Protein function and classification - PowerPoint Presentation

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Protein function and classification

Hsin

-Yu Chang

www.ebi.ac.uk

Slide2

Classifying proteins into families and identifying

protein homologues can help scientists

to

characterise

unknown proteins

.

Slide3

Greider and Blackburn discovered telomerase in 1984 and were awarded Nobel prize in 2009. Which model organism they used for this study ?

1.

Tetrahymena

thermophila

2.

S

accharomyces

cerevisiae

3. Mouse

4. Human

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A single

T

etrahymena

thermophila

cell has 40,000 telomeres, whereas a human cell only has 92.1984Discovery of telomerase Greider and Blackburn1989Telomere hypothesis of cell senescenceSzostak1995 Clone hTR1995/1997 Clone hTERT1997 Telomerase knockout mouse1998 Ectopic expression of telomerase in normal human epithelial cells cause the extension of their lifespan

1999/2000…Telomerase/telomere dysfunctions and cancerGilson and Ségal-Bendirdjian, Biochimie, 2010.

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Can we identify human telomerase from T

etrahymea

protein sequence?

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Let’s pretend that human telomerase has not been identified and we only know the protein sequences of

Tetrahymena

telomerase. How can we find the human telomerase?

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BLAST (Basic Local Alignment Tool)

:

compares protein

sequences to sequence databases and calculates the statistical significance of matches.

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BLAST

Advantages:

Relatively

fast

User friendlyVery good at recognising similarity between closely related sequences Drawbacks:sometimes struggle with multi-domain proteinsless useful for weakly-similar sequences (e.g., divergent homologues)

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Using

Tetrahymena

telomerase protein sequences as a query in BLAST, you will find a few human proteins that have very low identity.

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Tetrahymena

and putative human telomerase (AAC51724.1) have poor protein sequence match.

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Can we presume this protein is a telomerase homologue from humans?

Can

we

find more information about

it before pursuing it further?

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Telomerase

ribonucleoprotein

complex - RNA binding domain

Reverse

transcriptase

domainSearch for protein signatures (such as domains) in AAC51724.1

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Plan

experiments and find out more!

AAC51724.1 shares 23% identity with

Tetrahymena

telomerase. It also contains the same domains as telomerase.

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But, where can we search for information about the protein domains?

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Structural

domains

Functional annotation of families/domains

Protein features 

(sites)

Hidden Markov Models

Finger printsProfilesPatterns

Protein databases that use signature approachesHAMAP

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Construction of protein signatures

Construction of a multiple sequence alignment (MSA) from characterised protein sequences.

Modelling the pattern of conserved amino acids at specific positions within a MSA.

Use these

models to infer relationships with the characterised sequences

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Three different protein signature approaches

Patterns

Single motif methods

Fingerprints

Multiple

motif methods

Profiles &

Hidden Markov Models (HMMs)Full alignment methodsSequence alignment

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Patterns

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Patterns

Sequence alignment

Motif

Pattern signature

[AC] – x -V- x(4) - {ED}

R

egular expression

PS00000Pattern sequencesALVKLISGAIVHESATCHVRDLSCCPVESTIS

Patterns are usually directed against functional sequence features such as: active sites, binding

sites, etc.

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PDOC00199

[SAG]-G-G-T-G-[SA]-G

Tubulin

signature

A conserved motif in tubulins

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Patterns

Advantages:

Strict

-

a pattern with very little variability and can produce highly accurate matchesDrawbacks:Simple but less flexible

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Fingerprints

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Fingerprints:

a

multiple motif approach

Sequence alignment

Motif 2

Motif 3

Motif 1

Define motifsFingerprint signaturePR00000

Motif

sequences

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

xxxxxx

Weight matrices

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Telomerase signature (PR01365)

Motif 1

Motif 2

Motif 3

Motif 4

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The significance of motif context

order

interval

Identify small conserved regions in proteins

Several motifs

 characterise family1

23

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G

ood

at modeling the often small differences between closely related proteins

D

istinguish

individual subfamilies within protein families, allowing functional characterisation of sequences at a high level of specificityFingerprintsAmino acids relatively well conserved across all chloride channel protein family members Amino acids uniquely conserved in chloride channel protein 3 subfamily members.

