Critical Assessment of Function Annotation, 2005

A “Fair and Balanced” Assessment of Protein Function Prediction Servers

Iddo Friedberg, Martin Jambon, Andrei Osterman and Adam Godzik

Outline

What is function?

Describing function

Describing function similarity

Selecting Targets

Assessing servers

Thoughts for the future

What is Function?

Biochemical

Pathway/location

Phenotypic

10 -6 m 1m 10 -9 m Mutation: Histidinemia (Mental retardation and speech defect)‏ Histidine amino lyase (HAL, Histidase)‏ L-histidine Urocanate +NH 3

Outline

What is function?

Describing function

Selecting Targets

Assessing servers

Describing Function: From English to Keywords “ HAL—which is the first enzyme in the degradation pathway of L-histidine—catalyzes the non-oxidative deamination of its substrate to trans-urocanic acid”. László Poppe, (2001) COCB “ HAL— which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans -urocanic acid ”.

Describing Function: From English to Keywords “ HAL—which is the first enzyme in the degradation pathway of L-histidine—catalyzes the non-oxidative deamination of its substrate to trans-urocanic acid”. László Poppe, (2001) COCB “ HAL— which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans -urocanic acid ”. “ HAL — which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans -urocanic acid ”. “ HAL — which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans -urocanic acid ”. “ HAL — which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans -urocanic acid ”.

Outline

What is function?

Describing function

Selecting Targets

Assessing servers

Keyword Similarity Measurements

Represent each document as a set of words

Use set theory to normalize a common word count

Problem: does not take into account the corpus word count

Represent each document as a vector of (weighted by frequency) words

Document similarity based on the angle between the vectors

Problem: dimensions not really orthogonal (co-dependence of words)‏

Keywords and Semantics “ HAL —which is the first enzyme in the degradation pathway of L-histidine — catalyzes the non-oxidative deamination of its substrate to trans-urocanic acid ”. László Poppe, (2001) COCB “ Histidase catalyzes the elimination of the alpha-amino group of histidine using a 4-methylidene-imidazole-5-one (MIO), which is formed autocatalytically from the internal peptide segment 142Ala-Ser-Gly.” Baedecker & Schultz, (2002) Eur J Biochem

Keyword based methods are semantically blind

What do we do if there are no shared keywords?

Ontology: beyond keywords

Ontologies serve to establish the semantic function of words

Ontologies are 'specifications of a relational vocabulary': sets of defined terms like the sort that you would find in a dictionary, but the terms are networked. (from the GO site)‏

EC1 oxidoreductases EC2 transferases EC3 hydrolases EC4 lyases EC5 isomaerase EC6 ligases EC4.1 carbon-carbon EC4.2 carbon-oxygen EC4.3 carbon-nitrogen EC4.5 phosphorus-oxygen EC4.99 others EC4.4 carbon-sulfur EC 4.3.1 ammonia lyases EC 4.3.1.1 aspartate ammonia lyase EC 4.3.1.2 met-aspartate ammonia lyase EC 4.3.1.3 histidine ammonia lyase EC 4.3.3 amine lyases Enzyme Commission Classification Enzyme

EC4 lyases EC4.3 carbon-nitrogen EC 4.3.1 ammonia lyases EC 4.3.1.3 histidine ammonia lyase Enzyme Commission Classification

E.C. Provides a semantically accurate description by

1) Using a controlled vocabulary

2) Going from the general to the specific

3) Defining the scope of interest

Gene Ontology: Function Beyond Enzymes

GO describes function beyond the enzymatic

Three functional aspects are described:

Molecular function

Biological process

Cellular location

Terms are related by “is-a” and “part-of” relationships

GO: molecular function of HAL Histidine ammonia-lyase activity (GO:003456)‏ ammonia-lyase activity C-N lyase activity lyase activity Molecular Function

GO: biological process of HAL Histidine biosynthesis Histidine family biosynthesis Histidine metabolism Physiological process Cellular process Biological process

Outline

What is function?

Describing function

Keywords

Ontology

Selecting targets

Assessing servers

The Gene Ontology

GO is a hierarchy of terms . A directed acyclic graph.

A node contains a single term which is one of the following aspects :

molecular function

cellular component

cellular process

Problem: how do we create a distance/similarity measure?

