A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap.

We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system. This system requires creation of a bait strain that stably expresses a fusion product of part of the fluorescent monomeric Kusabira-Green (mKG) protein to a protein of interest. A PiggyBac transposon vector is then introduced into this strain, and a sequence encoding the remainder of mKG is inserted into the genome and fused randomly with endogenous genes. The binding partner can be identified by isolating cells that fluoresce when BiFC occurs. Using this system, we screened for interactors of p65 (also known as RELA), an NF-κB subunit, and isolated a number of mKG-positive clones. 5'- or 3'-RACE to produce cDNAs encoding mKG-fragment fusion genes and subsequent reconstitution assay identified PKM, HSP90AB1, ANXA2, HSPA8, and CACYBP as p65 interactors. All of these, with the exception of CACYBP, are known regulators of NF-κB. Immunoprecipitation assay confirmed endogenously expressed CACYBP and p65 formed a complex. A reporter assay revealed that CACYBP enhanced 3κB reporter activation under TNFα stimulation. This screening system therefore represents a valuable method for identifying interacting factors that have not been identified by other methods.

Introduction

Elucidating protein–protein interactions is critical for analyzing protein function and identifying potential therapeutic targets. However, existing methods for identifying such interactions are limited in their utility. For example, the commonly used two-hybrid method only recognizes interactions in the nucleus and can generate false positives if the proteins of interest have transcriptional activity. Fluorescence resonance energy transfer (FRET) can be used to probe the dynamics of protein interactions in living cells. However, the visualization is real-time, the fluorescence does not accumulate, and requires the use of highly sensitive detection devices. Also, a background of fluorescent proteins is inevitable. The utility of mass spectrometry (MS) is also limited because one cannot know whether the interaction is direct or indirect, and high expression levels are required.

Here, we focused on Bimolecular Fluorescence Complementation (BiFC), a highly sensitive and simple technology that is not subject to the weaknesses listed above. BiFC occurs when two complementary fragments of a fluorescent protein, neither of which fluoresce on their own, come into proximity and form a native-like structure that reconstitutes fluorescence. By fusing two proteins of interest, one to each fragment of the fluorescent protein, the level of interaction between the proteins of interest can be evaluated by the degree of fluorescence [1, 2]. Although it may take longer for fluorescence to mature than in the case of the FRET system, the fluorescence accumulates and become stable [3], allowing for the detection of even weak interactions.

In order to screen for binding partners of a bait protein, it is necessary to prepare a library of prey proteins. We used a DNA transposon to achieve library creation and screening simultaneously [4, 5]. The DNA transposon can insert any DNA element into the genome [6], and one can easily obtain a cell library in which a fragment of the fluorescent protein is fused with a different gene in each cell. Compared to viral vector systems, the transposon system achieves greater randomness of insertion, is easier to use, and can incorporate sequences that would hinder viral packaging [7]. In this study, we used the hyperactive PiggyBac transposon for highly efficient genomic integration [8].

As a case study, we screened for novel protein interactors of p65, a component of the transcription factor NF-κB that forms a heterodimer with another NF-κB component such as p50 (also known as NFKB1). p65 plays important roles in the transcriptional regulation of immune responses and has attracted attention as a target for treatment of numerous diseases [9-14]. Details of the screening system, methods for identifying candidate genes, and their effects on NF-κB activity are described.

Materials and methods

BiFC kit

For BiFC screening system, we used the Coral Hue® Fluo-chase kit (MBL). This kit includes p65-mKGN and p50-mKGC, in which the N-terminal and C-terminal fragments of the fluorescent protein mKG (monomeric Kusabira Greeen) are fused to fragments of NF-κB components p65 and p50. When the complementary components are co-expressed, the chromophore of mKG is reconstituted and fluoresces. The bait strain used in the screening system of this study stably expressed p65-mKGN. The nucleotide and amino acid sequences of p65-mKGN and p50-mKGC are shown in S1 Appendix of S1 File.

