Volume 6 Supplement 7
Evaluation of function predictions by PFP, ESG, and PSI-BLAST for moonlighting proteins
© Khan et al.; licensee BioMed Central Ltd. 2012
Published: 13 November 2012
Advancements in function prediction algorithms are enabling large scale computational annotation for newly sequenced genomes. With the increase in the number of functionally well characterized proteins it has been observed that there are many proteins involved in more than one function. These proteins characterized as moonlighting proteins show varied functional behavior depending on the cell type, localization in the cell, oligomerization, multiple binding sites, etc. The functional diversity shown by moonlighting proteins may have significant impact on the traditional sequence based function prediction methods. Here we investigate how well diverse functions of moonlighting proteins can be predicted by some existing function prediction methods.
We have analyzed the performances of three major sequence based function prediction methods, PSI-BLAST, the Protein Function Prediction (PFP), and the Extended Similarity Group (ESG) on predicting diverse functions of moonlighting proteins. In predicting discrete functions of a set of 19 experimentally identified moonlighting proteins, PFP showed overall highest recall among the three methods. Although ESG showed the highest precision, its recall was lower than PSI-BLAST. Recall by PSI-BLAST greatly improved when BLOSUM45 was used instead of BLOSUM62.
We have analyzed the performances of PFP, ESG, and PSI-BLAST in predicting the functional diversity of moonlighting proteins. PFP shows overall better performance in predicting diverse moonlighting functions as compared with PSI-BLAST and ESG. Recall by PSI-BLAST greatly improved when BLOSUM45 was used. This analysis indicates that considering weakly similar sequences in prediction enhances the performance of sequence based AFP methods in predicting functional diversity of moonlighting proteins. The current study will also motivate development of novel computational frameworks for automatic identification of such proteins.
The ever growing genome sequencing data and the overwhelming development of genome sequencing technologies have boosted the development of computational techniques and resources for protein function prediction [1, 2]. The traditional sequence based functional annotation is based on the concept of homology [3, 4] or motif/domain searches [5–7]. Some recent Automatic Function Prediction (AFP) methods such as PFP [8, 9], ESG , Gotcha , GOFigure , and ConFunc  use the Gene Ontology (GO) hierarchy. On the other hand, SIFTER , FlowerPower  and Orthostrapper  employ phylogenetic trees to transfer functions to target genes in the evolutionary context. There are other function prediction methods that consider co-expression patterns [17–21], 3D structures of proteins [22–30] as well as protein-protein interaction networks [31–36].
Although existing AFP methods show numerous successful predictions, moonlighting proteins may pose a challenge as they are known to show more than one function that are diverse in nature [37–39]. The varied functional behavior of these proteins can be due to localization within the cell, expression by different cell types, binding of a cofactor, oligomerization, complex formation, or multiple binding sites. Moonlighting proteins have been found to be involved in molecular functions ranging from diseases and disorders [16, 40, 41] to immune systems [40, 41].
In this work, we have analyzed the ability of existing function prediction methods to correctly identify diverse functions of experimentally identified moonlighting proteins . We have collected Gene Ontology (GO) term annotations of these proteins from the UniProt database and manually classified these annotations into two distinct functions. Based on the GO annotations, we have examined the prediction performance of PSI-BLAST and two other major sequence based function prediction methods, the Protein Function Prediction (PFP) and the Extended Similarity Group (ESG) method.
Overall, PFP showed higher average recall than PSI-BLAST and ESG. ESG showed lower recall as compared with PFP and PSI-BLAST, although it has a higher precision. The results suggest that the functional diversity of the moonlighting proteins can be captured if weakly similar sequences are considered among a broad range of similar sequence sets.
Function prediction methods
In this section we briefly describe the three AFP methods we examined, PFP, ESG, and PSI-BLAST. Since PFP [8, 9] and ESG  have been published in earlier works, please refer to the original works for more details.
