1. SRI International Bioinformatics 1 Computing with Pathway/Genome Databases

2. SRI International Bioinformatics 2 Motivations for Understanding Pathway Tools Schema When writing complex queries to PGDBs, those queries must refer to classes and slots within the schema l Queries using Lisp, Perl, Java APIs l Queries using Query Page l Queries using Structured Advanced Query Form

3. SRI International Bioinformatics 3 Motivations for Understanding Schema Pathway Tools visualizations and analyses depend upon the software being able to find precise information in precise places within a Pathway/Genome DB A Pathway/Genome Database is a web of interconnected objects; each object represents a biological entity

4. SRI International Bioinformatics 4 Pathway Tools Implementation Details Platforms: l Macintosh, PC/Linux, and PC/Windows platforms Same binary can run as desktop app or Web server Production-quality software l Version control l Two regular releases per year l Extensive quality assurance l Extensive documentation l Auto-patch l Automatic DB-upgrade 420,000 lines of Lisp code

5. SRI International Bioinformatics 5 Pathway Tools Architecture Ocelot DBMS GFP API Pathway Genome Navigator Web Mode Desktop Mode Protein Editor Pathway Editor Reaction Editor Oracle or MySQL Disk File Lisp Perl Java

6. SRI International Bioinformatics 6 Ocelot Knowledge Server Architecture Frame data model l Classes, instances, inheritance l Frames have slots that define their properties, attributes, relationships l A slot has one or more values u Datatypes include numbers, strings, etc. Transaction logging facility Slot units define metadata about slots: l Domain, range, inverse l Collection type, number of values, value constraints

7. SRI International Bioinformatics 7 Ocelot Storage System Architecture Persistent storage via disk files, Oracle DBMS l Concurrent development: Oracle l Single-user development: disk files Oracle storage l Oracle is submerged within Ocelot, invisible to users l Frames transferred from DBMS to Ocelot u On demand u By background prefetcher u Memory cache u Persistent disk cache to speed performance via Internet Transaction logging facility

8. SRI International Bioinformatics 8 More Information Pathway Tools Web Site, Tutorial Slides l http://bioinformatics.ai.sri.com/ptools/ l http://bioinformatics.ai.sri.com/ptools/examples.lisp PerlCyc & JavaCyc API, includes some relationships l http://www.arabidopsis.org/tools/aracyc/perlcyc/ http://www.arabidopsis.org/tools/aracyc/perlcyc/ l http://www.arabidopsis.org/tools/aracyc/javacyc/ Pathway Tools User’s Guide l Appendix: Guide to the Pathway Tools Schema Curator's Guide l http://bioinformatics.ai.sri.com/ptools/curatorsguide.pdf http://bioinformatics.ai.sri.com/ptools/curatorsguide.pdf aic/pathway-tools/nav/12.0/lisp/relationships.lisp

9. SRI International Bioinformatics 9 References Ontology Papers section of http://biocyc.org/publications.shtml http://biocyc.org/publications.shtml l "An Evidence Ontology for use in Pathway/Genome Databases" l "An ontology for biological function based on molecular interactions" l "Representations of metabolic knowledge: Pathways" l "Representations of metabolic knowledge"

10. SRI International Bioinformatics 10 Data Exchange APIs: Lisp API, Java API, and Perl API : read & modify Cyclone Export to files l BioPAX Export: since Pathway Tools 9.0 Biopax.org l Export PGDB genome to Genbank format l Export entire PGDB as column-delimited and attribute-value file formats l Export PGDB reactions as SBML -- sbml.org l Import/Export of Pathways: between PGDBs l Import/Export of Selected Frames, for Spreadsheets l Import/Export of Compounds as Molfile, CML l Registering/Publishing PGDBs on WWW BioWarehouse : Loader for Flatfiles, SQL access l http://bioinformatics.ai.sri.com/biowarehouse/ http://bioinformatics.ai.sri.com/biowarehouse/ l BMC Bioinformatics 7:170 2006

11. SRI International Bioinformatics 11 Programmatic Access to BioCyc Common LISP Native language of Pathway Tools Interactive & Mature Environment Full Access to the Data & Many Utility Functions Source code is available for academics PerlCyc API of Functions, Exposed to Perl Communication through UNIX Socket JavaCyc API of Functions, Exposed to Java Communication through UNIX Socket Cyclone

12. SRI International Bioinformatics 12 Cyclone Developed by Schachter and colleagues from Genoscope http://nemo-cyclone.sourceforge.net/archi.php Cyclone is a Java-based system that: l Extracts data from a Pathway Tools PGDB l Converts it to an XML schema l Maps the data to Java objects and to a relational database l Changes made to the data on the Java side can be committed back to a Pathway Tools PGDB

13. SRI International Bioinformatics 13 Pathway Tools Data Model PGDBs are object-oriented databases Frame Representation System, named Ocelot Frame data model l PGDB = Knowledge base = KB = Database = DB l Frames l Slots PGDBs are stored in three possible ways Preloaded into binary executable Ocelot file: single-user RDBMS: MySQL-4 or Oracle-10 : multi-user, change-logging Query API: GFP (Generic Frame Protocol)

