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

2. The “omic” era Genome Sequencing Projects: Archaea : 74 species
In Progress:52
Bacteria: 973 species
In Progress: 2266 species
Complete-23
Draft Assembly–318
In Progress-359
Eukaryotic:
Update:
January 2010

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

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

5. The Human Interactome in STRING
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 No of Proteins with known structure: 57529
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. From the Protein Sequence to the Structure space
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
What is protein function?

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 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. GO function vocabulary:
The Ontologies
Cellular component
Biological process
Molecular function
GO function vocabulary:

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

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

20. Functional annotation in silico by homology search
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
Sequence identity  40 %
Similar structure and function (??)
Methods for similarity searches:
BLAST, Psi-BLAST ( sequence
Altschul et al., (1990) J Mol Biol 215:
Altschul et al., (1998) Nucleic Acids Res. 25:
Pfam ( sequence/structure
Bateman et al., (2000) Nucleic Acids Research 28:

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

23. PDB
The annotation process at UniProt

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