1. ArrayMAPPIT: high-throughput interactome analysis in mammalian cells Cytokine Receptor Lab Ghent University - Dept. Medicine and Health Sciences VIB - Dept. Medical Protein Research Belgium www.crl-mappit.be www.crl-mappit.be

2. MAmmalian Protein-Protein Interaction Trap mammalian two-hybrid based on functional complementation of the JAK-STAT signalling pathway

3. The MAPPIT toolbox MAPPIT (2H): protein-protein interaction ReverseMAPPIT (R2H): disruption of protein-protein interaction MASPIT (3H): compound-protein interaction

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

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

6. YYYYYY YYYYYY JAK Y Y P P P P P P P P JAK-STAT signalling

7. YYYYYY YYYYYY JAK Y Y P P P P P P P P Y Y STAT JAK-STAT signalling

8. YYYYYY YYYYYY JAK Y Y P P P P P P P P Y P Y P Y P Y P STAT responsive promoter cytoplasm nucleus JAK-STAT signalling

9. JAK2 FFFFFF FFFFFF Y Y Eyckerman et al. (2001) Nat. Cell Biol. cytokine receptor leptin receptor (Y>F) MAPPIT

10. ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

11. ligand JAK2 FFFFFF Y FFFFFF Y P P MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

12. ligand JAK2 FFFFFF Y FFFFFF Y P P bait MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

13. ligand JAK2 FFFFFF Y FFFFFF Y P P YYYYYYYY prey gp130 MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

14. ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P P P P P P MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

15. ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P STAT3 Y Y MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

16. ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P Y Y reporter gene rPAP1 promoter P P MAPPIT Y P Y P Eyckerman et al. (2001) Nat. Cell Biol.

17. ligand JAK2 FFFFFF Y FFFFFF Y YYYYYYYY P PP P P P Y P Y P reporter gene rPAP1 promoter Y P Y P MAPPIT Eyckerman et al. (2001) Nat. Cell Biol.

18. features operates in intact human cells = close to normal physiological context works in different cell types (epithelial, haematopoietic, neuronal) tight background control –interaction and effector zone are separated –ligand-inducible system easy to perform, simple readout, can be automated

19. pathway analysis of early steps in Toll-Like Receptor signalling

20. TLR signaling: TLR4 dimerisation *

21. TLR signaling: TLR4 adaptor interactions * *

22. TLR signaling: Mal-MyD88 homo- and heterodimerisation *

23. TLR signaling: Mal bridges between MyD88 and TLR4 *

24. TLR signaling: MyD88 associates with IRAK-4 *

25. TLR signaling: MyD88 bridges between MAL and IRAK-4 *

26. TLR signaling: IRAK-4 interacts with IRAK-1 *

27. pathway analysis toll-like receptor leptin receptor erythropoietin receptor growth hormone receptor granulocyte colony-stimulating factor receptor Lemmens et al., Nucleic Acids Res. 2003 Montoye et al., Blood 2005 Lavens et al., J. Cell Sci. 2006 Montoye et al., FEBS Lett. 2006 Piessevaux et al., J. Biol. Chem. 2006 Lavens et al., Biochem. J. 2007 Ulrichts et al., FEBS Lett. 2007 Erkeland et al., Oncogene 2007 Uyttendaele et al., Mol. Endocrinol. 2007 Wauman et al., Mol. Endocrinol. 2008 Piessevaux et al., J. Biol. Chem. 2008 Ulrichts et al., Immunol. Lett. 2008

28. validation of Y2H interactome maps Boxem et al. A protein domain-based interactome network for C. elegans early embryogenesis, Cell 2008 Yu et al. High accuracy high throughput binary interactome datasets for S. cerevisiae, Science 2008 Braun et al. An experimentally derived confidence score for binary protein-protein interactions, Nat. Methods, under revision Simonis et al. A high confidence C. elegans protein-protein interaction map, Nat. Methods., under revision Venkatesan et al. Empirical sizing of the human interactome by self-controlled binary mapping, Nat. Methods, under revision

29. validation of Y2H interactome maps Braun et al.

30. performance Braun et al.

31. performance Braun et al.

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

33. screening for Skp1 interaction partners SCF E3 ligase

34. screening for Skp1 interaction partners 1904 full length human ORFs from Vidal collection (GO ‘signal transduction’) luciferase read-out (average of duplicates normalized for plate median) LR(F3)-Skp1 bait expressing cell line -/+ leptin

35. screening for Skp1 interaction partners 10-fold induction

36. facs screen ?

37. comparing the effect of different treatments treatment A treatment B treatment C

38. assaying the effect of exogenous treatments on PPIs examples: p53 interactions – nutlin3 HIV Reverse Transcriptase dimerisation - efavirenz Glucocorticoid Receptor interactions - dexamethasone

40. Differential modulation of p53-mdm2/4 interactions p53 mdm2 p53 mdm4 p53 mdm4 p53 mdm2 Nutlin

41. Differential modulation of p53-mdm2/4 interactions

42. HIV Reverse Transcriptase heterodimer p66 p51 NNRTI binding site (allosteric) NRTI binding site (polymerase active site)

43. analysis of RT dimerization

44. screen for HIV RT(p66) binders

45. competition with wild type RT subunits

46. stabilization of RT dimerisation by efavirenz Kd (µM)0.30.0140.082300.4 µM efavirenz

47. dose-reponse curve of efavirenz stabilizing effect RTp66 bait + RTp51 prey

48. Glucocorticoid receptor signalling p53

49. DEX dependent interactions with the GR

50. prospects sensitive assay physiologically relevant background large prey collection automated screening >> analysis of the dynamics of interaction patterns in response to changes in intra-/extracellular environment

51. CRL Jan Tavernier Dominiek Catteeuw Els Pattyn Delphine Lavens Leentje De Ceuninck Isabel Uyttendaele Celia Bovijn Laura Icardi Margarida Maia Sylvie Seeuws Lennart Zabeau Irma Lemmens Anne-Sophie De Smet Frank Peelman Julie Piessevaux Peter Ulrichts José Van der Heyden Nele Vanderroost Annick Verhee Joris Wauman CCSB Marc Vidal & co

52. www.crl-mappit.be