1. Mapping and Visualization of Biochemical Networks Stephen Michnick Département de biochimie Université de Montréal

2. Mapping and Visualization of Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme II: A general approach to mapping biochemical Theme II: A general approach to mapping biochemical networks. networks. Design organization and paradoxes in PKB signaling Theme III: Design Modularity and Evolution of Networks Theme III: Design Modularity and Evolution of Networks Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells. networks in living cells.

3. C. J. Roberts et al. (2000) Science, 287:873-880. “Mapping” of Biochemical pathways

4. Why Protein Interactions Importance :Importance : Organizing-insulate pathwaysOrganizing-insulate pathways Most efficient machinery for reactions and regulation of reactionsMost efficient machinery for reactions and regulation of reactions Forming complex structures or enzymatic machineryForming complex structures or enzymatic machinery Source : Nature reviews, molecular cell biology, 2001, 2, 55-62

5. Mapping and Visualization of Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme II: A general approach to mapping biochemical Theme II: A general approach to mapping biochemical networks. networks. Design organization and paradoxes in PKB signaling Theme III: Design Modularity and Evolution of Networks Theme III: Design Modularity and Evolution of Networks The Rosetta Hypothesis and organization of networks The Rosetta Hypothesis and organization of networks Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells. networks in living cells. The MAP kinase pathways in S. cerevisiae.

6. Fraction folded Interaction-Assisted Protein Folding

7. PCA (Protein fragment Complementation Assays) A A B B Screening Quantitation Pharmacology Localization Trafficking Genes are transfected into cells. The cell makes proteins A and B which have reporter tags on either end (N- or C-terminal). A/B complex formation reconstitutes the reporter and generates a signal. Any gene, cell and reporter protein can be used. Screening Dynamics Pharmacology Localization Trafficking

8. DHFR AGPT GARTase  -lactamase GFP A world of PCAs Remy, I., et al. (2002). Protein-protein interactions: A molecular cloning manual Cold Spring Harbor Laboratory Press. Chapter 25, 449-475. Michnick, S.W. (2001). Curr Opin Struct Biol, 11: 472-477. Michnick, S.W., et al. (2000) Methods in Enzymology. 328, 208-230.

9. PCA in Network Mapping Molecular interactions are detected directly. Molecular interactions are detected directly. Genes are expressed in a relevant cellular context, in which Genes are expressed in a relevant cellular context, in which components of the underlying pathway exist. components of the underlying pathway exist. Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways. detected, linking specific interactions to specific pathways. Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously. determined unambiguously.

10. Dihydrofolate reductase (DHFR)  Fluorometirc assay based on binding of fluor-methotrexate to DHFR PNAS Vol. 95, pp. 12141-12146 PNAS Vol. 96, pp. 5394-5399, May 1999

11. Mechanisms of action of chemical agents That act on specific pathways Quantification (amplitude) pharmacological profiles Localization intracellular trafficing Spatial and temporal organization of the network Pharmacological profiles Mapping of Biochemical Networks with PCA Identification of Pathway Components Establishing physical relationships between components. I. Remy et S.W. Michnick, Proc. Natl. Acad. Sci. USA 98, 5394-5399 (2001)

12. Mapping of Signal Transduction Pathways controled by Insulin

13. The Integration of Biochemical Networks Cell cycle and DNA repair Cytokines Growth factorsCell suicide (Apoptose) Pathogenic virus

14. Design, organization and paradoxes in PKB signaling D. P. Brazil and B. A. Hemmings (2001) TiBs, 26: 657-664

15. Convergence of RTK and FRAP Pathways

16. Crosstalk between FRAP and PKB signaling

17. Crosstalk between FRAP and PKB signaling: When is a pathway a pathway?

18. Crosstalk between FRAP and PKB signaling: hidden phenotypes

19. Mapping and Validation

20. PCA in Network Mapping Molecular interactions are detected directly. Molecular interactions are detected directly. Genes are expressed in a relevant cellular context, in which Genes are expressed in a relevant cellular context, in which components of the underlying pathway exist. components of the underlying pathway exist. Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways. detected, linking specific interactions to specific pathways. Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously. determined unambiguously.

21. Directional cDNA library screening with the GFP PCA the GFP PCA Size-fractionated directional cDNA library from human brain Size-fractionated directional cDNA library from human brain (10 7 independent clones) Transiently co-transfected COS-1 cells Transiently co-transfected COS-1 cells

22. Systematic Screening of PKB (AKT1 and AKT2)

23. PCA strategy A general approach to mapping pathways that allows for both defining the organization of pathways. Can be used for pure description in an ontological sense (what, where and when) as well as more quantitative descriptions. The key to resolving specificity and organization of biochemical networks is CELL-BASED ASSAYS. Mapping biochemical networks

24. PCA in Network Mapping Molecular interactions are detected directly. Molecular interactions are detected directly. Genes are expressed in a relevant cellular context, in which Genes are expressed in a relevant cellular context, in which components of the underlying pathway exist. components of the underlying pathway exist. Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways. detected, linking specific interactions to specific pathways. Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously. determined unambiguously.

25. Mapping and Visualization of Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks Theme II: A general approach to mapping biochemical networks. Theme II: A general approach to mapping biochemical networks. The key to resolving specificity and organization of biochemical networks is CELL-BASED ASSAYS. PCA provides general approach to mapping pathways that allows for both defining the organization of pathways in terms of interactions and their dynamics. Theme III: Design, Modularity and Evolution of Networks Theme III: Design, Modularity and Evolution of Networks e.g. Rosetta Hypothesis: Predictions of interactions and functional e.g. Rosetta Hypothesis: Predictions of interactions and functional inferences provide crucial and testable hypotheses. Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells. networks in living cells. Mapping the interactions among all biochemical network components is the only way that we may ultimately know natures design. If there is one.

26. Thanks to… Financing CIHR, NIH, The Burroughs-Wellcome Fund, HFSP Génome Québec/Canada, Odyssey Pharmaceuticals Inc. F.-X. Campbell-ValoisGalia Ghaddar André GalarneauMartin Primeau Ingrid RemyJean-François Turcotte Alexis Vallée-Belisle Annie Montmarquette Dimitri Sans Geoffroy Denis Helen Yu Philippe Nissaire Jane Lamerdin Po Hein Ear Hugo Lavois Stéphane Le Crom Luciano Vidali Kirill Tarrasov Nathalie Bourassa Ludovic Fuzellier