Cell culture

HeLa and HEK293T were maintained in DMEM (Fujifilm WAKO, Tokyo, Japan) supplemented with 10% FBS (Nichirei Biosciences, Tokyo, Japan), 100 μg/mL streptomycin sulfate, 100 U/mL penicillin G potassium (SMPG) at 37°C under 5% CO2.

Bait cell line

After infecting HeLa cells with a retroviral expression vector (pMXs-p65-mKGN-IRES-blaR), the cells were selected in bulk with blasticidin and then single cell sorting was performed using a cell sorter SH800Z (Sony, Tokyo, Japan). A cell clone that was able to withstand repeated single cell sorting, had high protein expression of p65-mKGN, and high BiFC fluorescence with the introduction of p50-mKGC was selected. The cell line was named HeLa-p65-mKGN.

Screening of p65 interaction proteins by FACS

HeLa-p65-mKGN cells were seeded in a 12-well plate at 8 × 104 cells/well. After overnight culture, transposon donor vectors shown in Fig 1(A) were co-introduced using polyethyleneimine (PEI, Polysciences) with the hyPBase transposase expression vector. The DNA solution used for transfection was adjusted to 0.75 μg donor vector and 0.25 μg transposase (donor:transposase = 3:1). In the case of a vector having a splicing acceptor on the 5′ side of mKGC, it is expected that the N-terminal side of mKGC would fuse to the exon sequence of a gene and expressed. In the case of a vector having a splicing donor (SD) sequence on the 3′ side, it is expected that the C-terminal side of mKGC would fuse to the exon sequence of a gene. One day after transfection, the cells were passaged into a 10-cm dish/sample, and 7 days after transfection, mKG-positive cells were collected in bulk using a cell sorter. After propagation and passage to a 10-cm dish, cloning was performed by single cell sorting. After confirming the fluorescence of the clone with a flow cytometer, the expression of the mKGC fusion protein was confirmed by immunoblot analysis using an anti-mKGC antibody.

Fig 1

Establishment of a screening system for p65 interacting proteins using a PiggyBac transposon system and BiFC.

(a) PiggyBac transposon donor vectors are designed to express fusion proteins with mKGC using a splicing mechanism. Three frames were prepared for each of the vectors to be fused to the C-terminal or N-terminal fragment of mKG. TR, terminal repeat; TPS, transcription pause site; SD, splicing donor; SA, splicing acceptor. (b) A transposon donor vector encoding mKGC (Fig 1A) was introduced with hyPBase vector into a bait cell line (p65-mKGN, HeLa). After 7 days, the mKG-positive population was recovered using FACS. After expansion, mKG-positive cells were cloned by single-cell sorting. Clones with high fluorescence were selected, and the expression of the mKGC fusion protein was confirmed by immunoblotting. Genes were identified by 5′- or 3′-RACE (rapid amplification of cDNA end). The cDNA of the fusion protein was cloned and then returned to the bait strain to examine whether BiFC fluorescence was reconstituted.

RNA recovery and cloning of candidate genes

RNA was isolated from cells seeded on 6-cm dish using 1 mL of Isogen (Nippon Gene) according to the manufacturer’s protocol. For cloning the mKGC fusion gene, cDNA was synthesized from 5 μg of extracted RNA using Superscript III First-Strand Synthesis System (Life Technologies). The expression vector of each mKGC fusion gene was prepared by in-fusion reaction of PCR fragments and/or a DNA fragment treated with a restriction enzyme. The primers or the restriction enzyme to obtain each DNA fragment were shown in S1 Table of S1 File.

Identification of C-terminal fusion gene by 3′-RACE

cDNA was synthesized from 5 μg of RNA using 3′-Full RACE Core Set (Takara Bio) according to the manufacturer’s protocol. As the reverse transcription primer, ‘Oligo dT-3 sites Adapter Primer’ attached to the product was used. Coding sequence linked to mKGC was amplified using KOD FX neo polymerase (TOYOBO) (Tables 1 and 2). The PCR primer sequence is shown in Table 3.