Protein function prediction (PFP) algorithm
where N is the number of sequence hits considered in the PSI-BLAST hits up to E-value of 100, Nfunc(i) is the number of GO annotations for the sequence hit i, E_value(i) is the PSI-BLAST E_value for the sequence hit i, f j is the j-th annotation of the sequence hit i, and constant b takes value 2 (= log 10 100) to keep the score positive as retrieved sequences up to E_value of 100 are used (-log(E_value(i)) + b = -log10(100) + 2 = 0, when E_value = 100). The conditional probabilities P(f a |f j ) is to consider co-occurrence of GO terms in single sequence annotation, which is computed as the ratio of number of proteins co-annotated with GO terms f a and f j as compared with genes annotated with the term f j . To take into account the hierarchical structure of the GO, PFP transfers the raw score to the parental terms by computing the proportion of proteins annotated with f a relative to all proteins that belong to the parental GO term in the database. The score of a GO term computed as the sum of the directly computed score by Eqn. 1 and the ones from the parental propagation is called the raw score.
Extended Similarity Group (ESG) algorithm
ESG recursively performs PSI-BLAST searches from sequence hits obtained in the initial search from the target sequence, thereby performing multi-level exploration of the sequence similarity space around the target protein. Each sequence hit in a search is assigned a weight that is computed as the proportion of the -log(E_value) of the sequence relative to the sum of -log(E_value) from all the sequence hits considered in the search of the same level. This weight is assigned for GO terms annotating the sequence hit. The weights for GO terms found in the second level search are computed in the same fashion. Ultimately the score for a GO term is computed as the total weight from the two levels of the searches. The score for each GO term ranges from 0 to 1.0.
PSI-BLAST search is performed with a default setting with maximum of three iterations. Then the top hits with an E_value score better than 0.01 that have annotations is used for transferring annotation to the query sequence. The BLAST predictions were ranked according to -log(E_value)+2 for each of the prediction. In addition to the default BLOSUM62, which is the default amino acid similarity matrix, we also tested PSI-BLAST performance using BLOSUM45 and BLOSUM30.
We analyzed the performances of PFP, ESG, and PSI-BLAST in predicting the functional diversity of 19 moonlighting proteins. The 19 moonlighting proteins were taken from a review article . These proteins have two diverse and distinct functions. According to the verbal description of the two diverse functions of the proteins, we classified GO terms of these proteins in UniProt into four classes: Terms that belong to the major moonlighting function of the protein (Function 1); those which belong to the second moonlighting function (Function 2); terms which belong to both functions; and terms that do not belong to either of the functions. The list of the moonlighting proteins and their classified GO terms are made available at http://kiharalab.org/MoonlightingProtein_Dataset1/.
The raw score of PFP predictions has a large range of values. Up to 1000 GO term predictions were sorted by their raw score and plotted at an interval of 10. ESG predictions have a score range of 0 to 1.0, and 100 cutoffs are used within this range. PSI-BLAST predictions are ranked by -log(E_value)+2, and 100 score cutoffs are used from 4 (E_value of 0.01) to 45 (E_value of 10-43). To compare the prediction performances of the methods, we computed precision and recall. Precision is defined as TP/(TP+FP) and recall is defined as TP/(TP+FN), where TP and FP denote true and false positive, respectively, and FN denote false negative. All predictions by the three methods are propagated to the root of the GO hierarchy, so are the true annotations for the proteins.
Average Precision-Recall performance of PFP, ESG, and PSI-BLAST
In Figure 2B, the performance was evaluated where only the GO annotations for the two moonlighting functions (Function 1 and Function 2) are taken into account as the target annotations. The prediction performance for the moonlighting functions is essentially the same as those measured for the all GO term annotations (Figure 2A).
