1. Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

2. The “omic” era Update: January 2010 Archaea : 74 species In Progress:52 Bacteria: 973 species In Progress: 2266 species Complete-23 Draft Assembly–318 In Progress-359 Eukaryotic: http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html Genome Sequencing Projects:

3. The Data Bases of Biological Sequences and Structures >BGAL_SULSO BETA-GALACTOSIDASE Sulfolobus solfataricus. MYSFPNSFRFGWSQAGFQSEMGTPGSEDPNTDWYKWVHDPENMAAGLVSG DLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPQNFDE SKQDVTEVEINENELKRLDEYANKDALNHYREIFKDLKSRGLYFILNMYH WPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDE YSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSRRHMYNIIQAHARAYDGI KSVSKKPVGIIYANSSFQPLTDKDMEAVEMAENDNRWWFFDAIIRGEITR GNEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGCERNSVS LAGLPTSDFGWEFFPEGLYDVLTKYWNRYHLYMYVTENGIADDADYQRPY YLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFSMRFGLLKVDYNT KRLYWRPSALVYREIATNGAITDEIEHLNSVPPVKPLRH GenBank: 108,431,692 sequences 106,533,156,756 nucleotides SwissProt: 514,212 sequences 180,900,945 residues PDB:60,654 structures membrane proteins <2% NR(*): 10,381,779 sequences 3,542,056,219 residues Update: January 2009 (*) CDS translations+PDB+SwissProt+PIR+PRF  35,5 HGE!

4. From Genotype to Phenotype … code for proteins... >protein kinase acctgttgatggcgacagggactgtatgctgatct atgctgatgcatgcatgctgactactgatgtgggg gctattgacttgatgtctatc.... Genes in DNA... (about 30,000 in the human genome) Proteins interact … proteins correspond to functions... …when they are expressed From 5000 to 10000 proteins per tissue …with different effects depending on variability Over 20 millions of single mutations are known in genes ….in methabolic pathways

5. http://string.embl.de STRING 8—a global view on proteins and their functional interactions in 630 organisms- Jensen et al., 2009, Nucleic Acids Research, Vol 37. The Human Interactome in STRING 22,937 proteins and 1,482,533 interactions

6. One problem of the “omic era”: Protein functional annotation

7. The Protein Data Bank http://www.rcsb.org/pdb/home/home.do No of Proteins with known structure: 57529

8. SCOP: Structural Classification of Proteins Domains are hierarchically classified: - class - fold: proteins with secondary structures in same arrangement with the same topological connections - superfamily: structures and functional features suggest a common evolutionary origin - family: proteins with identities ≥30%; with identities <30% but with similar structures and functions

9. From the Protein Sequence to the Structure and Function space Lesk A., 2004

10. PDB Sequence comparison Sequence Identity (%) 0% 30% 100% Fold recognition Machine-learning aided alignment Threading New Folds Ab initio and de novo modelling Machine-learning prediction of structural features From the Protein Sequence to the Structure space

11. What is protein function? From the Protein Sequence to the Structure and Function space

12. What is a function? For enzymes: function can be defined on the basis of the catalysed molecular reaction. e.g. aspartic aminotransferase (AST)

13. In biochemistry, a transaminase or an aminotransferase is an enzyme that catalyzes a type of reaction between an amino acid and an α-keto acid. Specifically, this reaction (transamination) involves removing the amino group from the amino acid, leaving behind an α-keto acid, and transferring it to the reactant α-keto acid and converting it into an amino acid. The enzymes are important in the production of various amino acids, and measuring the concentrations of various transaminases in the blood is important in the diagnosing and tracking many diseases. Transaminases require the coenzyme pyridoxal-phosphate, which is converted into pyridoxamine in the first phase of the reaction, when an amino acid is converted into a keto acid. Enzyme-bound pyridoxamine in turn reacts with pyruvate, oxaloacetate, or alpha- ketoglutarate, giving alanine, aspartic acid, or glutamic acid, respectively. The presence of elevated transaminases can be an indicator of liver damage.