14. SRI International Bioinformatics 14 Frames Entities with which facts are associated Kinds of frames: l Classes: Genes, Pathways, Biosynthetic Pathways l Instances (objects): trpA, TCA cycle Classes: l Superclass(es), Subclass(es), Instance(s) A symbolic frame name (id, key) uniquely identifies each frame l Examples: EG10223, TRP, Proteins

15. SRI International Bioinformatics 15 Slots Encode attributes and properties of a frame Represent relationships between frames l The value of a slot is the identifier of another frame

16. SRI International Bioinformatics 16 Slots Number of values l Single valued l Multivalued: sets, bags Slot values l Any LISP object: Integer, real, string, symbol (frame name) Every slot is described by a “slot frame” in a KB that defines meta information about that slot l Datatype, classes it pertains to, constraints l Two slots are inverses if they encode opposite relationships u Slot Product in class Genes u Slot Gene in class Polypeptides

17. SRI International Bioinformatics 17 Pathway Tools Ontology / Schema Ontology classes: 1621 l Datatype classes: Define objects from genomes to pathways l Classification systems for pathways, chemical compounds, enzymatic reactions (EC system) l Protein Feature ontology l Controlled vocabularies: u Cell Component Ontology u Evidence codes Comprehensive set of 248 attributes and relationships

18. SRI International Bioinformatics 18 Root Classes in the Pathway Tools Ontology Chemicals-- All molecules Polymer-Segments-- Regions of polymers Protein-Features-- Features on proteins Paralogous-Gene-Groups Organisms Generalized-Reactions-- Reactions and pathways Enzymatic-Reactions-- Link enzymes to reactions they catalyze Regulation-- Regulatory interactions CCO-- Cell Component Ontology Evidence -- Evidence ontology Notes-- Timestamped, person-stamped notes Organizations People Publications

19. SRI International Bioinformatics 19 Use GKB Editor to Inspect the Pathway Tools Ontology GKB Editor = Generic Knowledge Base Editor Type in Navigator window: (GKB) or [Right-Click] Edit->Ontology Editor View->Browse Class Hierarchy [Middle-Click] to expand hierarchy To view classes or instances, select them and: l Frame -> List Frame Contents l Frame -> Edit Frame

20. SRI International Bioinformatics 20 Schema Overview

21. SRI International Bioinformatics 21 Principal Classes Class names are capitalized, plural, separated by dashes Genetic-Elements, with subclasses: l Chromosomes l Plasmids Genes Transcription-Units RNAs l rRNAs, snRNAs, tRNAs, Charged-tRNAs Proteins, with subclasses: l Polypeptides l Protein-Complexes

22. SRI International Bioinformatics 22 Principal Classes Reactions, with subclasses: l Transport-Reactions Enzymatic-Reactions Pathways Compounds-And-Elements

23. SRI International Bioinformatics 23 Principal Classes Regulation l Regulation-of-Enzyme-Activity l Regulation-of-Transcription u Regulation-of-Transcription-Initiation u Transcriptional-Attenuation

24. SRI International Bioinformatics 24 Slot Links Sdh-flavoSdh-Fe-SSdh-membrane-1Sdh-membrane-2 sdhA sdhB sdhCsdhD Succinate + FAD = fumarate + FADH 2 Enzymatic-reaction Succinate dehydrogenase TCA Cycle product component-of catalyzes reaction in-pathway

25. SRI International Bioinformatics 25 Example of a Single GFP Call The General Pattern: gfp-function(frame-ID slot-ID value...) (gfp-function frame-ID slot-ID value …) LISP (get-slot-values 'TRYPSYN-RXN 'LEFT) ==> (INDOLE-3-GLYCEROL-P SER)

26. SRI International Bioinformatics 26 Architecture of the API server – PerlCyc and JavaCyc Works on Unix (Solaris or Linux) only Start up Pathway Tools with the –api arg Pathway Tools listens on a Unix socket – perl program communicates through this socket Supports both querying and editing PGDBs Must run perl or java program on the same machine that runs Pathway Tools l This is a security measure, as the API server has no built-in security Can only handle one connection at a time

27. SRI International Bioinformatics 27 Obtaining PerlCyc and JavaCyc Download from http://www.sgn.cornell.edu/downloads/ PerlCyc written and maintained by Lukas Mueller at Boyce Thompson Institute for Plant Research. JavaCyc written by Thomas Yan at Carnegie Institute, maintained by Lukas Mueller. Easy to extend…

28. SRI International Bioinformatics 28 Examples of PerlCyc, JavaCyc Functions GFP functions (require knowledge of Pathway Tools schema): l get_slot_values l get_class_all_instances l put_slot_values Pathway Tools functions (described at http://bioinformatics.ai.sri.com/ptools/ptools-fns.html): http://bioinformatics.ai.sri.com/ptools/ptools-fns.html l genes_of_reaction l find_indexed_frame l pathways_of_gene l transport_p l getSlotValues l getClassAllInstances l putSlotValues l genesOfReaction l findIndexedFrame l pathwaysOfGene l transportP