Table 1

Composition of 3′-RACE PCR reaction.

Template cDNA	0.3 μL
2× PCR buffer	12.5 μL
2 mM dNTPs	5.0 μL
20 μM each primer mix	1.5 μL
KOD FX neo 1U/μL	0.5 μL
MQ             to 25 μL

Table 2

3′-RACE PCR condition.

94°C	2 min
98°C	10 s	×30
60°C	30 s
68°C	5 min
68°C	7 min
15°C	∞

Table 3

Primers.

Primers used for 3′-RACE	5′-CCACTACATCAGCCACAGGC-3′
	5′-CTGATCTAGAGGTACCGGATCC-3′

Identification of N-terminal fusion gene by 5′-RACE

cDNA was synthesized from 5 μg of RNA using SuperScript III Reverse Transcriptase (Thermo Fisher) according to the manufacturer’s protocol using mKGC gene specific primer 5′CTGTGGCTGATGTAG3′. Next, 2.5 U of RNaseH and 1 U of RNaseA were added to the reverse transcription solution and reacted at 37°C for 1 h to degrade RNA. The cDNA was purified using NucleoSpin Tissue (Takara) and eluted with 50 μL water. Ten microliters of the eluted cDNA was reacted in a tailing buffer (10 mM Tris-HCl (pH 8.4), 25 mM KCl, 1.5 mM MgCl2, 0.2 mM dCTP, 7 U of terminal deoxynucleotidyl transferase (Takara)) at 37°C for 1 h to add polyC oligo-nucleotides to the 5′ end, followed by heat inactivation at 80°C for 3 min. Next, nested PCR was conducted to amplify the specific sequence of the mKGC fusion gene using KOD FX neo (TOYOBO) (Tables 4 and 5) using PCR primers shown in Table 6.

Table 4

Composition of 5′-RACE PCR.

Template cDNA	5.0 μL
2× PCR buffer	25 μL
2 mM dNTPs	10 μL
10 μM each primer mix	2.0 μL
KOD FX neo 1 U/μL	1.0 μL
MQ            to 50 μL

Table 5

5′-RACE PCR condition.

94°C	2 min
98°C	10 s	×35
50°C	30 s
68°C	5 min
68°C	5 min
15°C	∞

Table 6

Primers.

Primers used for 5′-RACE 1st PCR	5′-GGCCACGCGTCGACTAGTACGGGGGGGGGGGGGGGG-3′
	5′-TGAACTGGCACTTGTGGTTG-3′
Primers used for 5′-RACE 2nd PCR	5′-GGCCACGCGTCGACTAGTAC-3′
	5′-TCCTGAACCACCACTACCAC-3′

Immunoblotting and immunoprecipitation

For analyses of protein expression, cells were collected in TNE buffer (10 mM Tris-HCl (pH7.8), 150 mM NaCl, 1 mM EDTA, 1% NP-40). Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). Collected protein lysates were mixed with SDS-PAGE loading buffer (0.15 M Tris-HCl (pH 6.8), 6% (w/v) SDS, 0.003% (w/v) Bromophenol blue, 30% (w/v) glycerol, 15% (w/v) β-mercaptoethanol), sonicated for 5 min and then boiled at 100°C for 5 min followed by SDS-PAGE and immunoblotting. For immunoprecipitation, cultured cells were washed once in PBS and collected with a scraper. After centrifugation, cells were lysed in TNE buffer for 10 min with occasional vortexing on ice. Cell lysates were centrifuged and the supernatants were precleared by incubation with protein-G Sepharose beads (GE healthcare). Cleared lysates were incubated at 4°C with antibodies for 100 min and then with the protein-G Sepharose beads for further 120min. Beads were collected by centrifugation and washed three times with TNF buffer. Protein lysates were mixed with SDS-PAGE loading buffer, and then boiled at 100°C for 10 min followed by SDS-PAGE and immunoblotting. Table 7 shows the antibodies and conditions used.