Solution 1: measure the shortest path distance between terms

His-NH 3 lyase Serine-NH 3 lyase NH 3 -lyase C-S lyase lyase activity hydrolase Catalytic activity d(His- NH 3 lyase,C-S lyase) = 3

(Lord et. al Bioinformatics 2003)‏

The Problem with Path Distance Some terms are more informative than others! General terms are less informative An absurd situation: Catalytic activity d(His-NH 3 lyase, C-S lyase) = 3 d(His-NH 3 lyase,catalytic activity) = 3 Need to provide a measure based upon term information content, not path distance

His-NH 3 lyase Serine-NH 3 lyase NH 3 -lyase C-S lyase lyase activity hydrolase

Information Content Based Distance

A concept occurs if each term, or its children, occurs.

The probability for each concept, p ( c ) increases as we move towards the root

Let the set shared parents of two terms be S ( c 1, c 2)‏

The probability of the minimal subsumer is:

p(c1, c2) = min({p(c)})‏

c ∈ S ( c 1, c 2)‏

sim(c1,c2) = -log 2 ( p ms ( c 1, c 2)) gosim (His-NH3 lyase,C-S lyase) = -log 2 (p(Lyase activity)) = 1.57

His-NH3 lyase p=0.000433 Serine-NH3 lyase p=0.0977 NH3-lyase p=0.124 C-S lyase p=0.0281 Lyase activity p=0.15 hydrolase p=0.102 Catalytic activity p=0.5 Molecular Function p=1

gosim (His-NH3 lyase, cat activity) = -log 2 (0.5)=0.7

Outline

What is function?

Describing function

Selecting targets

Assessing servers

Selecting Targets

Ideally targets should be:

Without obvious homologs (sequence or structure)‏

Function experimentally determined

Not yet published

Thanks to the target contributers: Adam Godzik TBI, JCSG Subramanian Sri Krishna , JCSG Andrei Osterman, TBI Michal Linial, UW / HUJI

Selecting Targets Cont'd

Thermotoga maritima : a hyperthermophilic deep ocean eubacteria.

Genome wide fold coverage organism of choice for JCSG 1878 protein coding genes

Very few, “feeler” assessment targets were picked.

Outline

What is function?

Describing function

Selecting targets

Assessing servers (target by target)‏

Assessed Servers

Server Scorecard (empty)‏ Spearmint ___ RuleBase ___ AnnoLite ___ PFP ___ PhydBac ___ ProKnow ___ Proteome Analyst __ GOPET ___

T7: an aspartate oxidase or dehydrogenase?

Novel enzyme, aspartate dehydrogenase, non-orthologous replacement for aspartate oxidase. TM1643 from T. maritima

Function experimentally verified: Yang Z et al., J Biol Chem. 2003 278(10):8804-8

in cluster FIG ID Function TM ID #1 ASPDH: Aspartate dehydrogenase [same functional role as] (EC 1.4.3.16 ) TM1643* #2 QSYN: Quinolinate synthetase (EC 4.1.99.-) TM1644 #3 QAPRT: Quinolinate phosphoribosyltransferase [decarboxylating] (EC 2.4.2.19 ) TM1645* *Structures solved Biological Process: NAD/NADP Biosynthesis Summary: Genomic cluster conserved between T,maritima and several methanogenic archaea contains a novel gene* for the first step of NAD biosynthesis I II ASPOX ASPDH NAD + NADH QSYN PRPP IV FAD + FADH III QAPRT PP i Experimentally confirmed by: Z.Yang et al. (Toronto)‏ “ Aspartate Dehydrogenase, a Novel Enzyme Identified from Structural and Functional Studies of TM1643”, J. Biol. Chem., Vol. 278,, 2003 Connecting intermediates I L-Aspartate II Iminoaspartate III Dihydroxyacetone-P IV Quinolinic acid TM1643

T7: GO Tree and Probabilities Oxidoreductase CH-NH 2 bonds Molecular Function Catalytic Oxidoreductase Oxidoreductase CH-NH 2 bonds NAD/NADP acceptor Oxidoreductase CH-NH 2 bonds Oxygen acceptor Aspartate Dehydrogenase Aspartate Oxidoreductase Spearmint : homoserine dehydrogenase A nnoLite : L-lactate dehydrogenase ProKnow: oxidoreductase activity p=1; gosim=0 p=0.26; gosim=192. p=0.03; gosim=484. p=8.65x10 -4 ; gosim=1017 p=4.74x10 -4 ; gosim=1104 PhydBac : nicotinate nuc. dephosphorylase PFP :3-5 nucleotide phosphodiesterase

T7: summary

A “new” enzyme: aspartate dehydrogenase

Non orthologous replacement of aspartate oxidase

Experimentally characterized two years ago

Not yet in annotation databases: do we need a standard deposition system like GenBank (Sequence) PDB (structure) MIAME (microarray)?

