1. Prediction of protein function Lars Juhl Jensen EMBL Heidelberg

2. Overview Part 1 –Homology-based transfer of annotation –Function prediction from protein domains Part 2 –Prediction of functional motifs from sequence –Feature-based prediction of protein function Part 3 –Prediction of functional interaction networks

3. Why do we need to predict function?

4. What do we mean by function? The concept “function” is not clearly defined –A structural biologist, a cell biologist, and a medical doctor will have very different views Many levels of granularity –For the overall definition of “function”, the knowledge and description can be more or less specific Functional categories are somewhat artificial –People like to put things in boxes …

6. Descriptions of protein function Controlled vocabularies –Gene Ontology –SwissProt keywords –KEGG pathways –EcoCyc pathways Interaction networks More accurate data models –Reactome –Systems Biology Markup Language (SBML)

7. Molecular function Molecular function describes activities, such as catalytic or binding activities, at the molecular level GO molecular function terms represent activities rather than the entities that perform the actions, and do not specify where or when, or in what context, the action takes place Examples of broad functional terms are catalytic activity or transporter activity; an example of a narrower term is adenylate cyclase activity

8. Biological process A biological process is series of events accomplished by one or more ordered assemblies of molecular functions An example of a broad GO biological process terms is signal transduction; examples of more specific terms are pyrimidine metabolism or alpha- glucoside transport It can be difficult to distinguish between a biological process and a molecular function

9. Cellular component A cellular component is just that, a component of a cell that is part of some larger object It may be an anatomical structure (for example, the rough endoplasmic reticulum or the nucleus) or a gene product group (for example, the ribosome, the proteasome or a protein dimer) The cellular component categories are probably the best defined categories since they correspond to actual entities

10. Homology-based transfer of annotation Lars Juhl Jensen EMBL Heidelberg

11. Detection of homologs Pairwise sequence similarity searches –BLAST (fastest) –FASTA –Full Smith-Waterman (most sensitive) Profile-based similarity searches –PSI-BLAST –Hidden Markov Models (HMMs) Sequence similarity should always be evaluated at the protein level

13. Sequence similarity, sequence homology, and functional homology Sequence similarity means that the sequences are similar – no more, no less Sequence homology implies that the proteins are encoded by genes that share a common ancestry Functional homology means that two proteins from two organisms have the same function Sequence similarity or sequence homology does not guarantee functional homology

14. Orthologs vs. paralogs

15. Functional consequences of gene duplication Neofunctionalization –One copy has retained the ancestral function and can be treated as a 1–to–1 ortholog (functional homolog) –The other copy have changed their function and behave much like paralogs Subfunctionalization –Each copy has taken on a part of the ancestral function –A functional homolog cannot be defined –Each ortholog typically has the same molecular function in a different sub-process or location

16. 1–to–1 orthology A single gene in one organism corresponds to a single gene in another organism These can generally be assumed to encode functionally equivalent proteins –Same molecular function –Same biological process –Same localization 1–to–1 orthology is fairly common in prokaryotes and among very closely related organisms

17. 1–to–many orthology A single gene in one organism corresponds to multiple genes in another organism Any mixture of neo- and sub-functionalizations can have occurred –Typically same molecular function –Often different biological process or sub-process –Often different sub-cellular localization or tissue 1–to–many orthology is very common between simple model organisms and higher eukaryotes

18. Many–to–many orthology Many genes in each organism have arisen from a single gene in their last common ancestor Different neo- and sub-functionalizations have likely taken place in each lineage –Typically same molecular function –Often different biological process or sub-process –Often different sub-cellular localization or tissue Many–to–many orthology is common between higher eukaryotes that are distantly related

19. Detection of orthologs Reconstruction of phylogenetic trees –The theoretically most correct way –Works for analyzing particular genes of interest Methods based on reciprocal matches –What currently works at the genomic scale Manual curation –Detection of very remote orthologs may require that knowledge on gene synteny and/or protein function is taken into account

20. Construction of gene trees Identify the relevant proteins –Sequence similarity and possibly additional information Construct a blocked multiple sequence alignment –Use, for example, Muscle and Gblocks Reconstruct the most likely phylogenetic tree –Use, for example, PhyML Orthologs and paralogs can be trivially extracted based on a gene tree