Table 7

Antibody list.

Antibody	Model number	Company
Anti-monomeric Kusabira Green C terminal fragment mouse mAb	M149-3M	MBL
Rabbit(DA1E) mAb IgG XP isotype control	#3900S	Cell signaling
Anti-p65 rabbit mAb (D14E12)	#8242S	Cell signaling
Anti-CACYBP rabbit mAb (D43G11)	#8225S	Cell signaling

Reporter assay

The Dual Luciferase Reporter Assay Kit (Promega) was used for the reporter assays. HeLa cells were seeded in a 12-well dish at 1.0 × 105 cells/well. The next morning, 1 μg of DNA 0.5 μg gene expression vector, 0.5 μg pGL4.32 (Promega) and 4 μg of PEI were mixed in 100 μL Opti-MEM and applied to the cells. Six hours later, the medium was replaced with culture medium. The next day, 20 ng/mL TNFα was added, and after the indicated time, cell lysates were recovered with 300 μL 1× Passive Lysis Buffer (Promega). Luciferase activity was measured using TriSTAR2S LB942 (BERTHOLD).

Results and discussion

Establishment of a screening system using PiggyBac transposon and BiFC

To establish a bait cell line, an N-terminal fragment (168 aa) of monomeric Kusabira-Green (mKG) was fused to a fragment of p65 (residues 190–291 of the full-length protein) and stably expressed in HeLa cells. The bait strain was cloned by FACS, and a clone was selected that had a high p65-mKGN expression and high fluorescence with the introduction of p50 (residues 247–352)-mKGC, a known binding partner. To fuse any fragment to mKGC, a total of 6 different PiggyBac transposon vectors (gene X is fused to C-terminal of mKG or N-terminal of mKG in each 3 frame-variations) were constructed (Fig 1A) and introduced into bait cells. Screening was performed by plating 8 × 104 cells/sample on a 12-well plate and introducing each of the six different vectors with the hyPBase transposase vector (Fig 1B). After 7 days, the mKG-positive cells were isolated by FACS. From 15 independent screenings, 9 different clones with high fluorescence compared to an empty control were isolated (Fig 2) and it was confirmed that the mKGC fusion protein was expressed in these cells (S1 Fig in S1 File).

Fig 2

Identification of positive clones by the BiFC screening system.

The fluorescence levels of reconstituted mKG by the BiFC mechanism within the cloned cells obtained by the screening were analyzed by flow cytometry.

In order to identify the C-terminal fusion gene in clones #1 to #5, 3′-RACE was performed revealing that exons from CACYBP (Clone#1&2), HSP90AB1 (Clone #3), PKM (Clone#4), and KRT8 (Clone #5) were fused with mKGC in frame (S2 Fig in S1 File). Similarly, the N-terminal fusion gene in clones #6 to #9 was analyzed by 5′-RACE and revealed that exons from ANXA2 (Clone #6) and HSPA8 (Clones #7, 8, 9) were fused to mKGC in frame (S2 Fig in S1 File).

Next, the candidate genes were cloned as mKGC fusions from each cell clone into the pPB-PCMV-IRES-PuroR vector, introduced into the bait strain, and analyzed by flow cytometry. We confirmed that all samples were mKG-positive (Fig 3A). Among these, KRT8 (clone #5) had the weakest fluorescence (Figs 2 and 3A), and we did not perform further analyses with this clone. p50 was not identified as a binding partner in our screening, possibly because the transposon had low preference for the p50 locus, or, depending on the way in which the exon is chosen, not all of the trapped p50 peptide could cause BiFC, due to factors such as lack of required area or conformation. It is also possible that the total coverage of genes trapped by the vector was not high enough.

Fig 3

Analyses of mKGC fusion genes identified by RACE.

