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Establishment and Systematic Benchmarking of Innovative Methods for Quantitative Interactome Mapping in Mammalian Cells

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Establishment and Systematic Benchmarking of Innovative Methods for Quantitative Interactome Mapping in Mammalian Cells Establishment and Systematic Benchmarking of Innovative Methods for Quantitative Interactome Mapping in Mammalian Cells Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by PHILIPP TREPTE from Zwenkau January 2016 The work was conducted from January 2011 until January 2016 under supervision of Prof. Dr. Erich E. Wanker at the Max Delbrueck Center for Molecular Medicine in the Helmholtz-Association. I hereby confirm that I have written the thesis independently using solely the aids mentioned and without illicit assistance from third parties. 1. Reviewer: Prof. Dr. Erich E. Wanker 2. Reviewer: Prof. Dr. Fritz G. Rathjen Thesis defense on 29.04.2016 Table of Contents 1. SUMMARY ....................................................................................................................... 1 2. ZUSAMMENFASSUNG .................................................................................................... 3 3. INTRODUCTION ............................................................................................................ 5 3.1. On the binding affinity of protein-protein interactions ................................................... 6 3.2. Quantitative detection of protein-protein interactions in vitro and in cells .................... 8 3.2.1. Available methods to detect protein-protein interactions in vitro .................................. 9 3.2.1.1. Isothermal titration calorimetry (ITC) ......................................................................... 9 3.2.1.2. Surface Plasmon Resonance (SPR) ........................................................................... 10 3.2.2. Available methods to detect protein-protein interactions in cells ................................ 12 3.2.2.1. Yeast two-hybrid (Y2H) ........................................................................................... 12 3.2.2.2. Bimolecular fluorescence/luminescence complementation assay (BiFC/BiLC) .......... 13 3.2.2.3. Luminescence-based Mammalian Interactome Mapping (LUMIER) .......................... 16 3.2.2.4. Fluorescence/Bioluminescence Resonance Energy Transfer (FRET/BRET) ................. 17 3.3. Unraveling the challenges of interactome-research. ..................................................... 23 4. AIMS OF THE STUDY .................................................................................................... 27 5. RESULTS ........................................................................................................................ 29 5.1. DULIP: A Dual Luminescence-based Co-Immunoprecipitation Assay for Interactome Mapping in Mammalian Cells .................................................................................................. 29 5.1.1. Generation of Gateway-compatible vectors suitable for systematic DULIP interaction screening ................................................................................................................................. 30 5.1.2. DULIP assays facilitate the detection of the known interaction between BAD and BCL2L1 .................................................................................................................................. 31 5.1.3. Y2H and FRET assays confirm the interaction between BAD and BCL2L1 .................. 35 5.1.4. Assessment of assay quality ........................................................................................ 36 5.1.5. DULIP is suitable to validate interactions detected with the Y2H ................................ 43 5.1.6. DULIP allows the detection of both low- and high-affinity interactions ....................... 45 5.1.7. Point mutations influence the detection of PPIs with DULIP assays ............................. 46 5.2. BRIP: A Bioluminescence Resonance Energy Transfer (BRET) and Co- Immunoprecipitation (IP)-based PPI detection Assay for Quantitative Interactome Mapping in Mammalian Cells .................................................................................................................... 51 5.2.1. Generation of control and Gateway-compatible vectors suitable for quantitative BRIP interaction screening ............................................................................................................... 52 5.2.2. Establishment of a BRIP assay suitable for high-throughput PPI mapping .................... 54 5.2.3. Detection of known interactions with the BRIP assay .................................................. 56 5.2.4. Estimating the sensitivity and specificity to detect PPIs with the BRIP assay ................ 60 5.2.5. Detecting interactions with known binding affinities in intact cells and after co- immunoprecipitation ............................................................................................................... 64 5.2.6. Quantification of binding affinities altered by missense mutations .............................. 66 5.2.7. Detecting the effects of small-molecules on PPIs using the BRIP assay ........................ 69 6. DISCUSSION ................................................................................................................. 73 6.1. DULIP assays provide quantitative interaction scores for systematic interactome research .................................................................................................................................. 73 6.2. BRIP combines the principles of two mammalian cell-based PPI detection methods in one assay ................................................................................................................................. 76 6.3. Detection of low-affinity PPIs with the DULIP and BRIP assays .................................... 78 6.4. Investigation of the effects of missense mutations on binary interactions using DULIP and BRIP assays ....................................................................................................................... 80 6.5. Application of the BRET component of the BRIP assay for the investigation of the effects of small molecules on PPIs ...................................................................................................... 81 6.6. The innovative PPI detection methods DULIP and BRIP are suitable for quantitative high-throughput PPI screening ................................................................................................ 82 7. MATERIALS AND METHODS ........................................................................................ 85 7.1. Plasmid construction ..................................................................................................... 85 7.2. Cell culture and transfection ......................................................................................... 88 7.3. MDC positive and negative reference set creation ........................................................ 88 7.4. Yeast two-hybrid based reference set creation ............................................................. 88 7.5. Affinity-based interaction reference set creation .......................................................... 89 7.6. DULIP assay .................................................................................................................. 89 7.7. DULIP data analysis ...................................................................................................... 90 7.8. FRET assay ..................................................................................................................... 91 7.9. Y2H assay ..................................................................................................................... 91 7.10. BRIP assay ................................................................................................................... 91 7.11. BRIP data analysis ....................................................................................................... 93 7.12. Western blots .............................................................................................................. 93 7.13. Live-cell imaging ......................................................................................................... 94 8. SUPPLEMENTARY INFORMATION ............................................................................... 95 9. REFERENCES ................................................................................................................ 113 10. CONTRIBUTIONS ....................................................................................................... 125 11. RELEVANT PUBLICATIONS ......................................................................................... 125 12. ACKNOWLEDGEMENTS .............................................................................................. 127 Summary 1 1. Summary Protein-protein interactions (PPIs) play a key role in probably all biological processes. Disease- causing missense mutations act through complicated genotype-to-phenotype associations often resulting in the perturbation of PPIs. Thus, the systematic discovery of PPIs is an important goal of network biology to understand the fundamental mechanisms of cellular function and dysfunction in health and disease. My aim was to establish and benchmark a new set of innovative mammalian cell-based PPI detection assays, DULIP and BRIP, that generate quantitative interaction scores and allow the identification of interactions at medium to high throughput.
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