29. SRI International Bioinformatics 29 Writing a PerlCyc or JavaCyc program Create a PerlCyc, JavaCyc object: perlcyc -> new (“ORGID”) new Javacyc (“ORGID”) Call PerlCyc, JavaCyc functions on this object: my $cyc = perlcyc -> new (“ECOLI”); my @pathways = $cyc -> all_pathways (); Javacyc cyc = new Javacyc(“ECOLI”); ArrayList pathways = cyc.allPathways (); Functions return object IDs, not objects. l Must connect to server again to retrieve attributes of an object. foreach my $p (@pathways) { print $cyc -> get_slot_value ($p, “COMMON-NAME”);} for (int i=0; I < pathways.size(); i++) { String pwy = (String) pathways.get(i); System.out.println (cyc.getSlotValue (pwy, “COMMON-NAME”); }

30. SRI International Bioinformatics 30 Sample PerlCyc Query Number of proteins in E. coli use perlcyc; my $cyc = perlcyc -> new (“ECOLI”); my @proteins = $cyc-> get_class_all_instances("|Proteins|"); my $protein_count = scalar(@proteins); print "Protein count: $protein_count.\n";

31. SRI International Bioinformatics 31 Sample PerlCyc Query Print IDs of all proteins with molecular weight between 10 and 20 kD and pI between 4 and 5. use perlcyc; my $cyc = perlcyc -> new (“ECOLI”); foreach my $p ($cyc->get_class_all_instances("|Proteins|")) { my $mw = $cyc->get_slot_value($p, "molecular-weight-kd"); my $pI = $cyc->get_slot_value($p, "pi"); if ($mw = 10 && $pI = 4) { print "$p\n"; }

32. SRI International Bioinformatics 32 Sample PerlCyc Query List all the transcription factors in E. coli, and the list of genes that each regulates: use perlcyc; my $cyc = perlcyc -> new (“ECOLI”); foreach my $p ($cyc->get_class_all_instances("|Proteins|")) { if ($cyc->transcription_factor_p($p)) { my $name = $cyc->get_slot_value($p, "common-name"); my %genes = (); foreach my $tu ($cyc->regulon_of_protein($p)) { foreach my $g ($cyc->transcription_unit_genes($tu)) { $genes{$g} = $cyc->get_slot_value($g, "common-name"); } print "\n\n$name: "; print join " ", values %genes; }

33. SRI International Bioinformatics 33 Sample Editing Using PerlCyc Add a link from each gene to the corresponding object in MY-DB (assume ID is same in both cases) use perlcyc; my $cyc = perlcyc -> new (“HPY”); my @genes = $cyc->get_class_all_instances (“|Genes|”); foreach my $g (@genes) { $cyc->add_slot_value ($g, “DBLINKS”, “(MY-DB \”$g\”)”); } $cyc->save_kb();

34. SRI International Bioinformatics 34 Sample JavaCyc Query: Enzymes for which ATP is a regulator import java.util.*; public class JavacycSample { public static void main(String[] args) { Javacyc cyc = new Javacyc("ECOLI"); ArrayList regframes = cyc.getClassAllInstances("|Regulation-of-Enzyme-Activity|"); for (int i = 0; i < regframes.size(); i++) { String reg = (String)regframes.get(i); boolean bool = cyc.memberSlotValueP(reg, “Regulator", "ATP"); if (bool) { String enzrxn = cyc.getSlotValue (reg, “Regulated-Entity”); String enzyme = cyc.getSlotValue (enzrxn, “Enzyme”); System.out.println(enz); } } } }

35. SRI International Bioinformatics 35 Simple Lisp Query Example: Enzymes for which ATP is a regulator (defun atp-inhibits () (loop for x in (get-class-all-instances '|Regulation-of-Enzyme-Activity|) ;; Does the Regulator slot contain the compound ATP, and the mode ;; of regulation is negative (inhibition)? when (and (member-slot-value-p x ‘Regulator 'ATP) (member-slot-value-p x ‘Mode “-”) ) ;; Whenever the test is positive, we collect the value of the slot Enzyme ;; of the Regulated-Entity of the regulatory interaction frame. ;; The collected values are returned as a list, once the loop terminates. collect (get-slot-value (get-slot-value x ‘Regulated-Entity) ‘Enzyme) ) ) ;;; invoking the query: (select-organism :org-id 'ECOLI) (atp-inhibits) (get-slot-values 'TRYPSYN-RXN 'LEFT) ==> (INDOLE-3-GLYCEROL-P SER)

36. SRI International Bioinformatics 36 Simple Perl Query Example: Enzymes for which ATP is a regulator use perlcyc; my $cyc = perlcyc -> new("ECOLI"); my @regs = $cyc -> get_class_all_instances("|Regulation-of-Enzyme- Activity|"); ## We check every instance of the class foreach my $reg (@regs) { ## We test for whether the INHIBITORS-ALL ## slot contains the compound frame ATP my $bool1 = $cyc -> member_slot_value_p($reg, “Regulator", "Atp"); my $bool2 = $cyc -> member_slot_value_p($reg, “Mode", “-"); if ($bool1 && $bool2) { ## Whenever the test is positive, we collect the value of the slot ENZYME. ## The results are printed in the terminal. my $enzrxn = $cyc -> get_slot_value($reg, “Regulated-Entity"); my $enz = $cyc -> get_slot_value($enzrxn, "Enzyme"); print STDOUT "$enz\n"; }