Recall at individual proteins
When all the GO terms are considered (Figure 3A), PFP showed higher recall than PSI-BLAST for almost all the cases (except for proteins 2 and 4, which are ties). ESG has similar recall of predictions as PSI-BLAST for proteins 14 and 17, slightly higher recall for proteins 6, 12 and 15 than PSI-BLAST (BLOSUM62), and a lower recall than PFP and PSI-BLAST for the rest of the proteins. Recall by PSI-BLAST improved when BLOSUM45 was used. In the head-to-head comparison against PFP, PSI-BLAST with BLOSUM45 showed a higher recall than PFP for eight proteins while PFP had a higher recall in ten cases (there was a tie). PSI-BLAST with BLOSUM30 failed to predict any GO terms above E_value of 0.01 for twelve proteins (Figure 3A). Overall, PFP and PSI-BLAST with BLOSUM45 showed higher recall than the rest of the methods. We see a similar performance pattern for the five methods when we consider only the GO terms belonging to moonlighting function 1 and function 2 of the proteins (Figure 3B). Again PSI-BLAST with BLOSUM45 showed comparable performance to PFP. PSI-BLAST with BLOSUM45 had a higher recall than PFP in seven cases while PFP was higher in ten cases (again there was a tie).
These results indicate that the PFP can find moonlighting GO terms that are missed by regular PSI-BLAST searches for quite a lot of cases. The strength of PFP is its coverage of a large number of sequences, by including weakly similar sequences into consideration for annotation transfer. On the other hand, ESG puts more weight on the consensus sequences that have strong similarity with the query protein among all the sequences that it encounters along multiple iterations. Thus, although ESG provides a higher precision on the predictions among all three methods (Figure 1), it fails to detect the functional variations in a number of cases. These results suggest that the functional diversity of the moonlighting proteins could be captured by taking weakly similar sequences into consideration among a broad range of similar sequences.
The identification of moonlighting functions of a protein is important for automatic function predictions. We have analyzed the performances of PFP, ESG, and PSI-BLAST in predicting the functional diversity of moonlighting proteins. PFP shows overall better performance in predicting diverse moonlighting functions as compared with PSI-BLAST and ESG. Recall by PSI-BLAST greatly improved when BLOSUM45 was used instead of BLOSUM62. This analysis indicates that considering weakly similar sequences in prediction enhances the performance of sequence based AFP methods in predicting functional diversity of moonlighting proteins.
This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R01GM075004, R01GM097528) and the National Science Foundation (EF0850009, IIS0915801, IOS1127027) and National Research Foundation of Korea Grant (NRF-2011-220-C00004). MC is supported by Bilsland Dissertation fellowship at Purdue University.
This article has been published as part of BMC Proceedings Volume 6 Supplement 7, 2012: Proceedings from the Great Lakes Bioinformatics Conference 2012. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcproc/supplements/6/S7.
- Hawkins T, Kihara D: Function prediction of uncharacterized proteins. Journal of bioinformatics and computational biology. 2007, 5: 1-30. 10.1142/S0219720007002503.View ArticlePubMed
- Hawkins T, Chitale M, Kihara D: New paradigm in protein function prediction for large scale omics analysis. Mol BioSyst. 2008, 4: 223-231. 10.1039/b718229e.View ArticlePubMed
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. Journal of molecular biology. 1990, 215: 403-410.View ArticlePubMed
- Pearson WR: Rapid and sensitive sequence comparison with FASTP and FASTA. Methods in enzymology. 1990, 183: 63-98.View ArticlePubMed
