1. www.reactome.org Zagreb 30 June 2011 1 A Database of biological pathways David Croft

2. www.reactome.org Zagreb 30 June 2011 2 What are pathways?

3. www.reactome.org Zagreb 30 June 2011 3 What are pathways?

4. www.reactome.org Zagreb 30 June 2011 4 What are pathways?

5. www.reactome.org Zagreb 30 June 2011 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 Zagreb 30 June 2011 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 Zagreb 30 June 2011 7 Reactome is… A 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 (but not tissue or developmental stage)

8. www.reactome.org Zagreb 30 June 2011 8 Reaction Example 1: Enzymatic

9. www.reactome.org Zagreb 30 June 2011 9 Reaction Example 2: Transport REACT_1338.3

10. www.reactome.org Zagreb 30 June 2011 10 Other Reaction Types Dimerization Phosphorylation

11. www.reactome.org Zagreb 30 June 2011 11 Reactions Connect into Pathways OUTPUT INPUT CATALYST OUTPUT INPUT CATALYST INPUT OUTPUT CATALYST

12. www.reactome.org Zagreb 30 June 2011 12 Reactome Data Model Events: Reactions Pathways Entities: Proteins Complexes Small compounds Modulation: Inhibition Activation

13. www.reactome.org Zagreb 30 June 2011 13 Where Reactome’s Data Comes From Expert and curator create outline of new pathway Expert fills in details Curator enters information into database Reviewer (another expert) checks biological correctness Curator (staff) Expert (recruited) Reviewer (recruited)

14. www.reactome.org Zagreb 30 June 2011 14 Data Expansion - Link-outs From Reactome GO Molecular Function Compartment Biological process KEGG, ChEBI – small molecules UniProt – proteins Sequence dbs – Ensembl, OMIM, Entrez Gene, RefSeq, HapMap, UCSC, KEGG Gene PubMed references – literature evidence for events

15. www.reactome.org Zagreb 30 June 2011 15 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

16. www.reactome.org Zagreb 30 June 2011 16 Species Selection

17. www.reactome.org Zagreb 30 June 2011 17 Exportable Protein-Protein Interactions Inferred from complexes and reactions Interactions between proteins in the same complex, reaction, or adjoining reaction Lists available from Downloads See Readme document for more details

18. www.reactome.org Zagreb 30 June 2011 18 Coverage – Current TOC Scroll down for lots more...

19. www.reactome.org Zagreb 30 June 2011 19 Planned Coverage – Editorial Calendar wiki.reactome.org/index.php/Reactome_Calendar

20. www.reactome.org Zagreb 30 June 2011 20 Reactome Tools Interactive Pathway Browser Pathway Mapping and Over-representation Expression overlay onto pathways Molecular Interaction overlay Biomart

21. www.reactome.org Zagreb 30 June 2011 21 BioMart Dataset PK FK Pathway Protein Full details of BioMart at: www.biomart.org Gene Small molecule

22. www.reactome.org Zagreb 30 June 2011 22 BioMart Advantages Query without needing to know data model Scales for big datasets Easy linking to other Marts (federation)

23. www.reactome.org Zagreb 30 June 2011 23 BioMart and Reactome: Datasets Main tables: Complex Pathway Reaction Interaction Dimension tables: Gene Protein Small Molecule Literature reference

24. www.reactome.org Zagreb 30 June 2011 24 Tutorial

25. www.reactome.org Zagreb 30 June 2011 25 Front Page http://www.reactome.org Main text Navigation bar

26. www.reactome.org Zagreb 30 June 2011 26 Exercise 1 1.What’s the latest news item on the Reactome homepage? 2.How many human proteins are represented in Reactome? 3.What date is the next release? What is the first new topic that will be included?

27. www.reactome.org Zagreb 30 June 2011 27 The Pathway Browser Species selector Search & Analyze bar Sidebar Pathway Diagram Panel Details Panel (hidden) Thumbnail

28. www.reactome.org Zagreb 30 June 2011 28 Pathways tab – pathway hierarchy Pathway Reaction Black-box

29. www.reactome.org Zagreb 30 June 2011 29 Exercise 2 From the homepage, search for ‘Signaling by Notch’. 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 Notch 2 precursor transport to Golgi? Hint: If it’s not visible, open the Details pane at the bottom of the page by clicking on the blue triangle.

30. www.reactome.org Zagreb 30 June 2011 30 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

31. www.reactome.org Zagreb 30 June 2011 31 Exercise 3 From the Homepage, search for the pathway ‘Effects of PIP2 hydrolysis’ and open it in the Pathway Browser. 1.What symbol represents the reaction for ‘Binding of IP3 to the IP3 receptor’? 2.What symbol represents the reaction ‘Transport of Ca++ from platelet dense tubular system to cytoplasm’? What subtype of reaction is this? 3.What is the catalyst (descriptive name) for ‘2-AG hydrolysis to arachidonate by MAGL’? Can you find its UniProt ID and name the two outputs of this reaction?

