EndoBind detects endogenous protein-protein interactions in real time

We present two high-throughput compatible methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer formation over an extended duration. Using examples of the Nrf2-KEAP1 and the CRAF-KRAS-G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of endogenous protein-protein interactions in real time.


Reviewers' comments:
Reviewer #1 (Remarks to the Author): Brief summary of the manuscript In their manuscript, Bill, A. et al. present two approaches enabling detection and monitoring of protein-protein interactions (PPIs) upon their ectopic (RT-Bind) and endogenous (EndoBind) expression. Both methods allow to continuously monitor PPIs in real time and over an extended duration. For the RT-Bind approach the authors employed an interaction between KEAP1 and NRF2 and developed a stable HEK293T cell line overexpressing both proteins tagged with the split luciferase subunits. Next, the authors used some known modulators of KEAP1/NRF2 interaction to demonstrate the suitability of this approach. In the second part of the study the authors introduced the split luciferase subunits and additional tags upstream endogenous sequences encoding for KRAS and CRAF (for this purpose a pancreatic cell line PATU8988T has been employed) and again used some inducers and modulators of the interaction between these two proteins to verify the EndoBind approach they had developed. The authors also examined how targeting either KRAS or CRAF with siRNA will affect their interaction.
Overall impression of the manuscript The approaches presented in the manuscript are novel. The article is well-written, welldocumented, well-presented and, with few exceptions, easy to follow. The research design is appropriate. The conclusions are fully supported by the rigorous, diverse and complementary experimental data. The results are convincing. The research methodology is comprehensively described so that the experiments can be easily reproduced. The strong points of the study include generation of stable transfectants, monitoring protein expression levels, using RNAi strategy and a particularly innovative approach involving CRISPR/Cas9-assisted tagging of endogenously expressed proteins with split luciferase subunits. I predict that the impact of the article on the field will be high as the RT-Bind and EndoBind approaches developed by the authors can be employed in a variety of studies and therefore should be of great interest to the scientific community. Moreover, the article is highly timely as the luminescence-based approaches are gaining more and more attention.

