the protein ontology (pro)

Welcome to PRO Annotation Jamboree

The Protein Ontology (PRO)

Natalia RobertsUniversity of [email protected] Pathways Tools Workshop October 28, 2010

1OutlineIntroduction to the PRotein OntologyConsortiumFramework Curation

PRO WebsitePRO EntrySearch and BrowseAnnotation



3PRO ConsortiumInitially PRO to address proteins

Cathy Wu

Barry Smith

Judith BlakeExtended to protein complexes

Carol BultPeter DEustachio

OutlineIntroduction to the PRotein OntologyConsortiumFramework Curation




67Ontology for semantic integration of heterogeneous biological dataOBO Foundry establishes rules and best practices to create a suite of orthogonal interoperable reference ontologiesPRO within OBO Foundry

Molecule (PRO)7OBO Foundry ontologies are organized along two dimensions: (1) granularity (from molecule to population); and (2) relation to time (objects, qualities, processes). In terms of this scheme, PROis a representation of protein objects at the single molecule level of granularity. Specifically, it treats the protein molecules themselves rather than some property of the molecules (such asfunction, location, molecular weight, etc.). Such properties are instead handled by other ontologies such as GO; PRO provides the objects to which such properties can be attached. PROencompasses a sub-ontology of proteins based on evolutionary relatedness (ProEvo), and a subontology of the multiple protein forms produced from a given gene locus (ProForm).PRotein Ontology (PRO)8Ontology for Protein Evolution (ProEvo)captures the protein classes reflecting evolutionary relationships at full-length protein levels. Ontology for protein complexes (ProComp)formally defines the protein complexes in terms of the specific components. Ontology for Protein Forms (ProForm)captures the different protein forms of a specific gene arising from genetic variations, alternative splicing, cleavage, post-translational modifications. ABComplex ZPAAPAPAPhas_part Protein A phospho 1Complex Zhas_part Protein BProtein AProtein A phospho 1Protein A cleavedProtein A phospho 2Protein A unmodifiedOntology to formally represent proteins and protein complexes

8PROEvo: The ProEvo part of PRO is meant to indicate the relatedness of proteins, not their evolution. That is why the relation of a child term to its parent term is is_a and notevolutionarily_derived_from. Nonetheless, the process that resulted in the relatedness (usually reflected in a protein classification) is indeed evolution, and we make every attempt to useevolutionarily-justified classes. Thus, PRO would not group together two proteins that merely share a common function (which might have arose by convergent evolution); instead, PROwould group together two proteins based on evolutionary relatedness, irrespective of function.Why PRO?Allow specification of relationships between PRO and other ontologies, such as GO and Disease Ontology

Provides a structure to support formal, computer-based inferences based on shared attributes among homologous proteins

Provides a stable unique identifier to any protein type

Provides formalization and precise annotation of specific protein forms/ classes, allowing accurate and consistent data mapping, integration and analysis9ProEvo

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Smadhmhmhmhhmmhmhm

MH1 domain(PF03165)MH2 domain(PF03166)Gene DuplicationSpeciationI-SmadsCo-SmadTGFBBMPSMAD5SMAD1SMAD9SMAD3SMAD2SMAD6SMAD7SMAD4In the ontology...hmOntology for Protein Evolution(PROEvo)

captures the protein classes reflecting evolutionary relationships at full-length protein levels. Family: a PRO term at this level refers to proteins that can trace back to a common ancestor over the entire length of the protein are part of the same family.Gene: a PRO term at this level refers to the protein products of a distinct gene.11POINT: Need to account for the possibility that a protein might not share the traits of its parent or siblings

If one were to follow the evolution of Smad proteins proteins in humans and mice, a few inferences can be made:1) The proteins are indeed related. If we follow the branches back in time, we see that they converge.2) The split between the two protein forms preceded the species split. Thus, SMAD5 from human and mouse are more alike than are SMAD5 and SMAD1 from humans.3) All four proteins are composed of the same set of domains, or parts of proteins

The final slide shows how all of this relates and integrates into one scheme.Functional classes are not described by PROCathepsinsproteases, most of the them become activated at the low pH found in lysosomes. Crystallinsconstituent of eye lens.