37. SRI International Bioinformatics 37 Getting started with Lisp pathway-tools –lisp (load “file”) (compile-file “file.lisp”) Emacs is a useful editor Pathway Tools source code is available: ask Lisp resources: http://bioinformatics.ai.sri.com/ptools/ptools-resources.html

38. SRI International Bioinformatics 38 Query Gotchas Study schema carefully :test #’fequal Cascade of slot-values: check for NIL

39. SRI International Bioinformatics 39 Semantic Inference Layer relationships.lisp Library of functions that encapsulate common query building blocks and intricacies of navigating the schema enzymes-of-gene reactions-of-gene pathways-of-gene genes-of-pathway pathway-hole-p reactions-of-compound top-containers(protein) all-rxns(type) (:metab-smm :metab-all :metab-pathways :enzyme :transport etc.) l (all-rxns :metab-pathways)

40. SRI International Bioinformatics 40 Pathway Tools Schema and Semantic Inference Layer Genes, Operons, and Replicons

41. SRI International Bioinformatics 41 Representing a Genome Classes: l ORG is of class Organisms l CHROM1 is of class Chromosomes l PLASMID1 is of class Plasmids l Gene1 is of class Genes l Product1 is of class Polypeptides or RNA ORG CHROM1 CHROM2 PLASMID1 Gene1 Gene2 Gene3 genome components Product1 product

42. SRI International Bioinformatics 42 (defun genes-of-chrom (chrom) (loop for x in (get-slot-values chrom ‘components) when (instance-all-instance-of-p x ‘|Genes|) collect x) )

43. SRI International Bioinformatics 43 Polynucleotides Review slots of COLI and of COLI-K12

44. SRI International Bioinformatics 44 Genetic-Elements Sequence is stored in a separate file or database table

45. SRI International Bioinformatics 45 Polymer-Segments Review slots of Genes

46. SRI International Bioinformatics 46 Complexities of Gene / Gene-Product Relationships The Product of a gene can be an instance of Polypeptides or RNAs An instance of Polypeptides can have more than one gene encoding it Sequence position: l Nucleotide positions of starting and ending codons specified in Left-End- Position and Right-End-Position (usually greater, except at origin) l Transcription-Direction + / - Alternative splicing: l Nucleotide positions of starting and ending codons specified in Left-End- Position and Right-End-Position l Intron positions specified in Splice-Form-Introns of gene product u (200 300) (350 400)

47. SRI International Bioinformatics 47 Gene Reaction Schematic

48. SRI International Bioinformatics 48 Substring Search Example Find all genes that contain a given substring within their common name or synonym list. (defun find-gene-by-substring (substring) (let (result) (loop for g in (get-class-all-instances '|Genes|) do (loop for name in (get-slot-values g 'names) when (search substring name :test #'string-equal) do (pushnew g result) ) ) result ) )

49. SRI International Bioinformatics 49 Proteins

50. SRI International Bioinformatics 50 Proteins and Protein Complexes Polypeptide: the monomer protein product of a gene (may have multiple isoforms, as indicated at gene level) Protein complex: proteins consisting of multiple polypeptides or protein complexes Example: DNA pol III l DnaE is a polypeptide l pol III core is DnaE and two other polypeptides l pol III holoenzymes is several protein complexes combined

51. SRI International Bioinformatics 51 Protein Complex Relationships

52. SRI International Bioinformatics 52 Slots of a protein (DnaE) catalyzes Is it a regulator/reactant/etc? comment component-of dblinks features (edited in feature editor) Many other attributes possible

53. SRI International Bioinformatics 53 A complex at the frame level (pol III) Most of the same attributes as polypeptide frame component-of and components l note coefficients

54. SRI International Bioinformatics 54 Protein Complex Relationships

55. SRI International Bioinformatics 55 Relationships are Defined in Many Places component-of comes from creating a complex appears-in-left-side-of comes from defining a reaction (as do modified forms) regulates comes from an enzymatic reaction or TU can only edit dna-footprint if protein has been associated with a TU

56. SRI International Bioinformatics 56 Semantic Inference Layer Reactions-of-protein (prot) l Returns a list of rxns this protein catalyzes Transcription-units-of-proteins(prot) l Returns a list of TU’s activated/inhibited by the given protein Transporter? (prot) l Is this protein a transporter? Polypeptide-or-homomultimer?(prot) Transcription-factor? (prot) Obtain-protein-stats l Returns 5 values u Length of : all-polypeptides, complexes, transporters, enzymes, etc…

57. SRI International Bioinformatics 57 Example Find all enzymes that use pyridoxal phosphate as a cofactor or prosthetic group l (loop for protein in (get-class-all-instances ‘|Proteins|) for enzrxn = (get-slot-value protein ‘enzymatic-reaction) when (and enzrxn (or (member-slot-value-p enzrxn ‘cofactors ‘pyridoxal_phosphate) (member-slot-value-p enzrxn ‘prosthetic-groups ‘pyridoxal_phosphate)) collect protein) (member-slot-value-p frame slot value) : T if Value is one of the values of Slot of Frame.