Major comments:
The work is sound but requires some improvements. I would ask for clarification of the following issues and recommend the following amendments: 1) Although the NanoBiT strategy is relatively novel, it has been employed in many studies so far. Could the authors reference 2-3 more articles that utilized this approach? 2) Tagging of proteins may alter their subcellular localization. Did the authors check, whether the staining pattern of KRAS and CRAF proteins tagged with the split luciferase subunits and additional epitopes is the same as that of the unmodified proteins? Providing images obtained via immunofluorescence staining using antibodies mentioned in the Methods section could greatly improve the corresponding part of the manuscript. 3) Could the authors substantiate the choice of the cell lines used? 4) Could the authors explain if there is any difference between KRAS and KRAS-G12V? Are these terms used interchangeably? 5) Could the authors explain, why there are two bands visible in Supplementary Figure 2B (the blot referring to KRAS) in the lane corresponding to the engineered cells? 6) Could the authors improve the quality of the pictures by increasing the size of the plots and/or the font size? Some of them (e.g. Fig. 2B, 2C and 2G) are very difficult to decipher. 7) Could the authors provide a little more explanation and a reference to the statement claimed in lines 106-109? 8) For the sake of clarity, could the authors provide an extended description of the transfectants and/or combinations of the expression constructs, e.g. HEK293T-SmB-NRF2/Keap1-LgB; HEK293T cells co-expressing NRF2 N-terminally tagged with SmB and Keap1 C-terminally tagged with LgB, somewhere in the text?
Minor comments: 1. In line 16 an abbreviation "KRAS_G12V" is used, whereas in the rest of the manuscript (e.g. line 107) it is "KRAS-G12V", please unify. 2. In line 32 a term "Nano-luciferase" is used, whereas in the rest of the manuscript this enzyme is being referred to as NanoLuciferase, please unify. 3. Line 34: there should be "furimazine" instead of "fumirazine". 4. I do not quite understand why NRF2 is written in capital letters, whereas Keap1 is not, should not these abbreviations be written in a uniform manner? 5. Line 41: please explain abbreviations "SmB" and "LgB" in the text and unify them throughout the text (e.g. line 56). 6. Line 45: a term "promotor" is used, whereas in the other fragments of the manuscript (e.g. line 94) there is "promoter", please unify. 7. A reference to Supplementary Figure 1 should be given earlier in the text, preferably in line 48. 8. A style used in the caption of Supplementary Figure 1 is inconsistent (A is followed by a full stop, whereas B and C are followed by commas) -please unify (this refers to other figure captions as well). 9. Line 49: please explain abbreviation "Cul-3" earlier in the text and unify the corresponding abbreviation between lines 49, 74 and 94. 10. Line 65: there should be "subsequently" instead of "subsequent". 11. Line 117: there is a double space between words "of" and "the". 12. Line 146: "both" and "and" should not be written in italics. 13. Line 151: there should be "EndoBind" instead of "Endobind". 14. Line 199: there should be "an EF1a" instead of "a EIF1a". 15. Lines 204-205: "Streptococcus pyogenes" should be written in italics. 16. Line 209: the degree symbol is missing. 17. Lines 211, 236, 240 -please use a consistent way of giving cell numbers. 18. Please use a consistent manner of giving units (sometimes a unit is separated from a numerical value by a space and sometimes it is not). 19. Please use the "µ" symbol instead of "u" to denote the "micro" unit prefix throughout the text. 20. Line 228: there should be "Puromycin selection" instead of "Puromycin-selection". I have read "EndoBind -Real-time detection of endogenous protein-protein interactions," by Dr. Bill et al. This work aims to apply the commercially available NanoLucierase system to measure the NRF2/KEAP1 interaction and the as well as a KRAS(V12)-CRAF interaction using a lentiviral integration or CRISPR mediated insertion into the endogenous gene loci. They demonstrate the commercial applicability for compound screening by performing measurements in 348 and 1536well plates.
Overall, the paper provides a useful demonstration of the use of the NanoLuc system to two practical examples. The data are clear and the experiments presented should be replicable. Editing is needed to improve the readability of the paper. Often the language uses vague reference, leaving the reader guessing at the meaning. For example, page 2, line 25 "Analyzing the statusquo at a specific time…" I have no idea what this is refereeing to. A more specific introduction of the problem and the approach would help. Moreover, no introduction or citation is given for smBit or lgBit, results page 5. While these are known by some, rigor in the introduction and citation would also help make the work more clear and accessible.
Please provide a reference and explanation for the use of NRF2-D29H mutation. A more cogent explanation for its effects in the results section would make this more readable.
Reviewer #3 (Remarks to the Author): In this manuscript, the King laboratory optimizes a technique designed to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. The authors' methods basically follow the manufacture's (Promega Corporation) instruction (NanoBiT® Protein:Protein Interaction System) except for the use of pro-substrate, which used in other assay kit in this company, RealTime-Glo. This optimization is useful, however, the optimization does not use particularly novel technologies, but instead simply employs existing methods or materials. Moreover, although the advantage of using the pro-substrate is the detection of signal for longer time (pro-substrate: over 2 days), certainly fumirazine has a short half-life of about 2 hours, but other substrates provided for this interaction assay by Promega, Vivazine or Endurazine, have longer half-lives (about 10-20 hours or several days, respectively). Therefore, it does not appear to be novel unless there are dramatic advantages over these substrates.
We thank the reviewers for taking the time to thoroughly review our manuscript and for their comments and suggestions to improve our manuscript. We have addressed the specific points raised by the Reviewers by adding additional data as well as any necessary corrections, clarification and/or discussion.