- Bru C, Courcelle E, Carrere S, Beausse Y, Dalmar S, Kahn D: The ProDom database of protein domain families: more emphasis on 3D. Nucleic acids research. 2005, 33: D212-D215.PubMed CentralView ArticlePubMed
- Finn RD, Mistry J, Schuster-Bockler B, Griffiths-Jones S, Hollich V, Lassmann T, et al: Pfam: clans, web tools and services. Nucleic acids research. 2006, 34: D247-D251. 10.1093/nar/gkj149.PubMed CentralView ArticlePubMed
- Hunter S, Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, et al: InterPro: the integrative protein signature database. Nucleic acids research. 2009, 37: D211-D215. 10.1093/nar/gkn785.PubMed CentralView ArticlePubMed
- Hawkins T, Luban S, Kihara D: Enhanced automated function prediction using distantly related sequences and contextual association by PFP. Protein Science. 2006, 15: 1550-1556. 10.1110/ps.062153506.PubMed CentralView ArticlePubMed
- Hawkins T, Chitale M, Luban S, Kihara D: PFP: automated prediction of gene ontology functional annotations with confidence scores using protein sequence data. Proteins: Structure, Function, and Bioinformatics. 2009, 74: 566-582. 10.1002/prot.22172.View Article
- Chitale M, Hawkins T, Park C, Kihara D: ESG: extended similarity group method for automated protein function prediction. Bioinformatics. 2009, 25: 1739-1745. 10.1093/bioinformatics/btp309.PubMed CentralView ArticlePubMed
- Martin D, Berriman M, Barton G: GOtcha: a new method for prediction of protein function assessed by the annotation of seven genomes. BMC Bioinformatics. 2004, 5: 178-194. 10.1186/1471-2105-5-178.PubMed CentralView ArticlePubMed
- Khan S, Situ G, Decker K, Schmidt CJ: GoFigure: automated Gene Ontology annotation. Bioinformatics. 2003, 19: 2484-2485. 10.1093/bioinformatics/btg338.View ArticlePubMed
- Wass MN, Sternberg MJ: ConFunc--functional annotation in the twilight zone. Bioinformatics. 2008, 24: 798-806. 10.1093/bioinformatics/btn037.View ArticlePubMed
- Engelhardt BE, Jordan MI, Muratore KE, Brenner SE: Protein molecular function prediction by Bayesian phylogenomics. PLoS Comput Biol. 2005, 1: e45-10.1371/journal.pcbi.0010045.PubMed CentralView ArticlePubMed
- Krishnamurthy N, Brown D, Sj+¦lander K: FlowerPower: clustering proteins into domain architecture classes for phylogenomic inference of protein function. BMC Evolutionary Biology. 2007, 7: S12-PubMed CentralView ArticlePubMed
- Storm CEV, Sonnhammer ELL: Automated ortholog inference from phylogenetic trees and calculation of orthology reliability. Bioinformatics. 2002, 18: 92-10.1093/bioinformatics/18.1.92.View ArticlePubMed
- Brown MPS, Grundy WN, Lin D, Cristianini N, Sugnet CW, Furey TS, et al: Knowledge-based analysis of microarray gene expression data by using support vector machines. Proceedings of the National Academy of Sciences. 2000, 97: 262-10.1073/pnas.97.1.262.View Article
- Eisen MB, Spellman PT, Brown PO, Botstein D: Cluster analysis and display of genome-wide expression patterns. Proceedings of the National Academy of Sciences. 1998, 95: 14863-10.1073/pnas.95.25.14863.View Article
- Gao L, Li X, Guo Z, Zhu M, Li Y, Rao S: Widely predicting specific protein functions based on protein-protein interaction data and gene expression profile. Sci China C Life Sci. 2007, 50: 125-134. 10.1007/s11427-007-0009-1.View ArticlePubMed
- Khatri P, Dr-âghici S: Ontological analysis of gene expression data: current tools, limitations, and open problems. Bioinformatics. 2005, 21: 3587-3595. 10.1093/bioinformatics/bti565.PubMed CentralView ArticlePubMed
- van Noort V, Snel B, Huynen MA: Predicting gene function by conserved co-expression. TRENDS in Genetics. 2003, 19: 238-242. 10.1016/S0168-9525(03)00056-8.View ArticlePubMed
- Gherardini PF, Helmer-Citterich M: Structure-based function prediction: approaches and applications. Briefings in functional genomics & proteomics. 2008, 7: 291-302. 10.1093/bfgp/eln030.View Article