21. Reciprocal matches Simple “best reciprocal match” is a bad choice –Can only deal with one-to-one orthology Detection of in-paralogs –Similarity higher with species than between species Orthologs can now be detected based on best reciprocal matches between in-paralogous groups One or more out-group organisms can optionally be used to improve the definition of orthologs

23. Orthologous groups Orthologs and paralogs are in principle always defined with respect to two organisms Orthologous groups instead try to encompass an entire set of organisms The “inclusiveness” of the orthologous groups depends on how broad a set of organisms the groups cover

24. Definition of orthologous groups

27. COGs, KOGs, and NOGs The COGs and KOGs were manually curated –These were automatically expanded to more species Tri-clustering –Detection of in-paralogs –Identification of triangles of best reciprocal matches –Merging of triangles that share an edge Broad phylogenetics coverage –COGs and NOGs cover all three domains of life –KOGs cover all eukaryotes

30. Clustering based on similarity All-against-all sequence similarity is calculated A standard clustering method is applied to define groups of homologous genes –TribeMCL –Hierarchical clustering These methods generally detect groups of homologous genes, but are not good for distinguishing between orthologs and paralogs

32. Meta-servers Since numerous methods exist for identifying groups of orthologous proteins, meta-servers have begun to emerge These can be very useful for “fishing expeditions” where one is looking for a remote ortholog of a particular protein of interest However, such meta-servers do not attempt to unify the different orthologous groups and are thus not useful for genome-wide studies

33. Function prediction from protein domains Lars Juhl Jensen EMBL Heidelberg

34. When homology searches fail Sometimes no orthologs or even paralogs can be identified by sequence similarity searches, or they are all of unknown function No functional information can thus be transferred based on simple sequence homology By instead analyzing the various parts that make up the complete protein, it is nonetheless often possible to predict the protein function

35. Protein domains Many eukaryotic proteins consist of multiple globular domains that can fold independently These domains have been mixed and matched through evolution Each type of domain contributes towards the molecular function of the complete protein Numerous resources are able to identify such domains from sequence alone using HMMs

44. Which domain resource should I use? SMART is focused on signal transduction domains Pfam is very actively developed and thus tends to have the most up-to-date domain collection InterPro is useful for genome annotation since the domains are annotated with GO terms CDD is conveniently integrated with the NCBI BLAST web interface

45. Predicting globular domains and intrinsically disordered regions Not all globular domains have been discovered and the databases are thus not comprehensive Methods exist for predicting from sequence which regions are globular and which are disordered –GlobPlot uses a simple propensity scale –DisEMBL, DISOPRED, and PONDR all use ensembles of artificial neural networks Many disordered regions are important for protein function and they should thus not be ignored

52. Summary Functional annotation –Molecular function vs. biological process –Inference of molecular function by sequence similarity –Biological process only transferable between orthologs Detection of orthologs –In-depth studies: phylogenetic trees –Automated analysis: InParanoid and COG/KOG/NOG Profile searches for protein domains –Each domains contributes a different molecular function

53. Acknowledgments Christian von Mering Christopher Creevey Ivica Letunic Rune Linding Tobias Doerks Francesca Ciccarelli Berend Snel Martijn Huynen Toby Gibson Rob Russell Peer Bork

54. Prediction of functional motifs from sequence Lars Juhl Jensen EMBL Heidelberg

55. Proteins – more than just globular domains Transmembrane helices Disordered regions Eukaryotic linear motifs (ELMs) –Modification sites, e.g. phosphorylation sites –Ligand peptides, e.g. SH3 binding sites –Targeting signals, e.g nuclear localization sequences The short functional motifs are as important as the globular domains

56. Insulin Receptor Substrate 1

57. Databases of functional motifs Fewer and smaller databases –General databases of motifs: ProSite and ELM –Phosphorylation sites: Phospho.ELM and PhosphoSite –These databases contain much fewer instances that protein domain databases Curation is more difficult –Protein domain databases can be constructed based on analysis of protein sequences alone –Short functional motifs must be curated based on experimental evidence