Heat shock proteinclass of functionally related proteins whose expression is increased when cells are exposed to elevated temperatures or other stress.

Protein Kinasean enzyme that modifies other proteins by chemically adding phosphate groups.(for now)ProFormAAPAPAP13GeneProtein FormDistinctive AttributesSMAD2Long isoform phosphorylated(PRO:000000468)NOT has_function GO:00036677 DNA bindingShort isoform phosphorylated (PRO:000000469)has_function GO:00036677 DNA bindingCUL1Unmodified form(PRO:000002507)NOT part_of GO:0019005 SCF ubiquitin ligase complexNeedylated form(PRO:000000542)part_of GO:0019005 SCF ubiquitin ligase complexCD14Membrane form(PRO:000002149)located_in GO:0005886 plasma membraneSoluble form(PRO:000002147)located_in GO:0005615 extracellular spaceROCK1

Full length(PRO:000002529)has_function GO:0004674 protein serine/threonine kinase activity Cleaved form(PRO:000000563)Increased has_function GO:0004674 protein serine/threonine kinase activity CREBBPVariant R -> P(1378) (PRO:000000266)agent_in MIM:180849, RUBINSTEIN-TAYBI SYNDROMESO:1000118, loss_of_function_of_polypeptideThe Need for Representation of Various Proteins FormsLocalizationAssociationDiseaseModificationFunction14There has been an incremental need of being more precise in the identification of proteins objects

I-SmadsCo-SmadTGFBBMPSmadSMAD9Isoform 1Isoform 1 unmodifiedIsoform 2Isoform 1 modified (PTM/Cleaved)Isoform 2 unmodifiedIsoform 2 modified (PTM/Cleaved)SMAD5SMAD1SMAD3SMAD2PROFormPROEvocapture different protein forms of a specific gene arising from genetic variations, alternative splicing, cleavage, post-translational modifications Sequence: a PRO term at this level refers to the protein products with a distinct sequence upon initial translation.Modification: a PRO term at this level refers to the protein products with some change that occurs after initial translation.15Categories: indication of how PRO is organizedFamily: a PRO term at this level refers to proteins that can trace back to a common ancestor over the entire length of the protein are part of the same family.Gene: a PRO term at this level refers to the protein products of a distinct gene.Sequence: a PRO term at this level refers to the protein products with a distinct sequence upon initial translation.Modification: a PRO term at this level refers to the protein products with some change that occurs after initial translation.Sequence-level distinction: Each PRO term at this level refers to the protein products with a distinct sequence upon initial translation. The sequence differences can arise from different alleles of a given gene, from splice variants of a given RNA, or from alternative initiation and ribosomal frameshifting during translation. One can think of this as a mature mRNA-level distinction. For example, smad2 encodes both a long splice form and a short splice form. The protein products from each isoform are separate PRO terms. Sequence-level distinction is the first (parent-most) node of the ProForm part of PRO.Modification-level distinction: Each PRO term at this level refers to the protein products derived from a single mRNA species that differ because of some change (or lack thereof) that occurs after the initiation of translation (co- and post-translational). This includes sequence differences due to cleavage and chemical changes to one or more amino acid residues. For example, the long isoform of smad2 can either be unmodified or be post-translationally modified to contain phosphorylated residues. Modification-level distinction is the leaf-most node of the ProForm part of PRO.

In the ontologyProCompABP17Complex where subunits are post-translationally modifiedE.g.Phosphorylated IRF3 dimerComplexes that differ in subunit composition (within or in different species). E.g. mCD14/LPS vs sCD14/LPS

In the ontology

Soluble CD14memb CD14Figure adapted from http://www.reactome.org/cgi-bin/eventbrowser?DB=gk_current&FOCUS_SPECIES=Homo%20sapiens&ID=166016&

