Mapping and Visualization of Biochemical Mapping and Visualization of Biochemical NetworksNetworks

Stephen MichnickStephen Michnick

Département de biochimieDépartement de biochimieUniversité de MontréalUniversité de Montréal

Mapping and Visualization of Biochemical NetworksMapping and Visualization of Biochemical Networks

•Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks

•Theme II: A general approach to mapping biochemical Theme II: A general approach to mapping biochemical networks.networks.Design organization and paradoxes in PKB signalingDesign organization and paradoxes in PKB signaling •Theme III: Design Modularity and Evolution of NetworksTheme III: Design Modularity and Evolution of Networks

•Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells.networks in living cells.

C. J. Roberts et al. (2000) Science, 287:873-880.C. J. Roberts et al. (2000) Science, 287:873-880.

““Mapping” of Biochemical pathwaysMapping” of Biochemical pathways

Why Protein Interactions

• Importance :Importance :• Organizing-insulate Organizing-insulate

pathwayspathways• Most efficient machinery Most efficient machinery

for reactions and for reactions and regulation of reactionsregulation of reactions

• Forming complex Forming complex structures or enzymatic structures or enzymatic machinerymachinery

Source : Nature reviews, molecular cell biology, 2001, 2, 55-62

Mapping and Visualization of Biochemical NetworksMapping and Visualization of Biochemical Networks

•Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks

•Theme II: A general approach to mapping biochemical Theme II: A general approach to mapping biochemical networks.networks.Design organization and paradoxes in PKB signalingDesign organization and paradoxes in PKB signaling •Theme III: Design Modularity and Evolution of NetworksTheme III: Design Modularity and Evolution of Networks The Rosetta Hypothesis and organization of networksThe Rosetta Hypothesis and organization of networks

•Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells.networks in living cells.The MAP kinase pathways in The MAP kinase pathways in S. cerevisiaeS. cerevisiae..

Fr

actio

n fo

lded

Interaction-Assisted Protein FoldingInteraction-Assisted Protein Folding

PCA(Protein fragment Complementation

Assays)

A

A BB

Screening

Quantitation Pharmacology

Localization Trafficking

Genes are transfected into cells.

The cell makes proteins A and B which have reporter tags on either end (N- or C-terminal).

A/B complex formation reconstitutes the reporter and generates a signal.

Any gene, cell and reporter protein can be used.

Screening

Dynamics Pharmacology

Localization Trafficking

DHFR

V

I V

I I

III

IV. Ras-F[1]

II. Raf-F[2]

IV. Ras-F[1] + Raf-F[2]

II. Ras-F[1] + zip-F[2]

III. zip-F[1] + Raf-F[2]

N

HO2C

CO2H O

H

NH

HN

HN NH

NH2

O

H

NH

OH OH

O OPO3

H2N

ONH

OH OH

O OPO3

O

N

HO2C

CO2H O

H

NH

NHHN

HN NH

NH2

O

H

2- 2-

A Survival PCA based on Glycinamide Ribonucleotide (GAR)Transformylase

GAR fTase

AGPT GARTase

Time (secs)0 1000 2000 3000 4000 5000

0

100

200

300

400Well C1-Lm1 C2-Lm1 C3-Lm1 C4-Lm1C1-Lm2 C2-Lm2 C3-Lm2 C4-Lm2

Vmax 13,323 13,606 14,995 15,294R^2 0,751 0,638 0,666 0,346

-lactamase

GFP

A world of PCAsRemy, I., et al. (2002). Protein-protein interactions: A molecular cloning manual Cold Spring Harbor Laboratory Press. Chapter 25, 449-475.Michnick, S.W. (2001). Curr Opin Struct Biol, 11: 472-477.Michnick, S.W., et al. (2000) Methods in Enzymology. 328, 208-230.

PCA in Network MappingPCA in Network Mapping

•Molecular interactions are detected directly.Molecular interactions are detected directly.

•Genes are expressed in a relevant cellular context, in whichGenes are expressed in a relevant cellular context, in which components of the underlying pathway exist.components of the underlying pathway exist.

•Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways.detected, linking specific interactions to specific pathways.

•Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously.determined unambiguously.

-Rapamycin +Rapamycin

TOR-F[1,2]+

FKBP-F[3]

Raf-F[1,2] + Ras-F[3]

+Rapamycin -Rapamycin

Dihydrofolate reductase (DHFR)Dihydrofolate reductase (DHFR)

Fluorometirc assay based on binding of fluor-methotrexate to DHFR

PNAS Vol. 95,pp. 12141-12146

PNAS Vol. 96, pp. 5394-5399, May 1999

Mechanisms of action of chemical agents That act on specific pathways

• Quantification (amplitude)• pharmacological profiles

• Localization• intracellular trafficing

Spatial and temporal organization of the network

Pharmacological profiles

Mapping of Biochemical Networks with PCA

• Identification of Pathway Components• Establishing physical relationships between components.

