27041, week 02

27041, Week 02Review of Week 01

27041, Introduction to Systems Biology2 CBS, Department of Systems Biology

The human genome sequencing project (HGP)


Systems Biology and emergent properties


Different model representations

Chen et al., Mol. Biol. Cell., 2004


Model

Generation

Systems Biology at a glance

Parts List

YER001W

YBR088C

YOL007C

YPL127C

YNR009W

YDR224C

YDL003W

YBL003C

…

YDR097C

YBR089W

YBR054W

YMR215W

YBR071W

YBL002W

YNL283C

YGR152C

…

• Sequencing

• Gene knock-out

• Microarrays

Interactions

• Protein-Protein interactions

• Protein-DNA interactions

• Subcellular Localization

Dynamics

• Microarrays

• Proteomics

• Metabolomics


Levels of organization


Networks in Molecular Biology

Barabasi & Oltvai, Nature Reviews, 2004

• Protein-Protein interactions

• Protein-DNA interactions

• Genetic interactions

• Metabolic reactions

• Text mining interactions

• Association Networks

• Etc.

Protein-protein interactions


Protein-protein interaction data is accumulating


30-40% Orphan Human Proteins


Protein-protein interactions: guilty-by-association

Protein-protein interaction network

Red protein: Unknown function

Yellow protein: RNA splicing

White protein: Other functional role


Classical methods for identifying protein-protein interactions• Co-immunoprecipitation

• Affinity chromatography / crosslinking

• Fluorescence energy transfer (FRET)

• Dominant negatives– Over-expression of a mutant form of protein X causes loss of function

despite the presence of native proteins. One explanation is that X forms a multimer that sequesters functional proteins.


High-throughput methods for measuring interactions• Phage display• SOS recruitment assay• Split-ubiquitin system• Dual-bait system• 2-hybrid• Protein complementation assay (PCA)• Co-immunoprecipitation• Protein arrays• ChIP-Chip/Chip-Seq


Yeast Two Hybrid (Y2H) Method• One problem with phage display and other in vitro technologies is that the

measured binding may not actually occur.• Y2H assays interactions in vivo.• Uses property that transcription factors generally have separable

transcriptional activation (AD) and DNA binding (DBD) domains.• A functional transcription factor can be created if a separately expressed

AD can be made to interact with a DBD.• A protein ‘bait’ B is fused to a DBD and screened against a library of

protein ‘preys’, each fused to a AD.


An activating transcription factor:

1. Binds to DNA using a DNA-binding domain (DBD)

2. Recruits the transcriptional machinery using a transcriptional activation domain (AD)

Transcription factor

27041, Introduction to Systems Biology17 CBS, Department of Systems BiologyCausier, Mass spectrometry Reviews, 2004

Y2H assays interactions in vivo.

Uses property that transcription factors generally have separable transcriptional activation (AD) and DNA binding (DBD) domains.

A functional transcription factor can be created if a separately expressed AD can be made to interact with a DBD.

A protein ‘bait’ B is fused to a DBD and screened against a library of protein ‘preys’, each fused to a AD.

Yeast Two-Hybrid Method

Animation!

http://www.youtube.com/watch?v=OKxRx0ctrl0


Y2H goes global


Y2H Random Library a Approach

Bait B1 X Genomic fragment library

Protein

Selected fragments (prey)

Interacting Domain


692 Interactions

Uetz et al. : 6144 prey X 5345 baits

Two large-scale Y2H studies: Uetz et al.

Uetz et al, Nature 2000


841 Interactions

Ito et al. : ~ 6200 prey X ~ 6200 baits

Two large-scale Y2H studies: Ito et al.

Ito et al., PNAS 2001


841 Interactions

Ito et al. : ~ 6200 prey X ~ 6200 baits

692 Interactions

Uetz et al. : 6144 prey X 5345 baits

141551 700

Overlap

Reproducibility in Y2H


Protein Complementation Assay (PCA)


Affinity Purification followed by Mass Spectrometry

(AP/MS)


General strategy

Affi

nity

Purifi

catio

n S

tep

http://www.youtube.com/watch?v=Hb791WsC78s


Affinity Chromatography

Load affinity column with antigen (or antibody)

Designed to purify a protein from a complex mixture

Proteins sieve through matrix of affinity beads

Proteins react with different affinities


Affinity Chromatography (2)

Wash off proteins that do not bind Elute and collect bound proteins


General strategy

Affi

nity

Purifi

catio

n S

tep

Mass

Spect

rom

etr

y S

tep


Mass spectrometry• Mass spectrometers consist of three essential parts:

– Ionization source: Converts peptides into gas-phase ions (MALDI + ESI)

– Mass analyzer: Separates ions by mass to charge (m/z) ratio (Ion trap, time of flight, quadrupole)

– Ion detector: Current over time indicates amount of signal at each m/z value

For details on Proteomics, see Aebersold & Mann, Nature, 2003


Mass spectrometry

Aebersold & Mann, Nature 2003


Two large-scale mass spec experiments

Gavin et al. Ho et al.

589 protein complexes

(232 distinct)

Gavin et al. : 1167 baits

3617 interactions among 1578

proteins

Ho et al. : 725 baits



proteins

Ho et al. : 725


proteins

Gavin et al. : 1167 baits

1983007 3419

Overlap

1151052 (454)

610 (493)

Overlap in baits

Reproducibility in AP/MS


Recent HTP (binary) PPI networks

Y2H by Yu et al. 2008 : 2018 proteins, 2930 interactions

PCA by Tarassov et al. 2008 : 1124 proteins, 2770 interactions

Scoring protein-protein interactions


Topology based scoring of interactions

Low confidence (4 unshared interaction partners)

High confidence (1 unshared interaction partners)

A B C

Yeast two-hybrid

Low confidence (rarely purified together)

High confidence (often purified together)

Complex pull-downs

D

de Lichtenberg et al., Science 2005


Issues with Y2H• Strengths

– high sensitivity (transient & permanent PPIs)– takes place in vivo– independent of endogenous expression

• Weaknesses: False positive interactions– Auto-activation– ‘sticky’ prey– detects “possible interactions” that may not take place under real

physiological conditions– may identify indirect interactions (A-C-B)

• Weaknesses: False negatives interactions – Similar studies often reveal very different sets of interacting proteins

(i.e. False negatives)– may miss PPIs that require other factors (e.g. ligands, proteins, PTMs)


Affinity Purification & mass spectrometryStrengths

• high specificity

• well suited for detecting permanent or strong transient interactions (complexes)

• detects real, physiologically relevant PPIs

Weaknesses

• less suited for detecting weaker transient interactions (low sensitivity)

• may miss complexes not present under the given experimental conditions (low sensitivity)

• may identify indirect interactions (A-C-B)


Filtering by subcellular localization

de Lichtenberg et al., Science, 2005

27041, week 02

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