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Profiles & HMMs

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Sequence alignment

Entire domain

Define coverage

Whole protein

Use

entire alignment of domain or protein family

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxBuild model (Profile or HMMs)

Profile or HMM signature

Profiles & HMMs

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Profiles

Start with a multiple sequence alignment

Amino acids at each position in the alignment are scored according to the frequency with which they occur

Scores are weighted according to evolutionary distance using a BLOSUM matrix

Good at identifying homologues

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HMMs

Amino acid frequency at each position in the alignment and their transition probabilities are encoded

Insertions and deletions are also modelled

Start with a multiple sequence alignment

Very good at identifying evolutionarily distant homologues

Can model very divergent regions of alignment

Advantages

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Three different protein signature approaches

Patterns

Single motif methods

Fingerprints

Multiple

motif methods

Profiles &

HMMshidden Markov models Full alignment methods

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www.ebi.ac.uk/interpro

Fingerprints

Patterns

Profiles &

HMMs

hidden Markov models

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Structural

domains

Functional annotation of families/domains

Protein features 

(sites)

Hidden Markov Models

Finger printsProfilesPatterns

HAMAP

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The aim of InterPro

Family entry:

description, proteins matched and more information.

Domain entry:

description, proteins matched and more information.

Site entry: description, proteins matched and more information. Protein sequences

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What is

InterPro

?

I

nterPro

is an integrated sequence analysis resourceIt combines predictive models (known as signatures) from different databasesIt provides functional analysis of protein sequences by classifying them into families and predicting domains and important sites

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First release in 1999

11 partner databases

Add annotation to

UniProtKB

/TrEMBL Provides matches to over 80% of UniProtKBSource of >85 million Gene Ontology (GO) mappings to >24 million distinct UniProtKB sequences50,000 unique visitors to the web site per month> 2 million sequences searched online per month. Plus offline searches with downloadable version of softwareFacts about InterPro

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Signatures are provided by member databases

They are scanned against the

UniProt

database to see which sequences they match

Curators manually inspect the matches before integrating the signatures into

InterProInterPro signature integration processInterPro curators

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InterPro

signature integration process

Signatures representing the same entity are integrated

together

Relationships between entries are traced, where

possibleCurators add literature referenced abstracts, cross-refs to other databases, and GO terms

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http://www.ebi.ac.uk/interpro/

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Search using protein sequences

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Family

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Type

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InterPro entry types

Proteins share a common evolutionary origin, as reflected in their related functions, sequences or

structure. Ex.

T

elomerase family.

FamilyDistinct functional, structural or sequence units that may exist in a variety of biological contexts. Ex. DNA binding domain.Domain

Short sequences typically repeated within a protein. Ex. Tubulin binding repeats in microtubule associated protein Tau. Repeats

PTMActive Site

Binding

Site

Conserved

Site

Sites

Ex. Phosphorylation sites, ion binding sites, tubulin conserved site.

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Type

Name

Identifier

Contributing signatures

Description

GO termsReferences

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Type

Name

Identifier

Contributing signatures

Description

ReferencesRelationships

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InterPro

family and domain relationships

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Family relationships in InterPro

:

Interleukin-15/Interleukin-21 family

(

IPR003443)

Interleukin-15 (IPR020439)Interleukin-15Avian (IPR020451)Interleukin-15Fish(IPR020410)Interleukin-15Mammal(IPR020466)

Interleukin-21(IPR028151)

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Relationships

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InterPro

relationships: domains

Protein kinase-like

domain

Protein kinase

domain

Serine/threoninekinase catalyticdomain

Tyrosinekinase catalyticdomain

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Gene Ontology

Allow

cross-species and/or cross-database

comparisons

Unify the representation of gene and gene product attributes across species

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The Concepts in GO

1. Molecular Function

2. Biological Process

3. Cellular Component

protein kinase activity

insulin receptor activity

Cell cycleMicrotubule cytoskeleton organisation

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GO:0003677 DNA binding

GO:0003721

telomeric

template RNA

reverse transcriptase activityGO:0005634 Nucleus

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Search using keywords

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Summary

Protein

classification could help scientists to gain information about protein functions.