Modified from http://www.reactome.org/cgi-bin/eventbrowser?DB=gk_current&FOCUS_SPECIES=Homo%20sapiens&ID=166016&18IIITGF-beta receptorSmad 4NucleusDNA binding and transcription regulationMAPKKKShcSPYPSPYPYPSPYPSPSPTPXIAPP38 MAPKpathwayJNKcascadeIIISPYPYPSPYPSPSPTPSmad 7STRAPTAK1KUDegradationTAK1Smad 2SPSPSkiShcSmad 2Smad 4Smad 2SPSPSmad 4Smad 2SPSPSmad 4Smad 2SPSPXERK1/2CaMSmad 2SPSPXPSmad 2TPSPSPSPSPTPFurinTGF-bTGF-bTGF-bLAPCytoplasmCa2+Growth signalsSPTPYPKUPhosphorylation (P) at Serine (S), Threonine (T)Tyrosine (Y)Ubiquitination (U) at Lysine (K)TGF-beta signaling comparison between PID and Reactome Growth signalsOnly included in ReactomeCommon in both Reactome & PID* All others are in PID. Not all components in the pathway from both databases are listedStress signalsMEKK1TPTPSmad 2SPSPXPXPRO:000000523PRO:000000650PRO:000000651PRO:000000410PRO:000000616PRO:000000652PRO:000000366PRO:000000481PRO:000000618PRO:000000397PRO:000000650PRO:000000366PRO:000000650PRO:000000366PRO:00000046819Framework20Categories/ Levels of DistinctionFamily: a PRO term at this level refers to proteins that can trace back to a common ancestor over the entire length of the protein are part of the same family.Gene: a PRO term at this level refers to the protein products of a distinct gene.Sequence: a PRO term at this level refers to the protein products with a distinct sequence upon initial translation.Modification: a PRO term at this level refers to the protein products with some change that occurs after initial translation.

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I-SmadsFamilyCo-SmadTGFBBMPSmadSMAD9Isoform 1Isoform 1 unmodifiedIsoform 2Isoform 1 modified (PTM/Cleaved)Isoform 2 unmodifiedIsoform 2 modified (PTM/Cleaved)SMAD5SMAD1SMAD3SMAD2Gene productSequenceModificationCategories in ProEvo & ProFormOrganism-geneOrganism-sequenceOrganism-modificationPROFormPROEvoProtein22Categories: indication of how PRO is organizedFamily: a PRO term at this level refers to proteins that can trace back to a common ancestor over the entire length of the protein are part of the same family.Gene: a PRO term at this level refers to the protein products of a distinct gene.Sequence: a PRO term at this level refers to the protein products with a distinct sequence upon initial translation.Modification: a PRO term at this level refers to the protein products with some change that occurs after initial translation.