(a) Each candidate fusion gene was cloned by PCR, incorporated into the pPB-PCMV-vector and introduced into the bait strain (p65-mKGN). Clones were analyzed by flow cytometry to verify whether BiFC reconstitution occurred. (b) The lysates of the clones obtained from the screen and the lysate of the cell in which the cDNA of the mKGC fusion protein from the cloned cell was expressed were immunoblotted with an anti-mKGC antibody to compare their molecular weights.

Immunoblot analyses (Fig 3B) revealed that the trapped mKGC fusion protein expressed in each clone and the proteins expressed from the isolated cDNAs were consistent for CACYBP, PKM, HSP90AB1, ANXA2, and HSPA8. Among these, PKM [15], ANXA2 [16], HSPA8 [17] and HSP90AB1 [18, 19] are already known as regulators of NF-κB, indicating that the screening system worked correctly.

Regulation of NF-κB by CACYBP

Among the genes identified from the isolated clones, CACYBP, identified from clones #1 and #2, is the only one not previously known to interact with p65. p65 is known to be localized in the cytoplasm in an inactive state. We first analyzed the localization of BiFC fluorescence with a confocal microscope for clone #1 cells. While the majority of the signal was seen homogeneously in the cytoplasm, some formed unidentified speckles (Fig 4A). The nature of the speckles is currently unknown. To confirm the binding of endogenous p65 to CACYBP, immunoprecipitation assay was performed. As expected, endogenous CACYBP was detected in the coprecipitated complex of endogenous p65 in lysates from HeLa wild-type strain (WT) or clones # 1 and # 2 (Fig 4B and 4C). In addition, a band was detected at the expected molecular weight of mKGC-CACYBP only for the lysates of clones # 1 and # 2 (Fig 4B). CACYBP is involved in cell growth and differentiation by controlling dephosphorylation and ubiquitination of target proteins [20], but there are no reports that it regulates NF-κB activity. We therefore examined the effects of CACYBP on NF-κB activation using a 3κB-Fluc reporter assay. We found that CACYBP overexpression significantly enhanced the fold change of NF-κB activity induced by TNFα (Fig 4D). This suggests that CACYBP is a previously unrecognized regulator of NF-κB. It is known that p65 translocates to the nucleus by stimulation with TNFα. In order to investigate whether the complex localization was changed by the stimulation of TNFα, confocal microscopic analysis was conducted. There were no changes in TNFα stimulation at 0, 15, 30, and 60 min, and at 0, 4, and 24 h for both fluorescence signals (S3 Fig in S1 File). This is consistent with the fact that p65-mKGN, which contains only part of p65 (aa residues 190–291), does not have the ability to translocate into the nucleus. This result also indicated that mKGC-CACYBP does not release binding to p65-mKGN upon TNFα stimulation.

Fig 4

Characterization of CACYBP.

A, Confocal microscopic analysis of BiFC fluorescence of clone #1 (Bait: p65-mKGN) where mKGC-CACYBP is expressed. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus. B, Structures of endogenous CACYBP (upper) and CACYBP fused with mKGC (lower) in trapped clones #1 and #2. The latter was revealed by sequence analysis after cloning the cDNA of the fusion gene. C, Immunoprecipitation assay for binding between p65 and CACYBP. The antibody that recognizes endogenous p65 (but does not recognize p65-mKGN) was used for immunoprecipitation. IP, immunoprecipitation; IB, immunoblot; WT, HeLa wild type strain. D, Identification of NF-κB-regulating activity of CACYBP. A full-length CACYBP expression vector and a 3 kB-Fluc reporter vector were co-introduced into HeLa cells prior to addition of TNF-α. Cells were collected at each time shown and luciferase assays performed. Relative values were calculated with the mean value at 0 h as 1. A statistical processing t-test was performed (n = 3). *, p < 0.05, **, p < 0.01. This figure is representative of three independent experiments. n.s., not significant.

In conclusion, we have established a transposon- and BiFC-based convenient screening system for identifying protein–protein interactions. This system identified various binding factors by random capture of the peptide of the endogenous gene, using a simple procedure, without the need to prepare a library. The method is straightforward and easy to implement, and could be readily applied to other proteins, to explore new binding partners.

