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MAPPIT Analysing Protein Interactions in Human Cells VIB/UGent Workshop October 2009

MAPPIT Analysing Protein Interactions in Human Cells VIB/UGent Workshop October 2009. JAK-STAT signalling. type I cytokine receptor. extracellular. intracellular. Y Y Y. Y Y Y. Y. JAK. JAK. JAK kinase. Y. JAK-STAT signalling. P. Y Y Y. Y Y Y. Y. JAK. JAK. Y. P.

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MAPPIT Analysing Protein Interactions in Human Cells VIB/UGent Workshop October 2009

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  1. MAPPITAnalysing Protein Interactions in Human CellsVIB/UGent Workshop October 2009

  2. JAK-STAT signalling type I cytokine receptor extracellular intracellular Y Y Y Y Y Y Y JAK JAK JAK kinase Y

  3. JAK-STAT signalling P Y Y Y Y Y Y Y JAK JAK Y P cytokine

  4. JAK-STAT signalling P Y Y Y Y Y Y Y JAK JAK Y P P P P P P P

  5. JAK-STAT signalling Y JAK JAK Y P P Y P P P P Y P Y Y Y Y Y Y P STAT

  6. JAK-STAT signalling Y JAK JAK Y P P Y Y P P P P P P Y Y P P P Y Y Y Y Y Y P cytoplasm nucleus STAT responsive promoter

  7. MAPPIT cytokine receptor F F F F F F Y JAK2 JAK2 Y leptin receptor (Y>F)

  8. MAPPIT P P ligand F F F F F F Y JAK2 JAK2 Y

  9. MAPPIT P P ligand F F F F F F Y JAK2 JAK2 Y

  10. MAPPIT P P ligand F F F F F F Y JAK2 JAK2 Y bait

  11. MAPPIT P P ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y gp130 prey

  12. MAPPIT ligand P F F F F F F Y JAK2 JAK2 Y P Y Y Y Y P P P P

  13. MAPPIT P P Y P P P Y P ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y STAT3

  14. MAPPIT P P P Y Y P P P Y Y P ligand F F F F F F Y JAK2 JAK2 Y P Y Y Y Y P P rPAP1 promoter reporter gene

  15. MAPPIT P P P Y Y P P P Y Y P P P ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y P rPAP1 promoter reporter gene

  16. Epo F F F F F F P Y JAK2 JAK2 Y P Y Y Y Y EpoR EpoR Y Y P P CIS MAPPIT example Montoye et al., Blood 105, 4264-4271 (2005) Lavens et al., J. Cell Sci. 119, 2214-2224 (2006) Montoye et al., FEBS Lett. 580, 3301-3307 (2006) Piessevaux et al., J. Biol. Chem. 281, 32953-66 (2006) Lavens et al., Biochem. J., 401 257-267 (2007) Ulrichts et al., FEBS Lett. 581, 629-636 (2007) Erkeland et al., Oncogene 26, 1984-1995 (2007) Uyttendaele et al., Mol. Endocrinol. 21, 2821-2831 (2007) Wauman et al., Mol. Endocrinol. 22, 965-977 (2008) Ulrichts et al., Immunol. Lett. 115, 141-148 (2008) Piessevaux et al., J. Biol. Chem. 283, 21334-21346 (2008) Pattyn et al., J. Virol. Methods 153, 7-15 (2008) Lemmens et al., Biochem Soc. Trans 36, 1448-1451 (2008) Piessevaux et al., Cytokine Growth Factor Rev. 19, 371-381 (2008) Lievens et al., J. Proteome Res. 8, 877-886 (2009) Ulrichts et al., Meth. Mol. Biol., 517, 133-144 (2009). Lievens et al., TiBS, in press. De Ceuninck et al., J. Biol. Chem., submitted Ulrichts et al.,J. Cell Sci., submitted. Lavens et al., PLoS Pathogens, submitted.

