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Transcript of Mapping and Visualization of Biochemical Networks Stephen Michnick Département de biochimie...
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..
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
O
NH
OH OH
O OPO3
O
N
HO2C
CO2H O
H
NH
NH
HN
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-Lm1
C1-Lm2 C2-Lm2 C3-Lm2 C4-Lm2
Vmax 13,323 13,606 14,995 15,294
R^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)
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
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
PCA strategy
A 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…
FinancingFinancing
CIHR, NIH, The Burroughs-Wellcome Fund, HFSPGénome 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