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26 Aug 2020

PONE-D-20-15261

A new screening system for identifying interacting proteins using biomolecular fluorescence complementation

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Reviewer #1: The authors present a methodology based on bimolecular fluorescence complementation (BiFC) and validate it by analyzing interactors of the p65 subunit of NF-kappaB. While this approach may prove to be an interesting addition to existing methodologies to study protein-protein interactions, the manuscript needs strengthening in order to be more convincing about its general applicability and usefulness.

Major points:

1. The authors often mention their method as being novel, but is just a different implementation of an established procedure (BiFC), by using different fluorescent molecules. It should be more clearly stated in the manuscript that this is a variation of an existing method.

2. The introductory remarks about FRET are not entirely correct. FRET intensity depends on various factors, such as orientation of the molecules and proximity of the molecules, so over-expression in not necessarily required. FRET might not be able to detect weak/transient interactions, but in contrast to BiFC it can probe the dynamics of a system. In general, limitations of BiFC should be properly introduced together with its strengths, such as the time required for the complex to fold and fluoresce (it is NOT a real-time approach), the fact that it is irreversible (cumulative signal - hence no real time information), and that negative controls are not so straight forward.

3. In discussing the lack of interaction of p65 with p50, the authors suggest that the trapped p50 peptide may not conformationally cause BiFC. However, they mention early in the results that they had a high fluorescence signal with the introduction of p50 (residues 247–352)-mKGC. This needs to be clarified further by tagging whole p50 and seeing if they get complementation upon transfection together with tagged p65. How does this compare with other BiFC systems? Previous work (Hu et al. 2002) has shown an interaction between the two using BiFC with different fluorescent tags. There is no further investigation or discussion of these discrepancies.

4. The overall impression from this manuscript is that it needs strengthening, either in the characterization of the novel p65 interactor that was identified (very little is done in this respect) or in the direct comparison with existing BiFC approaches, to reveal any advantages of the methodology presented here (what is the rational for using the piggyBac system over others, is the folding time faster, does it give a stronger signal, can it be used for proteins present in not easily accessible subcellular compartments, etc).

Minor point

In the abstract, 5 p65 interactors are reported, while 6 are shown in the results.

Reviewer #2: Miyakura H et al. reported a new screening system by combining BiFC and transposon-mediated library creation. The authors found CACYBP as a novel binding partner of p65. The screening system that the authors established is interesting and useful to identify protein-protein interaction in vivo. This manuscript would have broad interest to readers of PLoS One. This reviewer lists a few comments below to further strengthen the manuscript.

1. The authors should confirm the interaction between p65 and CACYBP by a method other than BiFC such as immunoprecipitation.

2. It will be informative to present subcellular localization of CACYBP along with p65 before and after TNF stimulation. In addition, where is mKG fluorescence observed, in nucleus, cytosol, or both?

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25 Mar 2021

> Journal Requirements:

>

> When submitting your revision, we need you to address these additional requirements.

>

>

>

> 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

>

> https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

>

> https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

>

I have revised the manuscript to meet the specified journal format.

>

> 2. We note that your Methods section is included in the supporting information file. Please include this methods section within the main manuscript file.

>

As requested, the Methods section was moved to the main manuscript file.

>

> 3. One of the noted authors is a group or consortium [Japan Biological Informatics Consortium (JBiC)]. In addition to naming the author group, please list the individual authors and affiliations within this group in the acknowledgments section of your manuscript. Please also indicate clearly a lead author for this group along with a contact email address.

>

The main author belongs to one of the laboratories within the Consortium. I have changed the citation to reflect this situation, and make the authorship clearer.

>

> 4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

The data were not a core part of the research, so we removed the phrase from the manuscript.

