1. www.reactome.org March 2013 1 A Database of human biological pathways Steve Jupe - sjupe@ebi.ac.uk

2. www.reactome.org March 2013 2 Background Cells contain thousands of molecules, –proteins and small chemical compounds They work together to perform tasks. –provide energy –produce materials –respond to the environment. Processes require coordinated efforts –like a factory production line. –Biologists refer to a chain of connected molecular events as a pathway. ELLS 2011

3. www.reactome.org March 2013 3 Different types of pathways Metabolic networks – provide the energy and materials Signaling networks – sense the outside, coordinate activities within and between cells (Gene) regulatory networks – control processes, set limits, control the molecular composition of cells ELLS 2011

4. www.reactome.org March 2013 4 The scale of pathways Early pathways were pieced together by studying biochemical reactions of individual proteins, measuring the activity, learning the substrates and products, joining them to the next enzyme in the pathway. New technologies allow simultaneous measurement of tens of thousands of different molecules. This reveals that many biological pathways are interconnected, more complicated than the typical textbook examples, with fluid boundaries. They can work together or against each other (feedback and regulation). Scientists who study or interpret these large scale interactions sometimes refer to the field as ‘systems biology’ When multiple biological pathways interact = a biological network. When all possible interactions are added, pathways become extremely difficult to understand Biologists often work with ‘canonical’ pathways, those that represent the well-understood part of the entire network. ELLS 2011

5. www.reactome.org March 2013 5 Rationale – Journal information Nature 407(6805):770-6.The Biochemistry of Apoptosis. “Caspase-8 is the key initiator caspase in the death-receptor pathway. Upon ligand binding, death receptors such as CD95 (Apo-1/Fas) aggregate and form membrane- bound signalling complexes (Box 3). These complexes then recruit, through adapter proteins, several molecules of procaspase-8, resulting in a high local concentration of zymogen. The induced proximity model posits that under these crowded conditions, the low intrinsic protease activity of procaspase-8 (ref. 20) is sufficient to allow the various proenzyme molecules to mutually cleave and activate each other (Box 2). A similar mechanism of action has been proposed to mediate the activation of several other caspases, including caspase-2 and the nematode caspase CED-3 (ref. 21).” How can I access the pathway described here and reuse it?

6. www.reactome.org March 2013 6 Nature. 2000 Oct 12;407(6805):770-6. The biochemistry of apoptosis. Rationale - Figures A picture paints a thousand words… but…. Just pixels Omits key details Assumes Fact or Hypothesis?

7. www.reactome.org March 2013 7 Reactome is… Free, online, open-source curated database of pathways and reactions in human biology Authored by expert biologists, maintained by Reactome editorial staff (curators) Mapped to cellular compartment

8. www.reactome.org March 2013 8 Extensively cross-referenced Tools for data analysis – Pathway Analysis, Expression Overlay, Species Comparison, Biomart… Used to infer orthologous events in 20 other species Reactome is…

9. www.reactome.org March 2013 9 human PMID:5555PMID:4444 mouse cow Direct evidence Indirect evidence PMID:8976 PMID:1234 Using model organism data to build pathways – Inferred pathway events

10. www.reactome.org March 2013 10 Theory - Reactions Pathway steps = the “units” of Reactome = events in biology TRANSPORT CLASSIC BIOCHEMICAL BINDING DISSOCIATION DEGRADATION PHOSPHORYLATION DEPHOSPHORYLATION

11. www.reactome.org March 2013 11 Reaction Example 1: Enzymatic

12. www.reactome.org March 2013 12 Reaction Example 2: Transport REACT_945.4 Transport of Ca++ from platelet dense tubular system to cytoplasm

13. www.reactome.org March 2013 13 Other Reaction Types Binding Dimerization Phosphorylation

14. www.reactome.org March 2013 14 Reactions Connect into Pathways OUTPUT INPUT CATALYST OUTPUT INPUT CATALYST INPUT OUTPUT CATALYST

15. www.reactome.org March 2013 15 Human pathway PMID:5555PMID:4444 mouse cow Direct evidence Indirect evidence PMID:8976 PMID:1234 Evidence Tracking – Inferred Reactions

16. www.reactome.org March 2013 16 Species Selection

17. www.reactome.org March 2013 17 Data Expansion – Projecting to Other Species A + ATP A + ADP -P B Human A + ATP A + ADP -P B Mouse B A Drosophila Reaction not inferred No orthologue - Protein not inferred + ATP

18. www.reactome.org March 2013 18 Exportable Protein-Protein Interactions Inferred from complexes and reactions (more on this later) Interactions between proteins in the same complex, reaction, or adjoining reaction Lists available from Downloads See Readme document for more details

19. www.reactome.org March 2013 19 Coverage – Content, TOC And many more...