58. SRI International Bioinformatics 58 Sample Find all proteins without a comment anywhere

59. SRI International Bioinformatics 59 RNAs

60. SRI International Bioinformatics 60 RNAs PGDBs only represent RNAs that are “terminal gene products” l tRNAs l rRNAs l Regulatory RNAs l Miscellaneous small RNAs Slots similar to proteins tRNAs can have an anticodon

61. SRI International Bioinformatics 61

62. SRI International Bioinformatics 62 The RNA Ontology

63. SRI International Bioinformatics 63 Compounds / Reactions / Pathways

64. SRI International Bioinformatics 64 Compounds / Reactions / Pathways Think of a three tiered structure: l Reactions built on top of compounds l Pathways built on top of reactions Metabolic network defined by reactions alone; pathways are an additional “optional” structure Some reactions not part of a pathway Some reactions have no attached enzyme Some enzymes have no attached gene

65. SRI International Bioinformatics 65 Compounds Relatively few aspects of a compound defined within the compound editor l MW, formula calculated from edited structure Most aspects defined in other editors l “Pathway reactions” comes from reaction editing followed by pathway editing l Activator, etc come from the protein editor

66. SRI International Bioinformatics 66

67. SRI International Bioinformatics 67 (print-frame ‘TRP) -- Instance TRP --- Types: |Amino-Acid|, |Aromatic-Amino-Acids|, |Non-polar-amino-acids| APPEARS-IN-LEFT-SIDE-OF: RXN0-287, TRANS-RXN-76, TRYPTOPHAN-RXN, TRYPTOPHAN--TRNA-LIGASE-RXN APPEARS-IN-RIGHT-SIDE-OF: RXN0-2382, RXN0-301, TRANS-RXN-76, TRYPSYN-RXN CHEMICAL-FORMULA: (C 11), (H 12), (N 2), (O 2) COMMON-NAME: "L-tryptophan" DBLINKS: (LIGAND-CPD "C00078" NIL |kaipa| 3311532640 NIL NIL), (CAS "6912-86-3"), (CAS "73-22-3") NAMES: "L-tryptophan", "W", "tryptacin", "trofan", "trp", "tryptophan", "2-amino-3-indolylpropanic acid" SMILES: "c1(c(CC(N)C(=O)O)c2(c([nH]1)cccc2))" SYNONYMS: "W", "tryptacin", "trofan", "trp", "tryptophan", "2-amino-3-indolylpropanic acid" ____________________________________________

68. SRI International Bioinformatics 68 Where is diphosphate in the ontology?

69. SRI International Bioinformatics 69 Semantic Inference Layer Reactions-of-compound (cpd) Pathways-of-compound (cpd) Is-substrate-an-autocatalytic-enzyme-p (cpd) Activated/inhibited-by? (cpds slots) l Returns a list of enzrxns for which a cpd in cpds is a modulator (example slots: activators-all, activators-allosteric) All-substrates (rxns) l All unique substrates specified in the given rxns Has-structure-p (cpd) Obtain-cpd-stats l Returns two values: u Length of :all-cpds, cpds with structures

70. SRI International Bioinformatics 70 Miscellaneous things…. History List l Back/Forward and History buttons l Default list is 50 items Show frame (print-frame ‘frame)

71. SRI International Bioinformatics 71

72. SRI International Bioinformatics 72 Queries with Multiple Answers Navigator queries: l Example: Substring search for “pyruvate” l Selected list is placed on the Answer list l Use “Next Answer” button to view each one of them Lisp queries: Example : Find reactions involving pyruvate as a substrate u (get-class-all-instances ‘|Compounds|) ( loop for rxn in (get-class-all-instances ‘|Reactions|) when (member ‘pyruvate (get-slot-values rxn ‘substrates) collect rxn) (replace-answer-list * )

73. SRI International Bioinformatics 73 Reactions

74. SRI International Bioinformatics 74 Reactions Represents information about a reaction that is independent of enzymes that catalyze the reaction Connected to enzyme(s) via enzymatic reaction frames Classified with EC system when possible Example: 2.7.7.7 – DNA-directed DNA polymerization l Carried out by five enzymes in E. coli

75. SRI International Bioinformatics 75 Reaction Ontology

76. SRI International Bioinformatics 76 Where is 2.7.7.7 in the Ontology?

77. SRI International Bioinformatics 77 Slots of Reaction Frames Balance-state EC-number Enzymatic-reaction l Generated in protein or reaction editor In-pathway l Generated in pathway editor Left and Right (reactants / products) l Can include modified forms of proteins, RNAs, etc here l Not all reactants/products need to be frames

78. SRI International Bioinformatics 78

79. SRI International Bioinformatics 79 Reaction relationships

80. SRI International Bioinformatics 80 Enzymatic Reactions (DnaE and 2.7.7.7) A necessary bridge between enzymes and “generic” versions of reactions Carries information specific to an enzyme/reaction combination: l Cofactors and prosthetic groups l Alternative substrates l Links to regulatory interactions Frame is generated when protein is associated with reaction (via protein or reaction editor)