> 5. Review Comments to the Author

> Reviewer #1: The authors present a methodology based on bimolecular fluorescence complementation (BiFC) and validate it by analyzing interactors of the p65 subunit of NF-kappaB. While this approach may prove to be an interesting addition to existing methodologies to study protein-protein interactions, the manuscript needs strengthening in order to be more convincing about its general applicability and usefulness.

>

> Major points:

>

> 1. The authors often mention their method as being novel, but is just a different implementation of an established procedure (BiFC), by using different fluorescent molecules. It should be more clearly stated in the manuscript that this is a variation of an existing method.

>

Thank you for your suggestions. To avoid misunderstanding, we removed "new" from the title. The full text of the title was changed to “A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap’. The words ‘novel method’ in abstract was replaced with ‘valuable method”. To avoid giving the impression that the BiFC method itself was developed in this work, we replaced the statement “We have established a new screening system for identifying interacting proteins using biomolecular fluorescence complementation (BiFC).” in abstract with the statement “We have established a new screening system for identifying interacting proteins by combining biomolecular fluorescence complementation (BiFC) and a transposon gene trap system”

> 2. The introductory remarks about FRET are not entirely correct. FRET intensity depends on various factors, such as orientation of the molecules and proximity of the molecules, so over-expression in not necessarily required. FRET might not be able to detect weak/transient interactions, but in contrast to BiFC it can probe the dynamics of a system. In general, limitations of BiFC should be properly introduced together with its strengths, such as the time required for the complex to fold and fluoresce (it is NOT a real-time approach), the fact that it is irreversible (cumulative signal - hence no real time information), and that negative controls are not so straight forward.

We appreciate your comments. In response to this advice, the description “Fluorescence resonance energy transfer (FRET) can be used to visualize protein interactions in living cells, but high levels of expression are required otherwise FRET cannot be detected even when proteins bind.” was changed to “Fluorescence resonance energy transfer (FRET) can be used to probe the dynamics of protein interactions in living cells. However, the visualization is real-time, the fluorescence does not accumulate, and requires the use of highly sensitive detection devices.” The description “The reconstituted fluorescent protein is very stable, allowing for detection of even weak interactions.” was changed to the statement “Although it may take longer for fluorescence to mature than in the case of the FRET system, the fluorescence accumulates and becomes stable, allowing for the detection of even weak interactions.”

>

> 3. In discussing the lack of interaction of p65 with p50, the authors suggest that the trapped p50 peptide may not conformationally cause BiFC. However, they mention early in the results that they had a high fluorescence signal with the introduction of p50 (residues 247–352)-mKGC. This needs to be clarified further by tagging whole p50 and seeing if they get complementation upon transfection together with tagged p65. How does this compare with other BiFC systems? Previous work (Hu et al. 2002) has shown an interaction between the two using BiFC with different fluorescent tags. There is no further investigation or discussion of these discrepancies.

Thank you for your detailed review. I corrected the part that was lacking in the statements. In this discussion, we described possible reasons why p50 was not identified. One possibility is that we have not obtained a p50 fragment with a suitable binding region at which BiFC can occur, such as residues 247–352 as well as other constructs, including those suggested by the reviewer (Hu et al.)). It may depend on how the exon is chosen upon gene trapping by the trap vector. The assumption that not all trapped fragments cause BiFC may not be wrong, due to differences in factors such as folding and conformation, required for binding, and there are no discrepancies. It is also possible that the total coverage of genes trapped by the vector was not high enough, in which case not all known p65-interacting proteins would be detected using this method. However, in this study, PKM, HSP90AB1, ANXA2, and HSPA8, which are known to bind to p65, were identified using this method. The relevant description has been corrected as follows: “p50 was not identified as a binding partner in our screening, possibly because the transposon had low preference for the p50 locus, or, depending on the way in which the exon is chosen, not all of the trapped p50 peptide could cause BiFC, due to factors such as lack of required area or conformation. It is also possible that the total coverage of genes trapped by the vector was not high enough.”

