 1999, 17 (7), 683−90. (54) Tarassov, K.; Messier, V.; Landry, C. R.; Radinovic, S.; Serna Molina, M. M.; Shames, I.; Malitskaya, Y.; Vogel, J.; Bussey, H.; Michnick, S. W. An in vivo map of the yeast protein interactome. Science 2008, 320 (5882), 1465−1470. (55) Diss, G.; Gagnon-Arsenault, I.; Dion-Cote, A. M.; Vignaud, H.; Ascencio, D. I.; Berger, C. M.; Landry, C. R. Gene duplication can impart fragility, not robustness, in the yeast protein interaction network. Science 2017, 355 (6325), 630−634. (56) Sung, M. K.; Lim, G.; Yi, D. G.; Chang, Y. J.; Yang, E. B.; Lee, K.; Huh, W. K. Genome-wide bimolecular fluorescence complementation analysis of SUMO interactome in yeast. Genome Res. 2013, 23 (4), 736−46. (57) Mattiazzi Usaj, M.; Brloznik, M.; Kaferle, P.; Zitnik, M.; Wolinski, H.; Leitner, F.; Kohlwein, S. D.; Zupan, B.; Petrovic, U. Genome-Wide Localization Study of Yeast Pex11 Identifies Peroxisome-Mitochondria Interactions through the ERMES Complex. J. Mol. Biol. 2015, 427 (11), 2072−87. (58) Lepur, A.; Kovacevic, L.; Beluzic, R.; Vugrek, O. Combining Unique Multiplex Gateway Cloning and Bimolecular Fluorescence Complementation (BiFC) for High-Throughput Screening of Protein-Protein Interactions. J. Biomol. Screening 2016, 21 (10), 1100−1111. (59) Kolkhof, P.; Werthebach, M.; van de Venn, A.; Poschmann, G.; Chen, L.; Welte, M.; Stuhler, K.; Beller, M. A Luciferase-fragment Complementation Assay to Detect Lipid Droplet-associated ProteinProtein Interactions. Mol. Cell. Proteomics 2017, 16 (3), 329−345. (60) Acerra, N.; Kad, N. M.; Mason, J. M. Combining intracellular selection with protein-fragment complementation to derive Abeta interacting peptides. Protein Eng., Des. Sel. 2013, 26 (7), 463−70. (61) Ronan, T.; Macdonald-Obermann, J. L.; Huelsmann, L.; Bessman, N. J.; Naegle, K. M.; Pike, L. J. Different Epidermal Growth Factor Receptor (EGFR) Agonists Produce Unique Signatures for the Recruitment of Downstream Signaling Proteins. J. Biol. Chem. 2016, 291 (11), 5528−40. (62) Cannaert, A.; Vasudevan, L.; Friscia, M.; Mohr, A. L. A.; Wille, S. M. R.; Stove, C. P. Activity-Based Concept to Screen Biological Matrices for Opiates and (Synthetic) Opioids. Clin. Chem. 2018, 64 (8), 1221−9. (63) Ashkenazi, S.; Plotnikov, A.; Bahat, A.; Dikstein, R. Effective cell-free drug screening protocol for protein-protein interaction. Anal. Biochem. 2017, 532, 53−59. (64) Omachi, K.; Kamura, M.; Teramoto, K.; Kojima, H.; Yokota, T.; Kaseda, S.; Kuwazuru, J.; Fukuda, R.; Koyama, K.; Matsuyama, S.; Motomura, K.; Shuto, T.; Suico, M. A.; Kai, H. A Split-LuciferaseBased Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome. Cell Chem. Biol. 2018, 25 (5), 634−643. (65) Botvinnik, A.; Wichert, S. P; Fischer, T. M; Rossner, M. J Integrated analysis of receptor activation and downstream signaling with EXTassays. Nat. Methods 2010, 7 (1), 74−80. (66) Galinski, S.; Wichert, S. P.; Rossner, M. J.; Wehr, M. C. Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts. Sci. Rep. 2018, 8 (1), 8137. (67) Hattori, M.; Tanaka, M.; Takakura, H.; Aoki, K.; Miura, K.; Anzai, T.; Ozawa, T. Analysis of temporal patterns of GPCR-betaarrestin interactions using split luciferase-fragment complementation. Mol. BioSyst. 2013, 9 (5), 957−64. (68) Djannatian, M. S.; Galinski, S.; Fischer, T. M.; Rossner, M. J. Studying G protein-coupled receptor activation using split-tobacco etch virus assays. Anal. Biochem. 2011, 412 (2), 141−52. (69) Yu, H.; West, M.; Keon, B. H.; Bilter, G. K.; Owens, S.; Lamerdin, J.; Westwick, J. K. Measuring drug action in the cellular K


Reviews

Journal of Proteome Research (87) Xue, M.; Hou, J.; Wang, L.; Cheng, D.; Lu, J.; Zheng, L.; Xu, T. Optimizing the fragment complementation of APEX2 for detection of specific protein-protein interactions in live cells. Sci. Rep. 2017, 7 (1), 12039. (88) De Munter, S.; Gornemann, J.; Derua, R.; Lesage, B.; Qian, J.; Heroes, E.; Waelkens, E.; Van Eynde, A.; Beullens, M.; Bollen, M. Split-BioID: a proximity biotinylation assay for dimerization-dependent protein interactions. FEBS Lett. 2017, 591 (2), 415−424. (89) Schopp, I. M.; Amaya Ramirez, C. C.; Debeljak, J.; Kreibich, E.; Skribbe, M.; Wild, K.; Bethune, J. Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes. Nat. Commun. 2017, 8, 15690. (90) Croucher, D. R.; Iconomou, M.; Hastings, J. F.; Kennedy, S. P.; Han, J. Z.; Shearer, R. F.; McKenna, J.; Wan, A.; Lau, J.; Aparicio, S.; Saunders, D. N. Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers. Sci. Signaling 2016, 9 (436), No. ra69. (91) Morell, M.; Ventura, S.; Aviles, F. X. Protein complementation assays: approaches for the in vivo analysis of protein interactions. FEBS Lett. 2009, 583 (11), 1684−91. (92) Luker, K. E.; Smith, M. C.; Luker, G. D.; Gammon, S. T.; Piwnica-Worms, H.; Piwnica-Worms, D. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl. Acad. Sci. U. S. A. 2004, 101 (33), 12288−93. (93) Hu, C.-D.; Chinenov, Y.; Kerppola, T. K. Visualization of Interactions among bZIP and Rel Family Proteins in Living Cells Using Bimolecular Fluorescence Complementation. Mol. Cell 2002, 9, 10. (94) Wang, L.; Li, J. J.; Guo, L. Y.; Li, P.; Zhao, Z.; Zhou, H.; Di, L. J. Molecular link between glucose and glutamine consumption in cancer cells mediated by CtBP and SIRT4. Oncogenesis 2018, 7 (3), 26.

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