81. SRI International Bioinformatics 81

82. SRI International Bioinformatics 82 Regulation of Enzyme Activity

83. SRI International Bioinformatics 83 Semantic Inference Layer Genes-of-reaction (rxn) Substrates-of-reaction (rxn) Enzymes-of-reaction (rxn) Lacking-ec-number (organism) l Returns list of rxns with no ec numbers in that database Get-reaction-direction-in-pathway (pwy rxn) Reaction-type(rxn) u Indicates types of Rxn as: Small molecule rxn, transport rxn, protein-small-molecule rxn (one substrate is protein and one is a small molecule), protein rxn (all substrates are proteins), etc. All-rxns(type) l Specify the type of reaction (see above for type) Obtain-rxn-stats l Returns six values u Length of : all-rxns, transport, non-transport, etc…

84. SRI International Bioinformatics 84 Find all small-molecule reactions that have no enzyme but are not spontaneous (“orphan” reactions) (defun orphan-reactions (&optional (verbose? t)) (loop for r in (all-rxns :small-molecule) when (and (not (slot-has-value-p r 'enzymatic-reaction)) (not (get-slot-value r 'spontaneous?))) collect r) )

85. SRI International Bioinformatics 85 Reaction Direction Left/Right reflect direction of reaction as written by Enzyme Commission l Reflects systematic direction for different reaction classes Left/Right do not necessarily correspond to physiological direction of a reaction Get-rxn-direction(rxn) l Returns :L2R or :R2L or :BOTH or NIL l Integrates all available info about direction of this reaction u Direction(s) it occurs in all pathways in the PGDB u Direction(s) as specified in Enzymatic-Reactions

86. SRI International Bioinformatics 86 Pathways

87. SRI International Bioinformatics 87 Outline Pathways l Representation of Pathways l Querying Pathways Programmatically l How Pathway Diagrams are Generated l Future Work: Signalling Pathways Cellular Overview Diagram l New Functionality l Under the Hood l How Overview Diagram is Generated l Using Overview Diagram for Global Queries

88. SRI International Bioinformatics 88 What is a Pathway? An ordered set of interconnected, directed biochemical reactions Reactions form a coherent unit, e.g. l Regulated as a single unit l Evolutionarily conserved across organisms as a single unit l When combined, perform a single cellular function l Historically grouped together as a unit Includes metabolic pathways and signalling pathways Evidence for all reactions in a single organism Pathways can be linear, cyclical, branched, or some combination

89. SRI International Bioinformatics 89 Internal Representation of Pathways REACTION-LIST: unordered list of reactions that comprise the pathway PREDECESSORS: list of reaction pairs that define ordering relationships between reactions. E.g. R1 R2 C A B R3 D (R2 R1) : Predecessor of R2 is R1 (R3 R1) : Predecessor of R3 is R1 (R1) : R1 has no predecessor (can be omitted)

90. SRI International Bioinformatics 90 What is missing from Pathway Representation? Reaction directions l Some reactions are unidirectional, but many are reversible – how do we know in which direction to draw the reaction? Main vs. side substrates A B C D E F l Main compounds form the backbone of the pathway u substrates shared between connecting reactions u major inputs and outputs. l Side compounds omitted from pathway diagrams at low detail levels l Individual reactions do not necessarily have main and side compounds – a particular substrate may be either a main or a side depending on the pathway context.

91. SRI International Bioinformatics 91 Computing Directionality and Mains/Sides Our philosophy: Enable curator to specify as little as possible. Compute as much as possible. This reduces redundancy and potential for inconsistencies. Example: Reactions R1: A + B  C + D R2: B  E Predecessors: (R2 R1) Only substrate overlap is B B must be a main substrate A must be a side substrate, R1 must proceed from right to left R2 must proceed from left to right C + D  B  E A

92. SRI International Bioinformatics 92 Unfortunately, mains, sides and reaction directions are sometimes ambiguous: At beginnings and ends of pathways l Use heuristics to determine main/side substrates at beginnings, ends of pathways l Not always what the curator wants Substrate overlap with both sides of a reaction, e.g. A + B  C + D C + B  E Solution: Additional slot PRIMARIES, should only be populated when necessary: PRIMARIES: (R (A B) (C)) says that for reaction R, A and B are both main reactants, and C is a main product. But…

93. SRI International Bioinformatics 93 More Complications… ENZYME-USE: a reaction may be catalyzed by multiple enzymes, but not all the enzymes necessarily participate in a given pathway l Not present in the same compartment with rest of pathway enzymes l Down-regulated or not expressed under conditions in which pathway is active l ENZYME-USE slot tells us which enzymes catalyze reaction in pathway, if not all. LAYOUT-ADVICE: helps software draw pathway correctly, e.g. in a cyclical pathway, tells which substrate should be at the top. HYPOTHETICAL-REACTIONS: list of reactions in the pathway that are considered hypothetical (i.e. no direct experimental evidence)