32. www.reactome.org Zagreb 30 June 2011 32 Navigating in the Pathway Browser I Click here Highlights Details here Click here to open pathway diagram... Home and Analyze buttons Search

33. www.reactome.org Zagreb 30 June 2011 33 Highlights Details here Navigating in the Pathway Browser II Click here Zoom

34. www.reactome.org Zagreb 30 June 2011 34 Exercise 4 Open the pathway Apoptosis in the Pathway Browser. Select the sub-pathway box ‘Intrinsic Pathway in Apoptosis’. 1.How do you open the pathway diagram for this sub-pathway? 2.With this pathway diagram open, what happens if you click on the sub-pathway ‘BH3-only proteins associate with and inactivate anti- apoptotic BCL-2 members’? 3.What happens if you click on the reaction ‘Sequestration of tBID by BCL-2’ in the in the pathway hierarchy?

35. www.reactome.org Zagreb 30 June 2011 35 The Details Panel

36. www.reactome.org Zagreb 30 June 2011 36 Exercise 5 1.Find the reaction 'Activated type I receptor phosphorylates R- SMAD directly'. What pathway does it belong to? 2.In which cellular compartment does this reaction take place? 3.What is the associated GO molecular function? 4.What references verify this reaction? 5.Is this reaction predicted to occur in Canis familiaris? In Saccharomyces cerevisiae?

37. www.reactome.org Zagreb 30 June 2011 37 Pathway Analysis

38. www.reactome.org Zagreb 30 June 2011 38 Pathway Analysis – Overrepresentation ‘Top-level’ Reveal next level P-val

39. www.reactome.org Zagreb 30 June 2011 39 Exercise 6 Check this! 1.What is the most significantly over-represented top-level pathway for this dataset? 2.How many genes are in this pathway, and how many were represented in the dataset? 3.Why is the top-level pathway Chromosome Maintenance higher in the list than Signalling by Wnt when the latter has a more significant probability score? (Hint – use the Open All button) 4.Can you interpret these results in terms of the underlying biology? (Hint: good luck, there are many correct answers!)

40. www.reactome.org Zagreb 30 June 2011 40 Species Comparison I

41. www.reactome.org Zagreb 30 June 2011 41 Species Comparison II Yellow = human/rat Blue = human only Grey = not relevant Black = Complex

42. www.reactome.org Zagreb 30 June 2011 42 Exercise 7 Launch the Species Comparison and select the species Rattus norvegicus. When the results are displayed, open the pathway Complement Cascade. 1.Find Complement factor B (bottom left in the diagram) - what colour is it? What does that mean? 2.What other species is this protein inferred to be present in? Hint: You can answer this question without rerunning Species comparison. 3.Find Complement factor 2 (top middle) – why is it blue? 4.Find C3b (top left corner) – Why is it black? How many proteins contribute to this object? Are they predicted to exist in Rattus? 5.Why is Calcium grey?

43. www.reactome.org Zagreb 30 June 2011 43 Expression Analysis I

44. www.reactome.org Zagreb 30 June 2011 44 Expression Analysis II ‘Hot’ = high ‘Cold’ = low Step through Data columns

45. www.reactome.org Zagreb 30 June 2011 45 Exercise 8 Launch Expression Analysis and load the example dataset (The data was obtained from ArrayExpress and comes from the processed results of an experiment studying the differentiation of KG1 cells.). Click Analyse. When the results are displayed, find the pathway Nucleotide Excision Repair. 1.How many proteins are in this pathway? 2.How many had expression data? Click on the View button to see this pathway in the Pathway Browser. Use the Experiment Browser toolbar to cycle through the timepoints. 3.Which protein has the greatest change of expression? 4.Find the complex ‘Active Pol II complex with repaired DNA template:mRNA hybrid’ (top right of the diagram). Which component of the complex has the highest expression at 24h? 5.What was the probe ID used to measure expression of this component?

46. www.reactome.org Zagreb 30 June 2011 46 Molecular Interaction Overlay

47. www.reactome.org Zagreb 30 June 2011 47 Exercise 9 Open the pathway diagram for Netrin-1 Signaling. 1.Find the protein SHP2 (top left of the cytosol). Right click on it and select Display Interactors. How many are there? 2.How many times has the interaction between SHP2 Adapter protein GRB2 been documented? Hint: This detail is not in Reactome. 3.Find the protein NCK1 (to the right of SHP2). Display interactors for this protein. How many are there? Can you get a list of them? 4.Display interactors for UNC5B (bottom left of the cytosol). What happens and why? 5.What is the easiest way to remove interactors?

48. www.reactome.org Zagreb 30 June 2011 48 BioMart – selecting your dataset

49. www.reactome.org Zagreb 30 June 2011 49 BioMart – filters

50. www.reactome.org Zagreb 30 June 2011 50 BioMart – attributes Check to get attribute

51. www.reactome.org Zagreb 30 June 2011 51 BioMart – results

52. www.reactome.org Zagreb 30 June 2011 52 Exercise 10 Go to the Reactome BioMart page. 1.How would you select the “pathway” dataset? 2.If you were interested in the protein “Nucleolar transcription factor 1” (UniProt ID P17480), how would you identify pathways in Reactome involving that protein? How many pathways does your query find? 3.How would you find the UniProt IDs of the other proteins in the first of the pathways that you discovered? 4.Save the results list from 3) in tab-delimited format. How many lines of output are there, excluding column headers?

53. www.reactome.org Zagreb 30 June 2011 53 The End