Protein ontology framework

ProEvo, ProFormProComppro.oboPAF.txtannotationRoot level- protein (left panel)The levels of distinction are categories of PRO classes that provide some indication of how PRO is organized. The term distinction denotes that the terms at each level can be distinguished from one another as follows:Family-level distinction: Each PRO term at this level refers to protein products of a distinct gene family arising from a common ancestor. The leaf-most nodes at this level are usually families comprising paralogous sets of gene products (of a single or multiple organisms). For example, smad2 and smad3 both encode proteins that are TGF receptor-regulated while smad1, smad5, and smad9 are all BMP receptor-regulated. Thus, TGF-beta receptor-regulated smad protein and BMP receptor-regulated smad protein are terms denoting distinct families. Note that this level collectively refers to any such grouping at any level of similarity. For example, the two families indicated above can merge into a receptor-regulated smad protein class and further merge (with the protein products of smad4, smad6, and smad7) into the smad protein class. No merging occurs beyond what can be done to account for the evolution of the entire, full length protein. That is, all proteins that can trace back to a common ancestor over the entire length of the protein are part of the same family.Gene-level distinction: Each PRO term at this level refers to the protein products of a distinct gene. For example, smad2 and smad3 are two different genes, even though they are paralogs, and therefore have two different PRO entries at the gene level of distinction. The protein products of all alleles of what is recognized as smad2 in humans and what is recognized as smad2 in mouse thus fall under this single term. Thus, a single PRO framework descriptionLast Updated 04/23/2010term at the gene-level distinction collects the protein products of a subset of orthologs for that gene (the subset that is so closely related that its members are considered the same gene). Gene-level distinction is the leaf-most node of the ProEvo part of PRO.Sequence-level distinction: Each PRO term at this level refers to the protein products with a distinct sequence upon initial translation. The sequence differences can arise from different alleles of a given gene, from splice variants of a given RNA, or from alternative initiation and ribosomal frameshifting during translation. One can think of this as a mature mRNA-level distinction. For example, smad2 encodes both a long splice form and a short splice form. The protein products from each isoform are separate PRO terms. Sequence-level distinction is the first (parent-most) node of the ProForm part of PRO.Modification-level distinction: Each PRO term at this level refers to the protein products derived from a single mRNA species that differ because of some change (or lack thereof) that occurs after the initiation of translation (co- and post-translational). This includes sequence differences due to cleavage and chemical changes to one or more amino acid residues. For example, the long isoform of smad2 can either be unmodified or be post-translationally modified to contain phosphorylated residues. Modification-level distinction is the leaf-most node of the ProForm part of PRO.Rool level- protein complex (right panel)Within PRO, it is possible to capture the distinct complexes and sub-complexes as they exist in different species, defining them through their component proteins and annotating them through GO terms. It is also possible to assign separate terms for active and inactive forms, whether they are derived from post-translational modification of one or more components or from addition/subtraction of one or more components.The middle panel denotes the different ontologies or databases that are used to either (i) define a class, or (ii) annotate a property of an entity of the class. For example, at the modification-level, smad2 isoform 1 phosphorylated form can be defined as the intersection of PRO smad2 isoform1 and MOD:00696 phosphorylated residue, using the relation has_modification. One such phosphorylated form, smad2 isoform 1 phosphorylated 1, participates_in signal transduction. Such annotations are provided in the protein ontology association file (PAF) file. The arrows indicate to what levels these ontologies are applied.23Some conceptsOrtho-isoform: These are isoforms- encoded by orthologous genes that are believed to have arisen prior to speciation and divergence of the primary sequence.

Ortho-modified form: Post-translational modifications on equivalent residues in ortho-isoforms.

PRO:000000615 GTP-binding protein RhoA isoform 1 prenylated 1A GTP-binding protein RhoA isoform 1 prenylated form where a geranylgeranyl moiety has been added to the Cys residue within the C-terminal Cxxx motif. Example: UniProtKB:P61586-1, has_modification MOD:00113 S-geranylgeranyl-L-cysteine, Cys-190. [PMID:16773203, PRO:CNA] PRO:000000048 TGF-beta receptor type-2 isoform 1 (Also known as Isoform RII-1)





26What is curated in the ontology?Name: take name from data source but we follow established naming guidelines. Synonyms: imported from data source, add others by request and during curation.Definitions: we try to create standard definitions. A is a B that Cs Both text and logical definitions are created when possible

PRO:000025762 serine palmitoyltransferase complex A (mouse) def: "A serine palmitoyltransferase complex that is heterotrimeric and whose components are encoded in the genome of mouse." [PRO:CJB]

is_a: GO:0017059 ! serine C-palmitoyltransferase complex relationship: has_part PRO:000025361 {cardinality="1"} ! serine palmitoyltransferase 1 (mouse)relationship: has_part PRO:000025362 {cardinality="1"} ! serine palmitoyltransferase 2 (mouse)relationship: has_part PRO:000025363 {cardinality="1"} ! serine palmitoyltransferase 3 (mouse)relationship: only_in_taxon taxon:10090 ! Mus musculus

Cross-ref: when there is a database object that corresponds to the term.

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Example of how we define modified formsFeature section of UniProtKB record for human smad2Reference sectionSer-240/Ser-465/Ser-467Ser-465/Ser-46728

ProForm Curationby TGF-beta receptorthrough Ca++-mediated signalingSer-465/Ser-467Ser-240/Ser-465/Ser-467[Term]id: PRO:000000650name: smad2 isoform 1 phosphorylated 1def: "A smad2 isoform 1 phosphorylated form that has been phosphorylated in the last two Ser residues within the SSxS C-terminal motif by TGF-beta pathway activation." [PMID:8980228, PMID:9346966]comment: Category=modification.synonym: "TGF-beta receptor-activated smad2" RELATED []is_a: PRO:000000574 ! smad2 isoform 1 phosphorylated form[Term]id: PRO:000000652name: smad2 isoform 1 phosphorylated 3def: "A smad2 isoform 1 phosphorylated form that has been phosphorylated at a [S/T] residue within the MH1-MH2 domain linker region in response to decorin-induced Ca(2+) signaling. This form is also phosphorylated in the last two Ser residues within the SSxS C-terminal motif." [PMID:11027280]comment: Category=modification.is_a: PRO:000000574 ! smad2 isoform 1 phosphorylated form29What is Annotated?Domain, especially ProEvo level:

GO terms

PSI-MOD terms for protein modifications

SO for sequence variants

MIM for sequence variants

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PRO homepage

http://pir.georgetown.edu/pro/pro.shtml31PRO distribution filespro.oboOntology in OBO format

ftp://ftp.pir.georgetown.edu/databases/ontology/pro_obo/32PRO distribution files ftp://ftp.pir.georgetown.edu/databases/ontology/pro_obo/PAF_guidelines.pdfPAF.txtPRO association file (Tab delimited, similar to GAF)ColumnColumn TitleDescription1PRO_IDPRO identifier, mandatory2Object_termName of the PRO term3Object_synonymOther names by which the described object is known4ModifierFlags that modify the interpretation of an annotation 5RelationRelation to the corresponding annotation. 6Ontology_IDID for the corresponding annotation. 7Ontology_termTerm name for the corresponding ontology ID.8Relative_toModifiers increased, decreased and altered require an entry in this column to indicate what the change is relative to.9Interaction_withTo indicate binding partner. 10Evidence_sourcePubmed ID or database source for the evidence. 11Evidence_codeSame as evidence code for GO annotations12TaxonTaxon identifier for the species that the annotation is extracted from.13Inferred_fromUse only for evidence code: IPI and ISS for PRO. 14DB_IDOne or more unique identifiers for a single source cited as an authority for the attribution of the ontology term.15Protein_regionTo indicate part of the protein sequence.16Modiresidue(s), MOD_IDTo indicate the residue(s) that has a post-translational modification and the type of modification. 17DateDate on which the annotation was made.18Assigned_byThe database which made the annotation. 19Equivalent formsList the equivalent form in other organisms. 20CommentsCurator comments, free text.NOTcontributes_todecreasedincreasedalteredpart_oflocated_inhas_parthas_agenthas_functionparticipates_inagent_inhas_modification33PAF.txtPRO association file (Tab delimited, similar to GAF)

PRO distribution files ftp://ftp.pir.georgetown.edu/databases/ontology/pro_obo/PAF_guidelines.pdf34PRO scope and statisticsCurrent scope: human, mouse and E. coli proteins. Comprehensive coverage for gene level and general modified formsIn PRO Release 13.0, version 0 (link):

There are 25700 PRO terms # termsCategory281family 18184gene128organism-gene 1153sequence 5662modification13complex

AnnotationCurated papers: 929Annotation to GO terms:2006Annotation to MOD terms:36135OutlineIntroduction to the PRotein OntologyConsortiumFramework Curation




37PRO Entryhttp://purl.obolibrary.org/obo/PRO_000000563

1-Ontology2-Features3-Mapping4-Annotation38OutlineIntroduction to the PRotein OntologyConsortiumFramework Curation




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Browse

2-Sorting3-Information tabs4-Add text to find1-Number terms41

Find is exact text match to a termUse information tab to display information of interestInformation tabsAdd text to searchBrowse (cont)

Quick Browse allows to browse terms related toa given theme:-Terms for proteins with a given modification-Terms for saliva biomarkers-Terms with link to a given database-Terms with orthoisoforms

Quick Browse43

Quick Browse:OrthoisoformsQuick Browse:Saliva biomarkers

PRO homepage45Examples for search fields in: http://pir.georgetown.edu/pro/searchPRO.pdfPRO Advanced Search

Boolean searches: AND, OR, NOTNull/not null search46-All terms derived from a given gene

Link to family database. Proteins in this class are from vertebrates

Indicate level in the hierarchySave results as tab delimited fileShow the selected terms in the hierarchySearch for PRO terms that are modified forms that are annotated with GO term nucleusBoolean searches: AND, OR, NOThttp://pir.georgetown.edu/cgi-bin/pro/textsearch_pro