62. Prediction of ELMs Most functional motifs are “information poor” –Weak/short consensus sequences for ELMs –The typical ELM only has three conserved residues –Some variance is often allowed even for these ELMs are very hard to predict from sequence –Simply consensus sequences match everywhere –Even more advanced methods like PSSMs, ANNs, or SVMs give poor specificity –The full information is not in the site itself

67. Construction of data sets Compiling an initial data set –Positive examples can be obtained from existing databases or curated from the literature –Good negative examples are often harder to get Separate training and test sets –A method may be able to learn the training examples but to generalize to new examples Homology reduction! –It is crucial that there is no significant sequence similarity between examples in the training and test sets

68. Machine learning Numerous algorithms exist –Artificial neural networks –Support vector machines –Decision trees The choice of algorithm is not so important Providing the relevant input is important Having high-quality training data is crucial

71. Kinase-specific prediction of phosphorylation sites (NetPhosK) Artificial neural networks (ANNs) were trained several different kinases The sequence logos show only the positive examples Negative examples also provide information Also, ANNs and SVMs can capture correlations between positions

72. Prediction of signal peptides from sequence (SignalP) Function –Eukaryotic proteins are targeted to the ER –Prokaryotic proteins are targeted for secretion Architecture –Positively charged N- terminus –Hydrophobic core –Short, more polar region –Cleavage site Signal peptides can be accurately predicted

73. Machine learning can help identify errors in curated databases Some of the manually curated databases contain obvious errors that can be eliminated General “SIGNAL” errors –Wrong signal peptide cleavage site –The secreted protein is processed by proteases –Signal peptide include propeptide –Wrong start codon used

74. Signal peptide or propeptide

75. Propeptide cleavage Signal peptide cleavage

76. Wrong start codon

77. Use of short linear motifs for function prediction Only a few motifs (mostly localization signals) can be predicted with high accuracy –Even in these cases advanced machine learning methods are typically needed –These can be treated in the same way as domains Most motifs are weak, and predictions should be approached with care –To tell if these sites are likely to be true, one needs to consider the context –An experiment is needed to prove that it is functional

78. Feature-based prediction of protein function Lars Juhl Jensen EMBL Heidelberg

79. Function prediction from post translational modifications Proteins with similar function may not be related in sequence Still they must perform their function in the context of the same cellular machinery Similarities in features such like PTMs and physical/chemical properties could be expected for proteins with similar function Henrik Nielsen, CBS, DTU Lyngby

80. The concept of ProtFun

82. Function prediction on the human prion sequence ############## ProtFun 1.1 predictions ############## >PRIO_HUMAN # Functional category Prob Odds Amino_acid_biosynthesis 0.020 0.909 Biosynthesis_of_cofactors 0.032 0.444 Cell_envelope 0.146 2.393 Cellular_processes 0.053 0.726 Central_intermediary_metabolism 0.130 2.063 Energy_metabolism 0.029 0.322 Fatty_acid_metabolism 0.017 1.308 Purines_and_pyrimidines 0.528 2.173 Regulatory_functions 0.013 0.081 Replication_and_transcription 0.020 0.075 Translation 0.035 0.795 Transport_and_binding => 0.831 2.027 # Enzyme/nonenzyme Prob Odds Enzyme 0.250 0.873 Nonenzyme => 0.750 1.051 # Enzyme class Prob Odds Oxidoreductase (EC 1.-.-.-) 0.070 0.336 Transferase (EC 2.-.-.-) 0.031 0.090 Hydrolase (EC 3.-.-.-) 0.057 0.180 Isomerase (EC 4.-.-.-) 0.020 0.426 Ligase (EC 5.-.-.-) 0.010 0.313 Lyase (EC 6.-.-.-) 0.017 0.334

83. ProtFun data sets Labeling of training and test data –Cellular role categories: human SwissProt sequences were categorizes using EUCLID –Enzyme categories: top-level enzyme classifications were extract from human SwissProt description lines –Gene Ontology terms were transferred from InterPro The sequences were divided into training and test sets without significant sequence similarity Binary predictors were for each category

84. Prediction performance on cellular role categories

85. Prediction performance on enzyme categories

86. Predictive performance on Gene Ontology categories

87. Non-classical secretion Some proteins without N-terminal signal peptides are secreted via alternative secretion pathways –Several growth factors, i.e. FGF1 and FGF2 –Interleukine 1 beta –HIV-1 tat No consensus sequence motif is known Maybe they have some features in common with other secreted proteins …