Reviewer #1 (Remarks to the Author):
Brief summary of the manuscript

In their manuscript, Bill, A. et al. present two approaches enabling detection and monitoring of proteinprotein interactions (PPIs) upon their ectopic (RT-Bind) and endogenous (EndoBind) expression. Both methods allow to continuously monitor PPIs in real time and over an extended duration. For the RT-Bind approach the authors employed an interaction between KEAP1 and NRF2 and developed a stable HEK293T cell line overexpressing both proteins tagged with the split luciferase subunits. Next, the authors used some known modulators of KEAP1/NRF2 interaction to demonstrate the suitability of this approach. In the second part of the study the authors introduced the split luciferase subunits and additional tags upstream endogenous sequences encoding for KRAS and CRAF (for this purpose a pancreatic cell line PATU8988T has been employed) and again used some inducers and modulators of the interaction between these two proteins to verify the EndoBind approach they had developed. The authors also examined how targeting either KRAS or CRAF with siRNA will affect their interaction.
Overall impression of the manuscript The approaches presented in the manuscript are novel. The article is well-written, well-documented, wellpresented and, with few exceptions, easy to follow. The research design is appropriate. The conclusions are fully supported by the rigorous, diverse and complementary experimental data. The results are convincing. The research methodology is comprehensively described so that the experiments can be easily reproduced. The strong points of the study include generation of stable transfectants, monitoring protein expression levels, using RNAi strategy and a particularly innovative approach involving CRISPR/Cas9-assisted tagging of endogenously expressed proteins with split luciferase subunits. I predict that the impact of the article on the field will be high as the RT-Bind and EndoBind approaches developed by the authors can be employed in a variety of studies and therefore should be of great interest to the scientific community. Moreover, the article is highly timely as the luminescence-based approaches are gaining more and more attention.

Response:
We thank the Reviewer for pointing out the novelty of our approaches and the rigorous experimental design and data. We agree with the Reviewer that the presented techniques will be of high interest to the scientific community as they can be easily employed for multiple studies using standard laboratory equipment.

Reviewer #1 (Major comments):
The work is sound but requires some improvements. I would ask for clarification of the following issues and recommend the following amendments: 1) Although the NanoBiT strategy is relatively novel, it has been employed in many studies so far. Could the authors reference 2-3 more articles that utilized this approach?
2) Tagging of proteins may alter their subcellular localization. Did the authors check, whether the staining pattern of KRAS and CRAF proteins tagged with the split luciferase subunits and additional epitopes is the same as that of the unmodified proteins? Providing images obtained via immunofluorescence staining using antibodies mentioned in the Methods section could greatly improve the corresponding part of the manuscript.

Response:
We thank the Reviewer for this constructive comment. We performed a cellular fractionation experiment demonstrating that the tagged proteins in the PATU8988T-SmBKRAS-LgBCRAF cell line show the same localization as in the parental cell line. The data was added as Supplementary Figure 2C (see below) and the text in the manuscript has been updated as follows: "Introduction of the tags shifted the size of the endogenous proteins as expected while exerting no discernable effect on endogenous expression levels, localization of the tagged proteins and growth rate of the cells (Supplementary Figure  2B

4) Could the authors explain if there is any difference between KRAS and KRAS-G12V? Are these terms used interchangeably?
Response: We apologize for the confusion and thank the Reviewer for pointing it out. We updated the manuscript to make it more clear that we are referring to KRAS as the general protein and we pointed out the G12V mutation when introducing the cell line used. (see previous comment) 5

Response:
We apologize for the bad image quality. We transformed all figures to vector based graphics and include them as separate pdfs instead of inserting them into the manuscript text. 7) Could the authors provide a little more explanation and a reference to the statement claimed in lines 106-109?

Response:
We updated the manuscript to include more rational why overexpression of KRAS can be problematic and why an endogenous tagging strategy can be beneficial. "To test this particular hypothesis, we set out to measure the interaction of G12V-mutant KRAS and CRAF, two proteins that signal via forming complexes with each other and other proteins. Artificial overexpression of RAS can alter its physiological interactions and signaling behavior in cells, leading to increased cell growth, senescence or apoptosis (Birchler et al, 2012, Narita et al, 2005, Vartanian et al, 2013 and making an endogenous tagging approach highly desirable". (Page 4, lines 102-106) 8) For the sake of clarity, could the authors provide an extended description of the transfectants and/or combinations of the expression constructs, e.g. HEK293T-SmB-NRF2/Keap1-LgB; HEK293T cells coexpressing NRF2 N-terminally tagged with SmB and Keap1 C-terminally tagged with LgB, somewhere in the text?

Response:
We apologize for not providing enough description of the tagging in the cell lines used. We included additional information in the text to clearly indicate which tag position was used. "The cell line with N-terminal SmB-tag on Nrf2 and C-terminal LgB-tag on Keap1 (from herewith referred to as SmB-Nrf2/Keap1-LgB) was chosen for all further experiments..." (Page 3, lines 54-55) and "We sequentially introduced the SmBit at the N-terminus of KRAS and the LgBit at the N-terminus of CRAF via CRISPRknockin in PATU8988T cells" (Page 5, lines 108-110).