- Marti-Renom M, Rossi A, Al-Shahrour F, Davis F, Pieper U, Dopazo J, et al: The AnnoLite and AnnoLyze programs for comparative annotation of protein structures. BMC Bioinformatics. 2007, 8: S4-PubMed CentralView ArticlePubMed
- Martin ACR, Orengo CA, Hutchinson EG, Jones S, Karmirantzou M, Laskowski RA, et al: Protein folds and functions. Structure. 1998, 6: 875-884. 10.1016/S0969-2126(98)00089-6.View ArticlePubMed
- Pal D, Eisenberg D: Inference of protein function from protein structure. Structure. 2005, 13: 121-130. 10.1016/j.str.2004.10.015.View ArticlePubMed
- Ponomarenko JV, Bourne PE, Shindyalov IN: Assigning new GO annotations to protein data bank sequences by combining structure and sequence homology. Proteins: Structure, Function, and Bioinformatics. 2005, 58: 855-865. 10.1002/prot.20355.View Article
- Thornton JM, Todd AE, Milburn D, Borkakoti N, Orengo CA: From structure to function: approaches and limitations. nature structural biology. 2000, 7: 991-994.View ArticlePubMed
- Chikhi R, Sael L, Kihara D: Real-time ligand binding pocket database search using local surface descriptors. Proteins: Structure, Function, and Bioinformatics. 2010, 78: 2007-2028. 10.1002/prot.22715.View Article
- Sael L, Kihara D: Binding ligand prediction for proteins using partial matching of local surface patches. International Journal of Molecular Sciences. 2010, 11: 5009-5026. 10.3390/ijms11125009.PubMed CentralView ArticlePubMed
- Sael L, Chitale M, Kihara D: Structure- and sequence-based function prediction for non-homologous proteins. Journal of Structural and Functional Genomics. Journal of Structural and Functional Genomics. 2012, Ref Type: In Press
- Brun C, Chevenet F, Martin D, Wojcik J, Guenoche A, Jacq B: Functional classification of proteins for the prediction of cellular function from a protein-protein interaction network. Genome Biol. 2003, 5: R6.1-R6.13.View Article
- Chua HN, Sung WK, Wong L: Exploiting indirect neighbours and topological weight to predict protein function from protein-protein interactions. Bioinformatics. 2006, 22: 1623-1630. 10.1093/bioinformatics/btl145.View ArticlePubMed
- Letovsky S, Kasif S: Predicting protein function from protein/protein interaction data: a probabilistic approach. Bioinformatics. 2003, 19 (Suppl 1): i197-i204. 10.1093/bioinformatics/btg1026.View ArticlePubMed
- Nariai N, Kolaczyk ED, Kasif S: Probabilistic protein function prediction from heterogeneous genome-wide data. PLoS One. 2007, 2: e337.1-e337.7.View Article
- Sharan R, Ulitsky I, Shamir R: Network-based prediction of protein function. Mol Syst Biol. 2007, 3: 88-100.PubMed CentralView ArticlePubMed
- Deng M, Tu Z, Sun F, Chen T: Mapping gene ontology to proteins based on protein-protein interaction data. Bioinformatics. 2004, 20: 895-902. 10.1093/bioinformatics/btg500.View ArticlePubMed
- Jeffery CJ: Moonlighting Proteins. Trends in Biochemical Sciences. 1999, 24: 8-11. 10.1016/S0968-0004(98)01335-8.View ArticlePubMed
- Jeffery CJ: Moonlighting Proteins: old proteins learning new tricks. TRENDS in Genetics. 2003, 19: 415-417. 10.1016/S0168-9525(03)00167-7.View ArticlePubMed
- Gancedo C, Flores CL: Moonlighting proteins in yeasts. Microbiology and Molecular Biology Reviews. 2008, 72: 197-210. 10.1128/MMBR.00036-07.PubMed CentralView ArticlePubMed
- Jeffery CJ: Proteins with neomorphic moonlighting functions in disease. IUBMB Life. 2011, 63: 489-494. 10.1002/iub.504.View ArticlePubMed
- Ovadi J: Moonlighting Proteins in Neurological Disorders. IUBMB Life. 2011, 63: 453-456. 10.1002/iub.491.View ArticlePubMed
- Huberts DHEW, Klei IJvd: Moonlighting proteins: an intriguing mode of multitasking. Biochim Biophys Acta. 2010, 1803: 520-525. 10.1016/j.bbamcr.2010.01.022.View ArticlePubMed
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