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>Click apply to see the new column(s)Annotation column addedUse Display Option to add/remove columns

50Upcoming Functionalities:

Batch retrieval

PRO ID MGI ID -------- ------------------ PRO:000000124 1927343 PRO:000000123 1270849 PRO:000000121 107926ID mapping51OutlineIntroduction to the PRotein OntologyConsortiumFramework Curation




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AnnotationAnnotation tool54Annotation tool for PRO: RACE-PROObtain a PRO ID for the protein objects of interestDefine a protein object (based on literature, experimental data)Add annotation to that protein object

1 protein form=1 RACE-PRO

How does it work?Input your personal information (only for internal use)Complete form with sequence information and annotationSubmit when ready (otherwise you can save for later)PRO curation team will take the data, revise it, and create the corresponding PRO node in the ontologyUse will be informed through email about the new PRO IDs and when they will be public

55Annotation tool for PRO: RACE-PRO

1) Fill your personal information. This allows saving your data as well as future communication2) Define the protein object. This allows retrieving or pasting a sequence, defining a subsequence, and/or a post-translational modification.

O75475-2insert UniProtKB Accessions (including isoforms) and RetrieveMTRDFKPGDLIFAKMKGYPHWPARVDEVPDGAVKPPTNKLPIFFFGTHETAFLGPKDIFP YSENKEKYGKPNKRKGFNEGLWEIDNNPKVKFSSQQAATKQSNASSDVEVEEKETSVSKETDHEEKASNEDVTKAVDITTPKAARRGRKRKAEKQVETEEAGVVTTATASVNLKVOR paste a sequence56Annotation tool for PRO: RACE-PRO

Select a region (e.g. if a cleaved product)

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Annotation tool for PRO: RACE-PROSelect PTMs: use numbering in reference to the sequence displayed in the box 1

Use this section to indicate names assigned from this protein form as indicated in the source58

Annotation tool for PRO: RACE-PROModifiers: used to modify a relation between a PRO term and another term. It includes the GO qualifiers NOT, contributes_to plus increased, decreased, and altered (to be used with the relative to column). Relation to the specific annotation. For some database/ontology there is a single relation and that is displayedAdd ID for the specific database/ontology. 59

Save means you are not finished.Use the reference number to come back and finishSubmit means you are done, your entry will be reviewedWhat to expect when you are done?Receive an email with the ref number in the subject when your entry is under reviewed

A PRO curator will be assigned to review your entry and create the corresponding PRO node.You will receive an email with the PRO ID, and the terms for your final check

60How to link to PROTo link to PRO entry, please use persistent URLhttp://purl.obolibrary.org/obo/PRO_xxxxxxxxx where PRO_xxxxxxxxx is the corresponding PRO ID with an underscore (_) instead of colon (:).

Example: link to PRO:000000447 would be http://purl.obolibrary.org/obo/PRO_000000447.

61Ontology ProvidersDendritic Cell Ontology (DC_CL)Semantic ResourcesRoyal Society of Chemistry (RSC)Science Collaboration FrameworkSemantic Web Applications in Neuromedicine (SWAN)Process-Modeling ResourcesReactome, MouseCycEcoCycPathway LogicMolecule-Modeling ResourcesIntl Union of Basic and Clinical Pharmacology (IUPhar)

Some ongoing collaborationsPRO Consortium Team (so far)Principal InvestigatorsCathy Wu(UD/GU-PIR)Judith Blake(Jackson Lab-MGI)Carol Bult(Jackson Lab-MGI)Barry Smith(SUNY Buffalo-NCBO)Peter DEustachio(NYU/Reactome)

Co-Investigators, Curators and DevelopersDarren Natale (GU-PIR)Harold Drabkin (Jackson Lab-MGI)Alexei Evsikov(Jackson Lab-MGI) Michael Caudy (NYU)Cecilia Arighi(UD-PIR)Jian Zhang(GU-PIR)Hongzhan Huang(UD-PIR)Natalia Roberts(UD-PIR)Jules Nchoutmboube 63Funded by NIGMS

63Questions?6464

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