88. SecretomeP data sets Training and test set –Positive examples: 3321 extracellular mammalian proteins with their signal peptides removed –Negative examples: 3654 mammalian proteins from cytoplasm or nucleus Validation set –14 known non-classically secreted proteins

89. Secreted proteins are typically small

90. ROC plot for SecretomeP

91. Similar properties of classically and non-classically secreted proteins

93. A look into the black box Neural networks are often criticized for being a “black box” method However, there are several ways to investigate what a neural network ensemble has learned –Which fraction of the ensemble use a certain feature? –How good performance can be attained using each of the features individually? –How much does performance decrease if the neural networks are retrained without a certain feature (or combination of features)?

97. SecretomeP feature usage

98. ProtFun performance for other organisms Our predictors work in general for eukaryotes –Best performance on metazoan proteins Some categories work quite well for prokaryotes –Most metabolism categories –Transport and binding While other categories fail –Energy metabolism –Regulatory functions

99. Mapping category performances onto input features

100. Performance contribution of sequence derived features The correlations between features and function is conserved for eukaryotes Some correlations extend to archaea and bacteria –Physical/chemical properties –Secondary structure and transmembrane helices Other correlations only hold for eukaryotes –PTMs and subcellular localization features

101. Evolution conserves protein features and function Protein features are more conserved between orthologs than paralogs This leads to ProtFun predicting orthologs to be more likely to share function than paralogs That prediction is fully consistent with the notion that it is best to infer function from orthologous proteins

102. Conclusions Short linear motifs are likely equally important for protein function as the large well-studied domains These are much harder to predict from sequence –Reasonable accuracy can be obtained by applying machine learning methods on high-quality datasets Many classes of proteins can be predicted based on such sequence derived-protein features –These methods a not nearly as reliable as homology –However, often they are the only option

103. Acknowledgments Ramneek Gupta Can Kesmir Jannick Dyrløv Bendtsen Henrik Nielsen Nikolaj Blom Francesca Diella Rune Linding Damien Devos Alfonso Valencia Søren Brunak Toby Gibson

104. Prediction of functional interaction networks Lars Juhl Jensen EMBL Heidelberg

105. What is an interaction? Physical protein interactions –Proteins that physically touch each other –Members of the same stable complex –Transient interactions, e.g. a kinase and its substrate The pragmatic definition – whatever the assay in question can measure Functional interactions –Neighbors in metabolic networks –Members of the same pathway

106. The use of interaction networks for function prediction A functional interaction implies that two proteins are involved in the same biological process However, the networks do not divide proteins into a predefined set of functional classes such as the Gene Ontology terms Functional associations do not require homology to proteins of know function, and can complement the predictions even when homology is present

109. Functional interaction networks

110. Evidence types Genomic context methods –Phylogenetic profiles, gene neighborhood, and fusion Primary experimental data –Physical protein interactions and gene expression data Manually curated databases –Pathways and protein complexes Automatic literature mining –Co-ocurrence and Natural Language Processing

111. Phylogenetic profiles

115. Cell Cellulosomes Cellulose

116. Formalizing the phylogenetic profile method Align all proteins against all Calculate best-hit profile Join similar species by PCA Calculate PC profile distances Calibrate against KEGG maps

117. Gene neighbourhood

118. Gene neighborhood Identify runs of adjacent genes with the same direction Score each gene pair based on intergenic distances Score each gene pair based on intergenic distances Calibrate against KEGG maps Infer associations in other species Infer associations in other species

119. Gene fusion

120. Find in A genes that match a the same gene in B Exclude overlapping alignments Exclude overlapping alignments Calibrate against KEGG maps Calibrate against KEGG maps Calculate all-against-all pairwise alignments Calculate all-against-all pairwise alignments

121. Calibration of quality scores Different pieces of evidence are not directly comparable –A different raw quality score is used for each evidence type –Quality differences exist among data sets of the same type Solved by calibrating all scores against a common reference –The accuracy relative to a “gold standard” is calculated within score intervals –The resulting points are approximated by a sigmoid