  17. MAPPIT analysis of HIV Reverse Transcriptase interactions FLAG-gp130-RTp66 FLAG-gp130-RTp51 RTp51 bait RTp66 bait p51/p66=0.3µM p66/p66=4.0µM p51/p51=230µM

  18. MAPPIT as a tool for drug screening Efavirenz stabilizes the HIV RT dimer

  19. Interactomes Large-scale programs to map complete interactomes of an organism  collaboration with Marc Vidal group (DFCI, Harvard U, Boston): systematic matrix-based Y2H analysis Network quality : coverage (false negatives) accuracy (false positives) use of MAPPIT as an orthogonal assay

  20. Interactomes MAPPIT-based verification of Y2H interactome datasets: • Gateway-mediated transfer of ORF’s to MAPPIT vectors • Systematic MAPPIT analysis in 384-well format  high-confidence maps Yu et al., High quality binary protein interaction map of the yeast interactome network Science (2008) Simonis et al., Empirically-controlled mapping of the C. elegans protein-protein interactome network Nat. Methods (2009) Venkatesan et al., An empirical framework for binary interactome mapping, Nat. Methods (2009)  The data quality of high-throughput Y2H-based interactome datasets is high • Low coverage and detectability accounts for the low overlap between different large-scale datasets • The estimated size of the human interactome is around 130.000 binary interactions, current coverage is 8%

  21. MAPPIT performance Braun et al., An experimentally derived confidence score for binary protein-protein interactions.Nat. Methods (2009)

  22. Comparison of interactomes Yu et al., Science, 2008

  23. ArrayMAPPIT screening assay prey plasmid (+reporter) transfection reagent reverse transfection mix MAPPIT bait cell line MAPPIT prey array human ORFeome collection activation MAPPIT prey collection luciferase read-out

  24. Y Y Y Y ? ArrayMAPPIT screening: Skp1 interactions 10-fold induction Lievens et al., J. Proteome Res. 2009.

  25. ArrayMAPPIT screening: EloC interactions 10-fold induction 5-fold induction 3-fold induction

  26. ArrayMAPPIT – towards the dynamics of PPI networks treatment B treatment C treatment A

  27. ArrayMAPPIT – Glucocorticoid Receptor interactions P P ligand F F F F F F Y JAK2 JAK2 Y GR GR

  28. ArrayMAPPIT – Glucocorticoid Receptor interactions P P -Dex ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y GR GR Hsp90

  29. ArrayMAPPIT – Glucocorticoid Receptor interactions P P +Dex ligand F F F F F F Y JAK2 JAK2 Y Y Y Y Y Y Y Y Y GR GR Hsp90 p53

  30. ArrayMAPPIT – Glucocorticoid Receptor interactions GR bait Skp1 bait

  31. ArrayMAPPIT – Glucocorticoid Receptor interactions GR bait Skp1 bait

  32. ArrayMAPPIT – Glucocorticoid Receptor interactions GR bait Skp1 bait

  33. ArrayMAPPIT – Glucocorticoid Receptor interactions GR bait Skp1 bait

  34. rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening Hek293 Flp-In T-Rex 44 cells mEcoR SV FRT lacZeoR Lievens et al. (2004) Methods Mol. Biol.

  35. Y F F F JAK2 F F F JAK2 Y bait bait CMV EpoR/LR-LRF3 bait rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening mEcoR Lievens et al. (2004) Methods Mol. Biol.

  36. Y F F F JAK2 F F F JAK2 Y bait bait 5’LTR y gp130 prey CMV EpoR/LR-LRF3 bait Y Y Y Y Y Y Y Y prey prey rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening retroviral prey cDNA library mEcoR Lievens et al. (2004) Methods Mol. Biol.

  37. ligand ligand ligand ligand ligand P Y F F F JAK2 F F F JAK2 Y P Y F F F JAK2 bait bait F F F JAK2 Y bait bait 5’LTR y gp130 prey CMV EpoR/LR-LRF3 bait Y Y Y Y Y Y Y Y Y Y Y Y prey prey prey rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening mEcoR Lievens et al. (2004) Methods Mol. Biol.

