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

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

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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

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

Rita Casadio

BIOCOMPUTING GROUPUniversity of Bologna, Italy

Prediction of protein function from sequence analysis

Page 2: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

The “omic” era

Update: January 2010

Archaea : 74 speciesIn Progress:52

Bacteria: 973 species In Progress: 2266 species

Complete-23Draft Assembly–318

In Progress-359

Eukaryotic:

http://www.ncbi.nlm.nih.gov/genomes/static/gpstat.html

Genome Sequencing Projects:

Page 3: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

The Data Bases of Biological Sequences and Structures

>BGAL_SULSO BETA-GALACTOSIDASE Sulfolobus solfataricus.MYSFPNSFRFGWSQAGFQSEMGTPGSEDPNTDWYKWVHDPENMAAGLVSGDLPENGPGYWGNYKTFHDNAQKMGLKIARLNVEWSRIFPNPLPRPQNFDESKQDVTEVEINENELKRLDEYANKDALNHYREIFKDLKSRGLYFILNMYHWPLPLWLHDPIRVRRGDFTGPSGWLSTRTVYEFARFSAYIAWKFDDLVDEYSTMNEPNVVGGLGYVGVKSGFPPGYLSFELSRRHMYNIIQAHARAYDGIKSVSKKPVGIIYANSSFQPLTDKDMEAVEMAENDNRWWFFDAIIRGEITRGNEKIVRDDLKGRLDWIGVNYYTRTVVKRTEKGYVSLGGYGHGCERNSVSLAGLPTSDFGWEFFPEGLYDVLTKYWNRYHLYMYVTENGIADDADYQRPYYLVSHVYQVHRAINSGADVRGYLHWSLADNYEWASGFSMRFGLLKVDYNTKRLYWRPSALVYREIATNGAITDEIEHLNSVPPVKPLRH

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!

Page 4: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

From Genotype to Phenotype

…code for proteins...

>protein kinase

acctgttgatggcgacagggactgtatgctgatctatgctgatgcatgcatgctgactactgatgtgggggctattgacttgatgtctatc....

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

variabilityOver 20 millions of single mutations are

known in genes

….in methabolic pathways

Page 5: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

http://string.embl.de

STRING 8—a global view on proteins and theirfunctional 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

Page 6: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

One problem of the “omic era”:

Protein functional annotation

Page 7: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

The Protein Data Bankhttp://www.rcsb.org/pdb/home/home.do

No of Proteins with known structure: 57529

Page 8: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

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

Page 9: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

From the Protein Sequence to the Structure and Function space

Lesk A., 2004

Page 10: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

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

Page 11: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

What is protein function?

From the Protein Sequence to the Structure and Function space

Page 12: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

What is a function?

For enzymes: function can be defined on the basis of the catalysed molecular reaction.

e.g. aspartic aminotransferase (AST)

Page 13: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

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.

Page 14: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

Enzyme Commission (E.C.) classification

A hierarchical classification for enzymes

Page 15: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

EC 2.6 Transferring nitrogenous groupsEC 2.6.1TransaminasesEC 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

Page 16: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

Problems:

Isoformse.g How to differentiate the function of the cytoplasmic aspartate amintransferase from that of mitochondrial isoform?

Non enzymatic proteins

Page 17: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

The Ontologies • Cellular component • Biological process• Molecular function

GO function vocabulary: http://www.geneontology.org/

Page 18: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

Gene Ontology classification:The human cytoplasmic aspartate transaminase

GO:0005829

GO:0006533

GO:0004069

Page 19: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

One BIG problem of the “omic era”:

Protein functional annotation

Page 20: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

Sequence identity 40 %

Functional annotation in silico by homology search

Similar structure and function (??)

ADH1_SULSO ----------MRAVRLVEIGKP--LSLQEIGVPKPKGPQVLIKVEAAGVCHSDVHMRQGRFGNLRIVEADH_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/) sequenceAltschul et al., (1990) J Mol Biol 215:403-410Altschul 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

Page 21: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

Function annotation transfer from sequence through homology

Transfer by inheritance:

Page 22: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

http://www.uniprot.org/

Page 23: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

The annotation process at UniProt

PDB

Page 24: Rita Casadio BIOCOMPUTING GROUP University of Bologna, Italy Prediction of protein function from sequence analysis

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