I. Remy et S.W. Michnick, Proc. Natl. Acad. Sci. USA 98, 5394-5399 (2001)

Mapping of Signal Transduction Pathways controled by Insulin

The Integration of Biochemical Networks

Cell cycle andDNA repair

Cytokines

Growth factors

Cell suicide(Apoptose)

Pathogenic virus

Design, organization and paradoxes in PKB signalingDesign, organization and paradoxes in PKB signaling

D. P. Brazil and B. A. Hemmings (2001) TiBs, 26: 657-664

WortmanninPDK1

p70S6K

S6 protein4E-BP1

FRAP

Control of mRNA translation initiation

PP2A

PKB

PP

P PI3K

growth factors

RTK

Calyculin ARapamycin/FKBP

nutrients/amino acids

RTK/FRAP convergent signal transduction pathways

Convergence of RTK and FRAP PathwaysConvergence of RTK and FRAP Pathways

GCN4/GCN4

NT I S S+R S+W S+C

PDK1

PKBp70S6K

S6 protein4E-BP1

serum/insulin

Wortmannin

FRAP

nutrients/amino acids

Rapamycin/FKBP

RTK pathway

NT I S S+R S+W NT I S S+R S+W

NT I S S+R S+W NT I S S+R S+W

NT I S S+R S+W

Crosstalk between FRAP and PKB signalingCrosstalk between FRAP and PKB signaling

Crosstalk between FRAP and PKB signaling:Crosstalk between FRAP and PKB signaling:When is a pathway a pathway?When is a pathway a pathway?

Crosstalk between FRAP and PKB signaling:Crosstalk between FRAP and PKB signaling:hidden phenotypeshidden phenotypes

Mapping and ValidationMapping and Validation

PCA in Network MappingPCA in Network Mapping

•Molecular interactions are detected directly.Molecular interactions are detected directly.

•Genes are expressed in a relevant cellular context, in whichGenes are expressed in a relevant cellular context, in which components of the underlying pathway exist.components of the underlying pathway exist.

•Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways.detected, linking specific interactions to specific pathways.

•Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously.determined unambiguously.

P K B G F P [ 2 ]

C m

A m p

G F P [ 1 ] c D N A

- C m

+ A m p

1

2 3

4

5

6

A m p

G F P [ 1 ] c D N A

Directional cDNA library screening withDirectional cDNA library screening with the GFP PCAthe GFP PCA

•Size-fractionated directional cDNA library from human brainSize-fractionated directional cDNA library from human brain(10(1077 independent clones) independent clones)

•Transiently co-transfected COS-1 cellsTransiently co-transfected COS-1 cells

Systematic Screening of PKB (AKT1 and Systematic Screening of PKB (AKT1 and AKT2)AKT2)

PCA strategyA general approach to mapping pathways that allows for both definingthe organization of pathways. Can be used for pure description in an ontological sense (what, where and when) as well as more quantitativedescriptions.

The key to resolving specificity and organization of biochemical networksis CELL-BASED ASSAYS.

Mapping biochemical networks

PCA in Network MappingPCA in Network Mapping

•Molecular interactions are detected directly.Molecular interactions are detected directly.

•Genes are expressed in a relevant cellular context, in whichGenes are expressed in a relevant cellular context, in which components of the underlying pathway exist.components of the underlying pathway exist.

•Events induced by any pathway purturbation can be Events induced by any pathway purturbation can be detected, linking specific interactions to specific pathways.detected, linking specific interactions to specific pathways.

•Subcellular locations of protein complexes can be Subcellular locations of protein complexes can be determined unambiguously.determined unambiguously.

Mapping and Visualization of Biochemical NetworksMapping and Visualization of Biochemical Networks•Theme I: Design and Modularity in Biochemical Networks Theme I: Design and Modularity in Biochemical Networks

•Theme II: A general approach to mapping biochemical networks.Theme II: A general approach to mapping biochemical networks.The key to resolving specificity and organization of biochemical networksis CELL-BASED ASSAYS. PCA provides general approach to mapping pathways that allows for both defining the organization of pathways in terms of interactions and theirdynamics.

•Theme III: Design, Modularity and Evolution of NetworksTheme III: Design, Modularity and Evolution of Networks e.g. Rosetta Hypothesis: Predictions of interactions and functional e.g. Rosetta Hypothesis: Predictions of interactions and functional inferences provide crucial and testable hypotheses.inferences provide crucial and testable hypotheses.

•Theme IV: Towards genome-wide mapping of biochemical Theme IV: Towards genome-wide mapping of biochemical networks in living cells.networks in living cells.Mapping the interactions among all biochemical network components is theMapping the interactions among all biochemical network components is theonly way that we may ultimately know natures design. If there is one. only way that we may ultimately know natures design. If there is one.

Thanks to…Thanks to…

FinancingFinancingCIHR, NIH, The Burroughs-Wellcome Fund, HFSP

Génome Québec/Canada, Odyssey Pharmaceuticals Inc.

F.-X. Campbell-Valois Galia GhaddarAndré Galarneau Martin PrimeauIngrid Remy Jean-François TurcotteAlexis Vallée-Belisle Annie MontmarquetteDimitri Sans Geoffroy DenisHelen Yu Philippe NissaireJane Lamerdin Po Hein EarHugo Lavois Stéphane Le CromLuciano Vidali Kirill TarrasovNathalie Bourassa Ludovic Fuzellier