Blast is fast and easy to use but has its drawbacks.

Alternative approach: protein signature

databases build models (protein signatures) by using different methods (patterns, fingerprints, profile and HMMs).InterPro integrates these signatures from 11 member databases. It serves as a sequence analysis resource that classifies sequences into protein families and predicts important domains and sites.

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Why use

InterPro

?

Large amounts of manually curated data

35,634

signatures integrated into 25,214 entriesCites 38,877 PubMed publicationsLarge coverage of protein sequence spaceRegularly updated~ 8 week release scheduleNew signatures addedScanned against latest version of UniProtKB

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Caution

We need your feedback!

missing/additional references

reporting problems

requests

InterPro is a predictive protein signature database - results are predictions, and should be treated as such InterPro entries are based on signatures supplied to us by our member databases....this means no signature, no entry!EBI support page.And one more thing…..

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The InterPro Team:

Amaia Sangrador

Craig

McAnulla

Matthew

Fraser

Maxim ScheremetjewSiew-Yit YongAlex MitchellSebastien Pesseat

SarahHunterGiftNukaHsin-YuChangwww.ebi.ac.uk/interproTwitter: @InterProDB

Slide66

Database

Basis

Institution

Built from

Focus

URLPfamHMMSanger InstituteSequence alignment

Family & Domain based on conserved sequencehttp://pfam.sanger.ac.uk/Gene3DHMMUCLStructure alignmentStructural Domainhttp://gene3d.biochem.ucl.ac.uk/Gene3D/SuperfamilyHMM

Uni. of BristolStructure alignmentEvolutionary domain relationshipshttp://supfam.cs.bris.ac.uk/SUPERFAMILY/SMARTHMMEMBL HeidelbergSequence alignmentFunctional domain annotationhttp://smart.embl-heidelberg.de/

TIGRFAM

HMM

J. Craig Venter Inst.

Sequence alignment

Microbial Functional Family Classification

http://www.jcvi.org/cms/research/projects/tigrfams/overview/

Panther

HMM

Uni. S. California

Sequence alignment

Family functional classification

http://www.pantherdb.org/

PIRSF

HMM

PIR, Georgetown, Washington D.C.

Sequence alignment

Functional classification

http://pir.georgetown.edu/pirwww/dbinfo/pirsf.shtml

PRINTS

Fingerprints

Uni. of Manchester

Sequence alignment

Family functional classification

http://www.bioinf.manchester.ac.uk/dbbrowser/PRINTS/index.php

PROSITE

Patterns & Profiles

SIB

Sequence alignment

Functional annotation

http://expasy.org/prosite/

HAMAP

Profiles

SIB

Sequence alignment

Microbial protein family classification

http://expasy.org/sprot/hamap/

ProDom

Sequence clustering

PRABI :

Rhône-Alpes Bioinformatics Center

Sequence alignment

Conserved domain prediction

http://prodom.prabi.fr/prodom/current/html/home.php

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Thank you!

www.ebi.ac.uk

Twitter: @

emblebi

Facebook: EMBLEBI

YouTube: EMBLMedia

Slide68

The

BLOSUM

(

BLO

cks

SUbstitution Matrix) matrix is a substitution matrix used for sequence alignment of proteins. BLOSUM matrices are used to score alignments between evolutionarily divergent protein sequences.

Slide69

The

BLOSUM

(

BLO

cks

SUbstitution Matrix) matrix is a substitution matrix used for sequence alignment of proteins. BLOSUM matrices are used to score alignments between evolutionarily divergent protein sequences.