Murcia - 3 February A Database of human biological pathways Bijay Jassal

Murcia - 3 February 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 maybe reuse it?

Murcia - 3 February Nature Oct 12;407(6805): The biochemistry of apoptosis. Rationale - Figures A picture paints a thousand words… but…. Just pixels Omits key details Assumes Fact or Hypothesis?

Murcia - 3 February 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

Murcia - 3 February Extensively cross-referenced Tools for data analysis – Pathway Analysis, Expression Overlay, Species Comparison, Biomart… Used to infer orthologous events in 20 non-human species Reactome is…

Murcia - 3 February Theory - Reactions Pathway steps = the “units” of Reactome = events in biology TRANSPORT CLASSIC BIOCHEMICAL BINDING DISSOCIATION DEGRADATION PHOSPHORYLATION DEPHOSPHORYLATION

Murcia - 3 February Reaction Example 1: Enzymatic

Murcia - 3 February Reaction Example 2: Transport REACT_945.4 Transport of Ca++ from platelet dense tubular system to cytoplasm

Murcia - 3 February Other Reaction Types Binding Dimerization Phosphorylation

Murcia - 3 February Reactions Connect into Pathways OUTPUT INPUT CATALYST OUTPUT INPUT CATALYST INPUT OUTPUT CATALYST

Murcia - 3 February Human pathway PMID:5555PMID:4444 mouse cow Direct evidence Indirect evidence PMID:8976 PMID:1234 Evidence Tracking – Inferred Reactions

Murcia - 3 February 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

Murcia - 3 February Species Selection

Murcia - 3 February 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

Murcia - 3 February 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

Murcia - 3 February Coverage – Current TOC And many more...

Murcia - 3 February Planned Coverage – Editorial Calendar

Murcia - 3 February Reactome Tools Interactive Pathway Browser Pathway Mapping and Over-representation Expression overlay onto pathways Molecular Interaction overlay Biomart

Murcia - 3 February Arabidopsis Reactome

Murcia - 3 February Tutorial

Murcia - 3 February Front Page Sidebar Main text Navigation bar

Murcia - 3 February Exercise 1

Murcia - 3 February The Pathway Browser Species selector Search & Analyze bar Sidebar Pathway Diagram Panel Details Panel (hidden) Zoom/move toolbar Thumbnail

Murcia - 3 February Pathways tab – pathway hierarchy Pathway Reaction Black-box

Murcia - 3 February Exercise 2 From the homepage, search for ‘Notch 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 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.

Murcia - 3 February The Pathway Browser - Pathway Diagrams Boxes are proteins, sets or complexes. Ovals are small molecules. Green boxes are proteins or sets, blue are complexes. Catalyst Input Outputs Compartment Reaction node Transition Binding Dissociation Omitted Uncertain Regulation +ve -ve

Murcia - 3 February 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’? 2.2. What symbol represents the reaction ‘Transport of Ca++ from platelet dense tubular system to cytoplasm’? What subtype of reaction is this? 3.3. 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?

Murcia - 3 February Navigating in the Pathway Browser I Click here Highlights Details here Click here to open pathway diagram... Home and Analyze buttons Search

Murcia - 3 February HighlightsDetails here Navigating in the Pathway Browser II Click hereZoom

Murcia - 3 February Exercise 4

Murcia - 3 February The Details Panel

Murcia - 3 February Exercise 5

Murcia - 3 February Pathway Analysis

Murcia - 3 February Pathway Analysis – Overrepresentation ‘Top-level’ Reveal next level P-val

Murcia - 3 February 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!)

Murcia - 3 February Species Comparison I

Murcia - 3 February Species Comparison II Yellow = human/rat Blue = human only Grey = not relevant Black = Complex

Murcia - 3 February Exercise 7

Murcia - 3 February Expression Analysis I

Murcia - 3 February Expression Analysis II ‘Hot’ = high ‘Cold’ = low Step through Data columns

Murcia - 3 February Exercise 8

Murcia - 3 February Molecular Interaction Overlay

Murcia - 3 February Exercise 9 Open the pathway diagram for Netrin-1 Signaling. 1.Find the protein FADK1 (top centre of the cytosol). Right click on it and select Display Interactors. How many are there? 2.How many times has the interaction between FADK1 and Tumor endothelial marker 6 been documented? Hint: This detail is not in Reactome. 3.Find the protein SRC (to the left of FADK1). 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?

Murcia - 3 February BioMart – selecting your dataset

Murcia - 3 February BioMart – filters

Murcia - 3 February BioMart – attributes Check to get attribute

Murcia - 3 February BioMart – results

Murcia - 3 February Exercise 10

Murcia - 3 February The End