Some servers' predictions are better: is GO the ideal similarity scale?

T1: the little Thiamine synthesis enzyme who couldn't (and neither could we)‏

T1: the Thiamine synthesis hypothesis Beck & Downs, JB 1998

T1 is not involved directly in the Thiamine pathway

ApbE is present in bacteria with and without the thiamine biosynthesis pathway

No correlation with genomic presence of thiamine transporters (ThiBPQ or YuAJ)‏

Not present in the thi loci

Lack of the ApbC or ApbE protein results in a defect in Fe-S cluster metabolism (Downs, JB 2003)‏

T1 Beck & Downs, JB 1998 ApbE

T1: a non oligomerizing T-fold T-fold: normally forms homo-oligomeric barrels Uricase T1 Active site Oligomerization region Catalytic site Cat. site Oligomerization region

T1: predictions

Cellular Process (Thiamine biosynthesis) found by: Spearmint, Rulebase, PFP, ProKnow

Molecular Function: PFP, ProKnow: oxidoreductase,

Molecular function PFP: transferring glycosyl groups

Cellular process aspect: mis-annotation due to outdated annotations in the reference databases (Pfam, KEGG)‏

We know the structure, active site, fold type, possible pathway

Surprise! We do not know the real function (yet)‏

T1 - conclusions

It is possible to have extensive knowledge of a protein, inc. structure, pathway yet know little about its actual biochemical function

T2

TM1622. ORFan.

MF: GTPase binding.

Evidence: structural similarity to GTPase binders: Mog1, PsbP

Genomic co-location with the LepA (elongation factor Tu), a GTPase.

T2 aligned to Ran-GTPASE MOVIE

T2 Predictions binding Protein binding Enzyme binding GTPase binding Small GTPase binding p=0.45; gosim=113 p=0.21 gosim=224 p=7.4x10 -3 gosim=707 gosim=1024 gosim=1122

AnnoLite:

actin binding

PIP(4,5) binding

PFP : Rab interactor

T4: Pantothenate Kinase

TM0883

Pantothenate kinase activity GO:0004594 E.C. 2.7.1.33

Evidence: experimental (Brand & Strauss, JBC May 2005)‏

New protein:CoaX

T4: Pantothenate Kinase, CoA synthesis Boxes: enzymes Circles: substrates/products Arrows: “preferred” reaction directionality Color: organism in which enzyme exists UNIVERSAL PATHWAY Fatty Acid metabolism Central Carbon metabolism CoA B5 PANK VI ATP ADP PPCS VIII CTP CMP, PP i VII Cysteine metabolism PPCDC IX CO 2 PPAT X PP i ATP DPCK ADP ATP PANK2 PANK3 PPAT2 present in H.sapiens present in both present in E.coli absent in both

Brand, L. A. et al. J. Biol. Chem. 2005;280:20185-20188 Cluster analysis of the predicted coaX gene in selected organisms

T4: server predictions

ProKnow : kinase activity; ATP binding

Many servers erroneously provided “Bortadella pertussis Bvg accessory factor family”: old Pfam annotation

The exact function can be inferred (almost) from genomic context:

T4 is clustered with other proteins in the CoA synthesis pathway in many organisms AND

There is no other recognizable pantothenate kinase in T. maritima 's genome

T5: Growth Arrest; GDNF Receptor SwissProt: GAS1_HUMAN

Contributed by Michal Linial

Cellular process:

induces caspase dependent apoptosis.

Prevents G 0 -> S cell-cycle transition

Location: membrane; rafts

Biochemistry: binds glial derived neurotrophic factors (GDNF); GPI binding (Personal communication M. Linial)‏

No structure (Robetta model)‏

T5: Growth Arrest; GDNF Receptor Signal transducer Receptor activity X-membrane receptor activity hematopoein/interferon-class cytokine receptor activity GDNF receptor activity p=0.134; gosim=289 p=0.072 gosim=379 p=0.052 gosim=426 p=2.32x10 -3 ; gosim=875 p=8.4x10 -5 ; gosim=1353