14. Enzyme Commission (E.C.) classification A hierarchical classification for enzymes

15. EC 2.6 Transferring nitrogenous groups EC 2.6.1Transaminases EC 2.6.1.1 Aspartate transaminase Other name(s): glutamic-oxaloacetic transaminase; glutamic-aspartic transaminase; transaminase A; AAT; AspT; 2- oxoglutarate-glutamate aminotransferase; aspartate α-ketoglutarate transaminase; aspartate aminotransferase; aspartate-2-oxoglutarate transaminase; aspartic acid aminotransferase; aspartic aminotransferase; aspartyl aminotransferase; AST; glutamate-oxalacetate aminotransferase; glutamate-oxalate transaminase; glutamic-aspartic aminotransferase; glutamic-oxalacetic transaminase; glutamic oxalic transaminase; GOT (enzyme); L-aspartate transaminase; L-aspartate-α-ketoglutarate transaminase; L-aspartate-2-ketoglutarate aminotransferase; L-aspartate- 2-oxoglutarate aminotransferase; L-aspartate-2-oxoglutarate-transaminase; L-aspartic aminotransferase; oxaloacetate-aspartate aminotransferase; oxaloacetate transferase; aspartate:2-oxoglutarate aminotransferase; glutamate oxaloacetate transaminase Systematic name: L-aspartate:2-oxoglutarate aminotransferase

16. Problems: Isoforms e.g How to differentiate the function of the cytoplasmic aspartate amintransferase from that of mitochondrial isoform? Non enzymatic proteins

17. The Ontologies Cellular component Biological process Molecular function GO function vocabulary : http://www.geneontology.org/

18. Gene Ontology classification: The human cytoplasmic aspartate transaminase GO:0005829 GO:0006533 GO:0004069

19. One BIG problem of the “omic era”: Protein functional annotation

20. Sequence identity  40 % Functional annotation in silico by homology search Similar structure and function (??) ADH1_SULSO ----------MRAVRLVEIGKP--LSLQEIGVPKPKGPQVLIKVEAAGVCHSDVHMRQGRFGNLRIVE ADH_CLOBE ----------MKGFAMLGINKLG---WIEKERPVAGSYDAIVRPLAVSPCTSDIHTVFEGA------- ADH_THEBR ----------MKGFAMLSIGKVG---WIEKEKPAPGPFDAIVRPLAVAPCTSDIHTVFEGA------- ADH1_SOLTU MSTTVGQVIRCKAAVAWEAGKP--LVMEEVDVAPPQKMEVRLKILYTSLCHTDVYFWEAKG------- ADH2_LYCES MSTTVGQVIRCKAAVAWEAGKP--LVMEEVDVAPPQKMEVRLKILYTSLCHTDVYFWEAKG------- ADH1_ASPFL ----MSIPEMQWAQVAEQKGGP--LIYKQIPVPKPGPDEILVKVRYSGVCHTDLHALKGDW------- Sequence comparison is performed with alignment programs BLAST, Psi-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) sequence Altschul et al., (1990) J Mol Biol 215:403-410 Altschul et al., (1998) Nucleic Acids Res. 25:3389-3402 Methods for similarity searches : Pfam (http://pfam.wustl.edu/hmmsearch.shtml) sequence/structure Bateman et al., (2000) Nucleic Acids Research 28:263-266

21. Function annotation transfer from sequence through homology Transfer by inheritance:

22. http://www.uniprot.org/

23. The annotation process at UniProt PDB

24. Open problems of “inheritance through homology “ Not all UniProt files are GO annotated The optimal threshold value of sequence identity for function transfer is not known Proteins contain multiple domains Proteins can share common domains and not necessarily the same function In proteins different combination of shared domains lead to different biological roles