20. www.reactome.org March 2013 20 Planned Coverage – Editorial Calendar

21. www.reactome.org March 2013 21 Reactome Tools Interactive Pathway Browser Pathway Mapping and Over-representation Expression overlay onto pathways Molecular Interaction overlay Biomart

22. www.reactome.org March 2013 22 Tutorial Tutorial handouts in your info packages…

23. www.reactome.org March 2013 23 Front Page http://www.reactome.org Sidebar Main text Navigation bar

24. www.reactome.org March 2013 24 Exercise 1 …see the Tutorial handouts

25. www.reactome.org March 2013 25 The Pathway Browser Species selector Diagram Key Sidebar Pathway Diagram Panel Details Panel (hidden) Zoom/move toolbar Thumbnail

26. www.reactome.org March 2013 26 Pathways tab – pathway hierarchy Pathway Reaction Black-box

27. www.reactome.org March 2013 27 Exercise 2 From the homepage, search for ‘PDGF signaling’. Click on the top pathway hit. This will open it in the Pathway Browser. Ignoring the diagram for now, look at the Pathways tab on the left. 1.How many sub-pathways does this pathway have? 2.How many reactions are in the first of these sub-pathways? 3.What reaction follows Processing of classical PDGFs? Hint: If it’s not visible, open the Details pane at the bottom of the page by clicking on the blue triangle. …see the Tutorial handouts

28. www.reactome.org March 2013 28 The Pathway Browser - Pathway Diagrams Boxes are proteins, protein sets, mixed sets or complexes. Ovals are small molecules (or sets of) Green boxes are proteins or sets, blue are complexes. Catalyst Input Outputs Compartment Reaction node Transition Binding Dissociation Omitted Uncertain Regulation +ve -ve

29. www.reactome.org March 2013 29 Exercise 3 …see the Tutorial handouts

30. www.reactome.org March 2013 30 Navigating in the Pathway Browser I Click here Highlights Details here Click here to open pathway diagram... Home and Analyze buttons

31. www.reactome.org March 2013 31 Details here Navigating in the Pathway Browser II Click hereZoomHighlights

32. www.reactome.org March 2013 32 Exercise 4 …see the Tutorial handouts

33. www.reactome.org March 2013 33 The Details Panel

34. www.reactome.org March 2013 34 Exercise 5 …see the Tutorial handouts

35. www.reactome.org March 2013 35 Pathway Analysis

36. www.reactome.org March 2013 36 Pathway Analysis – Overrepresentation ‘Top-level’ Reveal next level P-val

37. www.reactome.org March 2013 37 Exercise 6 Check this! …see the Tutorial handouts

38. www.reactome.org March 2013 38 Species Comparison I

39. www.reactome.org March 2013 39 Species Comparison II Yellow = human/rat Blue = human only Grey = not relevant Black = Complex

40. www.reactome.org March 2013 40 Exercise 7 …see the Tutorial handouts

41. www.reactome.org March 2013 41 Expression Analysis I

42. www.reactome.org March 2013 42 Expression Analysis II ‘Hot’ = high ‘Cold’ = low Step through Data columns

43. www.reactome.org March 2013 43 Exercise 8

44. www.reactome.org March 2013 44 Molecular Interaction Overlay

45. www.reactome.org March 2013 45 Exercise 9 …see the Tutorial handouts

46. www.reactome.org March 2013 46 New Look Browser

47. www.reactome.org March 2013 47 End

48. www.reactome.org March 2013 48 BioMart – selecting your dataset

49. www.reactome.org March 2013 49 BioMart – filters

50. www.reactome.org March 2013 50 BioMart – attributes Check to get attribute

51. www.reactome.org March 2013 51 BioMart – results

52. www.reactome.org March 2013 52 Exercise 10