  38. ligand ligand ligand ligand ligand P P Y F F F JAK2 F F F JAK2 Y Y F F F JAK2 F F F JAK2 P Y P bait bait P P bait bait 5’LTR y gp130 prey CMV EpoR/LR-LRF3 bait hIL5Rα-Δcyt Y Y Y Y Y Y Y Y Y Y Y Y prey prey prey rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening mEcoR Lievens et al. (2004) Methods Mol. Biol.

  39. Y Y ligand ligand ligand ligand ligand P P Y F F F JAK2 F F F JAK2 Y Y F F F JAK2 F F F JAK2 P Y P bait bait P P bait bait 5’LTR y gp130 prey CMV EpoR/LR-LRF3 bait hIL5Rα-Δcyt Y Y Y Y Y Y Y Y Y Y Y Y prey prey prey rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening MACS presort (enrich-deplete-enrich) anti-mIgG-PE Y anti-PE magnetobead Y anti-hIL5Rα mEcoR Lievens et al. (2004) Methods Mol. Biol.

  40. Y Y ligand ligand ligand ligand ligand hIL5Rα-Δcyt P P Y F F F JAK2 F F F JAK2 Y Y F F F JAK2 F F F JAK2 P Y P bait bait P P bait bait 5’LTR y gp130 prey CMV EpoR/LR-LRF3 bait hIL5Rα-Δcyt Y Y Y Y Y Y Y Y Y Y Y Y prey prey prey rPAP1 hIL5Rα-Δcyt FACS-based MAPPIT screening FACS single cell sort anti-mIgG-PE Y anti-PE magnetobead Y anti-hIL5Rα anti-mIgG-PE Y Y anti-hIL5Rα mEcoR Lievens et al. (2004) Methods Mol. Biol.

  41. ligand P F F F F F F Y P JAK2 JAK2 Y Y Y Y Y Skp1A Skp1A ? MAPPIT cDNA library screening assay hSkp1A-bait versus Hek293T prey cDNA library Lievens et al., J. Proteome Res., 2009.

  42. MAPPIT summary Operates in intact human cells = close to normal physiological context Easy to perform, simple readout High sensitivity and specificity Tight background control  interaction and effector zone are separated  ligand-inducible system Can detect  weak/transient interactions  indirect interactions  modification-dependent interactions (Heteromeric MAPPIT) High-throughput modus  ArrayMAPPIT runs on 96 and 384 well format on Tecan Evo robotics platform Applications in interactome mapping (20.000 PPI’s/year) and drug screening  cDNA library screens

  43. Limitations of MAPPIT • No real-time measurements  FRET/BRET • Subcellular localization  Designed for cytosolic proteins • Difficult for membrane proteins (e.g. GPCR’s, Ion Channels) • Split Ubiquitin Y2H

  44. MAPPIT summary Operates in intact human cells = close to normal physiological context Easy to perform, simple readout High sensitivity and specificity Tight background control  interaction and effector zone are separated  ligand-inducible system Subcellular localization Can detect  weak/transient interactions  indirect interactions  modification-dependent interactions (Heteromeric MAPPIT) High-throughput modus  ArrayMAPPIT runs on 384 well format on Tecan Evo robotics platform Applications in large-scale interactome mapping (20.000 PPI’s/year)  cDNA library screens

  45. Inducible MAPPIT: transient transfections • No preformed bait/prey complexes • Can the protein folding pathway be targeted by small molecules? Best configuration: pTREtight-prey (Clontech): inducible prey rtTA-Advanced (Clontech)

  46. Inducible mappit: transient Vif-ElonginC interaction 1. + Epo 4 3 1 2 Dox 1000 ng/ml + Epo 2. WB anti-flag 3. Dox 100 ng/ml + Epo 4. Dox 10 ng/ml + Epo WB anti-actin

  47. www.mappit.be

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