122. Data integration

123. Protein-protein interaction databases Imported databases –BIND, Biomolecular Interaction Network Database –DIP, Database of Interacting Proteins –GRID, General Repository for Interaction Datasets –HPRD, Human Protein Reference Database –MINT, Molecular Interactions Database Databases to be added –IntAct –PDB

124. Physical protein interactions Make binary representation of complexes Make binary representation of complexes Yeast two-hybrid data sets are inherently binary Yeast two-hybrid data sets are inherently binary Calculate score from number of (co-)occurrences Calculate score from number of (co-)occurrences Calculate score from non-shared partners Calculate score from non-shared partners Calibrate against KEGG maps Infer associations in other species Combine evidence from experiments

125. Binary representations of purification data

126. Topology based quality scores Scoring scheme for yeast two-hybrid data: –S1 = -log((N 1 +1)·(N 2 +1)) –N 1 and N 2 are the numbers of non-shared interaction partners –Similar scoring schemes have been published by Saito et al. Scoring scheme for complex pull-down data: –S2 = log[(N 12 ·N)/((N 1 +1)·(N 2 +1))] –N 12 is the number of purifications containing both proteins –N 1 is the number containing protein 1, N 2 is defined similarly –N is the total number of purifications Both schemes aim at identifying ubiquitous interactors

127. Mining microarray expression databases Re-normalize arrays by modern method to remove biases Re-normalize arrays by modern method to remove biases Build expression matrix Build expression matrix Combine similar arrays by PCA Combine similar arrays by PCA Construct predictor by Gaussian kernel density estimation Construct predictor by Gaussian kernel density estimation Calibrate against KEGG maps Calibrate against KEGG maps Infer associations in other species Infer associations in other species

128. Databases of curated knowledge Pathway databases –BioCarta –KEGG, Kyoto Encyclopedia of Genes and Genomes –Reactome –STKE, Signal Transduction Knowledge Environment Curated protein complexes –MIPS, Munich Information center for Protein Sequences Databases to be added –Gene Ontology annotation

129. Co-occurrence in the scientific texts Associate abstracts with species Identify gene names in title/abstract Count (co-)occurrences of genes Test significance of associations Calibrate against KEGG maps Infer associations in other species

130. Databases used for text mining Corpora –Medline –OMIM, Online Mendelian Inheritance in Man –SGD, Saccharomyces Genome Database –The Interactive Fly These text sources are all parsed and converted into a unified format Gene synonyms –Ensembl –SwissProt –HUGO –LocusLink –SGD –TAIR Cross references and sequence comparison is used for merging

131. Gene and protein names Cue words for entity recognition Verbs for relation extraction [ nxgene The GAL4 gene] [ nxexpr The expression of [ nxgene the cytochrome genes [ nxpg CYC1 and CYC7]]] is controlled by [ nxpg HAP1] Natural Language Processing

132. Multiple types of interactions

133. Transfer of evidence STRING “red” – COG mode –Each node in the network represents a COG –For each pair of COGs, the highest confidence score for each evidence type counts from each clade –The scores are combined using naïve Bayes STRING “blue” – protein mode –Each node in the network represents a single locus –Evidence from other organisms are transferred based on fuzzy orthology –The scores are combined using naïve Bayes

136. ? Source species Target species Evidence transfer based on “fuzzy orthology” Orthology transfer is tricky –Correct assignment of orthology is difficult for distant species –Functional equivalence is not guaranteed for paralogs These problems are addressed by our “fuzzy orthology” scheme –Functional equivalence scores are calculated from all-against-all alignment –Evidence is distributed across possible pairs

137. The power of cross-species transfer and evidence integration

143. The big challenge

144. Prediction of “mode of action”

145. Summary Functional interaction networks are useful for predicting the biological role of a protein Many algorithms and types of data can be used for predicting functional interactions –Each method must be benchmarked –The different types of evidence should be integrated in a probabilistic scoring scheme To make the most of the available data, evidence should also be transferred between organisms

146. Acknowledgments Christian von Mering Jasmin Saric Berend Snel Sean Hooper Rossitza Ouzounova Samuel Chaffron Julien Lagarde Mathilde Foglierini Isabel Rojas Martijn Huynen Peer Bork