94. SRI International Bioinformatics 94 Polymerization Pathways …  X [n] X [n+1] X [10] POLYMERIZATION-LINKS: specifies reactions that should be connected by a polymerization link (X R1 R1) --- REACTANT-NAME-SLOT: N-NAME --- PRODUCT-NAME-SLOT: N+1-NAME CLASS-INSTANCE-LINKS: specifies when a link should be drawn between a substrate class and some instance of it (necessary only if instance is not a member of some reaction, so no predecessor relationship can be defined) R1 --- PRODUCT-INSTANCES: X [10]

95. SRI International Bioinformatics 95 Super-Pathways Collection of pathways that connect to each other via common substrates or reactions, or as part of some larger logical unit Can contain both sub-pathways and additional connecting reactions Can be nested arbitrarily REACTION-LIST: a pathway ID instead of a reaction ID in this slot means include all reactions from the specified pathway PREDECESSORS: a pathway ID instead of a tuple in this slot means include all predecessor tuples from the specified pathway

96. SRI International Bioinformatics 96 Pathway Links Can be used as an alternative or in addition to defining super-pathways Link must be to or from some main substrate in the pathway Other end of link can be a pathway, a reaction, or an arbitrary text string Software automatically computes direction of link, but curator can override it

97. SRI International Bioinformatics 97 Querying Pathways Programmatically See http://bioinformatics.ai.sri.com/ptools/ptools-resources.html (all-pathways) (base-pathways) l Returns list of all pathways that are not super-pathways (genes-of-pathway pwy) (unique-genes-of-pathway pwy) l Returns list of all genes of a pathway that are not also part of other pathways (enzymes-of-pathway pwy) (substrates-of-pathway pwy) (variants-of-pathway pwy) l Returns all pathways in the same variant class as a pathway (get-predecessors rxn pwy), (get-successors rxn pwy) (get-rxn-direction-in-pathway pwy rxn) (pathway-inputs pwy), (pathway-outputs pwy) l Returns all compounds consumed (produced) but not produced (consumed) by pathway (ignores stoichiometry)

98. SRI International Bioinformatics 98 Example Queries Find all genes involved in metabolic pathways: (remove-duplicates (loop for p in (all-pathways) append (genes-of-pathway p))) Find all compounds that are unique to a single pathway: (loop for p in (base-pathways) append (loop for c in (substrates-of-pathway p) when (null (remove p (pathways-of-compound c))) collect (list c p)))

99. SRI International Bioinformatics 99 Regulation

100. SRI International Bioinformatics 100 Regulation Reorganization and expansion of regulation under way in Pathway Tools Initial application to EcoCyc Class Regulation with subclasses that describe different biochemical mechanisms of regulation Slots: l Regulator l Regulated-Entity l Mode l Mechanism

101. SRI International Bioinformatics 101 Regulation of Enzyme Activity Class Regulation-of-Enzyme-Activity Each instance of the class describes one regulatory interaction Slots: l Regulator -- usually a small molecule l Regulated-Entity -- an Enzymatic-Reaction l Mechanism -- One of: u Competitive, Uncompetitive, Noncompetitive, Irreversible, Allosteric, Other l Mode -- One of: +, - l Physiologically-relevant? – true/false

102. SRI International Bioinformatics 102 Transcription Initiation Class Regulation-of-Transcription-Initiation l Transcription factor binds to DNA binding site to regulate transcription initiation from a promoter Slots: l Regulator -- instance of Proteins or Complexes (a transcription-factor) l Regulated-Entity -- instance of Promoters l Mode -- One of: +, - l Associated-binding-site – a DNA-Binding-Site

103. SRI International Bioinformatics 103 Attenuation Class Transcriptional-Attenuation Several subclasses depending on type of attenuation Slots common to all: l Regulator -- Depends on subtype of attenuation l Regulated-Entity -- instance of Terminators l Mode -- One of: +, -

104. SRI International Bioinformatics 104 Attenuation Subtypes Ribosome-Mediated-Attenuation l E.g. trp operon – ribosome pauses based on levels of charged tRNA, determines formation of terminator or antiterminator RNA-Mediated-Attenuation l RNA (tRNA or sRNA) binds to transcript, determines formation of terminator or antiterminator Protein-Mediated-Attenuation l Protein binds to transcript, determines formation of terminator or antiterminator Small-Molecule-Mediated-Attenuation l Small molecule binds to transcript, determines formation of terminator or antiterminator Rho-Blocking-Antitermination RNA-Polymerase-Modification l Regulatory protein binds to site in transcription unit and interacts with RNA polymerase to determine termination

105. SRI International Bioinformatics 105 Transcriptional Regulation site001 pro001 trpE trpD trpC trpB trpA trpL TrpR*trpreg001 trpLEDCBA trp apoTrpR rxn001 term001reg002charged-tRNA*trp

106. SRI International Bioinformatics 106 Data Exchange

107. SRI International Bioinformatics 107 Data Exchange Java API and Perl API : read & modify BioPAX Export: since Pathway Tools 9.0 l Biopax.org Export of entire PGDB as Flatfiles Export of Reactions as SBML -- sbml.org Import/Export of Pathways: between PGDBs Import/Export of Selected Frames, for Spreadsheets Import/Export of Compounds as Molfile, CML Registering/Publishing PGDBs on WWW Export PGDB as Genbank BioWarehouse : Loader for Flatfiles, SQL access l http://bioinformatics.ai.sri.com/biowarehouse/