>

> 4. The overall impression from this manuscript is that it needs strengthening, either in the characterization of the novel p65 interactor that was identified (very little is done in this respect) or in the direct comparison with existing BiFC approaches, to reveal any advantages of the methodology presented here (what is the rational for using the piggyBac system over others, is the folding time faster, does it give a stronger signal, can it be used for proteins present in not easily accessible subcellular compartments, etc).

Thank you for your helpful suggestions. We do not think it is necessary to compare the methodology presented here with the conventional BiFC method, which is often used for proteins that have a fixed target to be analyzed. However, we utilized BiFC theory in combination with the properties of transposons to create a convenient screening system for searching for binding proteins. The transposons explore the genome in depth, and any protein fragments are fused randomly. This approach is therefore more straightforward to apply than normal library creation methods. These points were made in the previous submission. In this revised version, the conclusions section in the ‘Results and discussion’ was corrected and the major advantages were added as follows: “This system identifies various binding factors by random capture of the peptide of the endogenous gene, using a simple procedure, without the need to prepare a library. The method is straightforward and easy to implement, and could be readily applied to other proteins, to explore new binding partners.” Using this system we identified a new binding factor (CACYBP) for p65, without significant effort, and showed its function in enhancing the activation of NF-kB by TNFα. In a new experiment examining the binding of CACYBP to p65, added in this revision, the interaction between the endogenous proteins was confirmed by immunoprecipitation assays using p65 and CACYBP antibodies (Fig4 B and C). Also, as suggested by another reviewer, we added fluorescence image data from a confocal microscope (Fig4A, and S3 Figure). By adding these results to this revised edition, we believe that this method can be emphasized to be both simple and useful. Since the method is highly versatile, it can be readily applied to other proteins to explore new binding partners. The approach therefore has considerable potential to be valuable in future research.

>

> Minor point

>

> In the abstract, 5 p65 interactors are reported, while 6 are shown in the results.

>

Thank you for your detailed review. As described in the previous submission, KRT8 was weakest fluorescent signal detected by BiFC (Fig3A), and therefore should not necessarily be presented. However, its lysate was used in the immunoblot (Fig3B). Its description could be completely eliminated from the manuscript, but since it was included in the immunoblot image in Fig3B, we decided it needed to be described.

> Reviewer #2: Miyakura H et al. reported a new screening system by combining BiFC and transposon-mediated library creation. The authors found CACYBP as a novel binding partner of p65. The screening system that the authors established is interesting and useful to identify protein-protein interaction in vivo. This manuscript would have broad interest to readers of PLoS One. This reviewer lists a few comments below to further strengthen the manuscript.

>

> 1. The authors should confirm the interaction between p65 and CACYBP by a method other than BiFC such as immunoprecipitation.

Thank you for raising this issue. New immunoprecipitation experiments were conducted, and binding between endogenous p65 and either endo-CACYBP or mKGC-CACYBP was confirmed. These results are included in this revised edition (Fig 4B and C).

> 2. It will be informative to present subcellular localization of CACYBP along with p65 before and after TNF stimulation. In addition, where is mKG fluorescence observed, in nucleus, cytosol, or both?

Thank you for your suggestion. The localization was newly analyzed using a confocal microscope (Fig 4A). A signal was observed in the cytoplasmic region outside the nucleus. While the majority of the signal was seen homogeneously in the cytoplasm, some signal formed unidentified speckles. The nature of this speckle is currently not known. This pattern did not seem to change during the 0-24h period of TNFα stimulation. In this revised version, these data are shown in Fig 4A and S3 figures.

> 3. Figure legends. The authors should describe more detailed information in order for readers to easily understand figures. For instance, abbreviations used in figures should be defined in figure legends.

Thank you for your helpful comment. We have revised the relevant legends in the manuscript, according to this advice.

Submitted filename: Response to reviewers.doc

Click here for additional data file.

23 Apr 2021

A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap

PONE-D-20-15261R1

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27 Apr 2021

PONE-D-20-15261R1

A screening system for identifying interacting proteins using biomolecular fluorescence complementation and transposon gene trap

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