108. SRI International Bioinformatics 108 Dump PGDB into Flatfiles Export of entire PGDB as Flatfiles Format Description: UG v.I section 4.5 l Column delimited: 1 line per frame l Attribute-value: 1 record per frame Multiple slot values: l Column delimited: several values per column l Attribute-value: several lines for several values

109. SRI International Bioinformatics 109 Frame Import/Export Import/Export of Selected Frames, for Spreadsheets Frame selection, Slot selection GUI Format Description: UG v.I section 4.6.3 l Column delimited: 1 line per frame l Attribute-value: 1 record per frame Multiple slot values: l Column delimited: several values per column l Attribute-value: several lines for several values

110. SRI International Bioinformatics 110 BioWarehouse: A Bioinformatics Database Warehouse Peter D. Karp, Thomas J. Lee, Valerie Wagner Oracle or MySQL UniProt ENZYME Genbank Taxonomy BioCyc CMR KEGG BioWarehouse = Java-based Loader = C-based Loader Oracle (10g) or MySQL (4.1.11) UniProt ENZYME Genbank Taxonomy BioCyc BioPAX BioWarehous e GO MAGE-ML KEGG CMR Eco2DBase

111. SRI International Bioinformatics 111 Motivations Hundreds of bioinformatics DBs exist Important problems involve queries across multiple DBs

112. SRI International Bioinformatics 112 Technical Approach Multi-platform support: Oracle (10g) and MySQL Schema support for multitude of bioinformatics datatypes Create loaders for public bioinformatics DBs l Parse file format of the source DB l Semantic transformations l Insert DB contents into warehouse tables Provide Warehouse query access mechanisms l SQL queries via ODBC, JDBC, OAA Operate public BioWarehouse server: publichouse BMC Bioinformatics 7:170 2006

113. SRI International Bioinformatics 113 BioWarehouse Schema Manages many bioinformatics datatypes simultaneously l Pathways, Reactions, Chemicals l Proteins, Genes, Replicons l Sequences, Sequence Features l Organisms, Taxonomic relationships l Computations (sequence matches) l Citations, Controlled vocabularies l Links to external databases Each type of warehouse object implemented through one or more relational tables (currently 43)

114. SRI International Bioinformatics 114 Warehouse Schema Manages multiple datasets simultaneously l Dataset = Single version of a database Version comparison Multiple software tools or experiments that require access to different versions Each dataset is a warehouse entity Every warehouse object is registered in a dataset

115. SRI International Bioinformatics 115 BioWarehouse Loaders DatabaseLoader Language Input Format Comments BioCycCBioCyc attribute-valuePathway/Genome Databases BioPAXJavaBioPAX formatProtein interactions data CMRCCMR column-delimitedComprehensive Microbial Resource: 350+ microbial genomes Eco2DbaseJavaRelational table dumpsE. coli 2-D gel data ENZYMEJavaENZYME attribute-valueEnzyme Commission set of reactions GenbankJavaXML derived from ASN.1Bacterial subset of Genbank Gene OntologyJavaOBO XMLHierarchical controlled vocabulary KEGGCKEGG formatMetabolic pathway data MAGE-MLJavaMAGE-ML formatMicroarray gene expression data NCBI TaxonomyCTaxonomy formatOrganism taxonomy UniProtJavaUniProt XMLSWISS-PROT and TrEMBL

116. SRI International Bioinformatics 116 Acknowledgements SRI l Michelle Green, Ron Caspi, Ingrid Keseler, John Pick, Carol Fulcher, Markus Krummenacker, Alex Shearer EcoCyc Collaborators l Julio Collado-Vides, John Ingraham, Ian Paulsen MetaCyc Collaborators l Sue Rhee, Peifen Zhang, Hartmut Foerster, Chris Tissier BioCyc Collaborators l Christos Ouzounis and EBI CGG Funding sources: l NIH National Center for Research Resources l NIH National Institute of General Medical Sciences l NIH National Human Genome Research Institute l Department of Energy Microbial Cell Project l DARPA BioSpice BioCyc.org Learn more from BioCyc webinars: biocyc.org/webinar.shtml

117. SRI International Bioinformatics 117 Chokepoint Example For Antibiotic Target Development Find Strategic Essential Weak Links in Metabolism Many Compounds have just 1 Producing and consuming reaction (defun chokepoint-1 () (remove-duplicates (loop for cpd in (remove-if-not #'coercible-to-frame-p (all-substrates (all-rxns))) when (= 1 (length (get-slot-values cpd 'APPEARS-IN-LEFT-SIDE-OF)) (length (get-slot-values cpd 'APPEARS-IN-RIGHT-SIDE-OF))) collect (get-slot-value cpd 'APPEARS-IN-LEFT-SIDE-OF) and collect (get-slot-value cpd 'APPEARS-IN-RIGHT-SIDE-OF) ) :test #'fequal) ) ;;; invoking the query: (length (chokepoint-1)) ==> 348