Genome: 30.000 genes Transcriptome: 40-100.000 mRNAs Proteome: 100-400.000 proteins

1. Protein Interaction 106 Human Proteome 105 Transcripts Human Genome Genome:30.000 genes Transcriptome:40-100.000 mRNAs Proteome:100-400.000 proteins Interactome: >1.000.000 interactions

2. Identification, production and purification Identification methods Purification strategies Single gene expression and in vitro assembly: Co-expression techniques

3. Bait Prey Identification methods • In vitro • Co-immunoprecipitation • Far-western analysis • GST-pull down assays • Protein arrays • TAP-MS Bait – Prey model • In vivo • Yeast two-hybrid system • Phage display Physical interaction between protein binding domains

4. Co-immunoprecipitation (Eric Phizicky, et al. 1995)

5. GST gene X pGEX GST-pull down assays prepare protein extract from tissue express GST-fusion protein GST-fusion protein GST alone mix and incubate GST-affinity purification SDS-PAGE and MS analysis

6. Far-Western Based on western blotting but uses an non-antibody molecule for detection Analysis on protein-protein interactions Prey recognizes the bait after gel eletrophoresis and transfer

7. Protein array Basic idea identical as in cDNA arrays - HTP technology - substrate (protein, Ab, TFs,..) bound on the surface of the array - sample is introduced to array: binding - detection and analysis Used for various purposes - screening/profiling - protein/protein interaction - protein/small molecule interactions - kinase/substrate interaction Technique exists, some proteins are more sticky than others, normalization..

8. Antibody micro-arrays Functional protein array What is needed ? - expression library - methods for HTP protein purification - array technology

9. Cy3-labelled probes

10. Tandem Affinity Purification-Mass Spectrometry • Rapid purification of complexes without prior knowledge of the complex composition, activity, or function • Ability to purify low abundant proteins/protein complexes • Fusion of the TAP tag to the target protein • Complex retrieval from tissue culture • Large-scale studies

11. TAP-MS (Arnaud Droit, et al. 2005) (Guillaume Rigaut,et al. 1999)

12. TAP-TAG in yeast (Gavin, 2002)

13. TAP-TAG in Mammalian cells

16. G418 = geneticin (aminoglycoside) blocks protein synthesis Close but not identical to gentamicin A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.

21. TransfectionMethods

22. QUELLES INTERACTIONS SONT/NE SONT PAS IDENTIFIEES PAR LE TAP-TAG ? • Interactions permanentes  complexe • Faux-positifs 20% (estimation) • Complexes identifiés en conditions quasi physiologiques • (cellules, animaux entiers) • Variation de la composition des complexes (par exemple, en fonction d'un stimulus, de l'activation d'une voie…) • Pas d'interactions transitoires • Pb: conformation • gènes essentiels • très petites protéines • protéines non solubles

23. Peptide Mass finger printing Proteins separated by SDS/PAGE are stained and an excised gel slice containing the protein digested (with eg trypsin) and the resulting peptides masses determined using MALDI Searches performed using theoretical peptides digests from known protein databases. Proteins are generally identified if they are visualized by Coomassie brilliant blue staining and they are present in the database. Silver staining may also be employed, but sequence coverage may be limited.

25. Nano-LC / ESI-ION-TRAP MS ESI-QTOF MS MALDI-TOF MS ESI-Q-TRAP MALDI-TOF/TOF MS Mass spectrometers

26. Digester/spotter MALDI Multiprobe 2 (Packard/Perkin) • Data processing (LIMS) Robotics – Sample preparation • Spot picker proteineer SP (Bruker/Gilson)

27. ARN rapporteur ARN rapporteur DA DA ARN rapporteur Proie Y Proie Y ARN rapporteur ARN rapporteur ARN rapporteur ARN rapporteur ARN rapporteur Gène rapporteur AppâtX DF Yeast two-hybrid system • Detecting protein-protein interactions in yeast • Transcriptional regulator system • “prey”-”bait” model :fusion proteins with a transcriptional activating domain (AD, prey), a DNA-binding domain (DBD, bait) • Term “two-hybrid” derives from these two chimeric proteins. • Most commonly used method for large scale, high-throughput identification of potential protein-protein interactions

28. ARN rapporteur ARN rapporteur DA ARN rapporteur ARN rapporteur ARN rapporteur Proie Y ARN rapporteur ARN rapporteur ARN rapporteur Gène rapporteur Appât X DF Appât X DF Appât C DF Appât A DF Appât B DF La proie collante (interagit avec un très grand nombre d'appâts) L'appât auto-activateur (activation de la transcription en absence d'interacteur) QUELLES INTERACTIONS SONT IDENTIFIEES PAR UN CRIBLE DOUBLE HYBRIDE ? • Interactions permanentes • Interactions transitoires dont les interactions enzyme-substrat (ex: 10 à 40 % des interactions kinase-substrat). • Interactions qui n'existent pas physiologiquement •  Faux-positifs

29. QUELLES INTERACTIONS NE SONT PAS IDENTIFIEES PAR UN CRIBLE DOUBLE HYBRIDE ? - Les interactions impliquant des protéines qui présentent des problèmes: - structuraux (repliement) - stabilité - toxicité - mauvaise localisation (protéines membranaires) - modification post-traductionnelle • Estimation: 80 à 90 % des interactions sensées exister, n'auraient pas été détectées  Faux-négatifs  évolution des méthodes de double-hybrides pour palier à ces problèmes.

30. Surface display technologies • Linkage of genotype to phenotype • Selection. • Directed evolution • M13, 900 nm long; diameter 9 nm • 5 proteins, 6.5 kb ss DNA • After enterring the cell, replicates its genome • and synthetase its coat proteins which are secreted • to its outer membrane • - DNA is extruded into the protein capsules

32. Affinity selection • - Biospanning • - Ligands immobilized on a solid surface • Phage libraries (1010 clones) incubated with the support to allow binding • Unbound phages are washed away • Bound phages are eluted (low or high pH)

33. Applications • Limited size fo the libraries: • Antibody generation • Identification of peptide ligands • Enhance stability • Improve catalysis • …..

35. Databases BIND http://bond.unleashedinformatics.com/ DIP http://dip.doe-mbi.ucla.edu/ MINT http://mint.bio.uniroma2.it/HomoMINT/ STRING http://string.embl.de//

36. Données de la littérature Expériences Tap-TAG Spoke model Matrix model STRING (P. Bork, EMBL) Search Tool for the Retrieval of Interacting Proteins

38. Complexes involved in transcription: Yeast interaction networks derived from String centered around the mediator complex Med1 • Stable complexes: • TFIIH • mediator • RNA polII • TFIID • SAGA • Transient complexes: • Associated proteins • Shared subunits: TFIID/SAGA THIIH SAGA mediator TFIID SnfI/Cat1 PAF GTF RNA polymerase

40. Kin28/cdk7 TFIIH subunit Cell cycle regulation Transcription, Functional Physical TFIIH

41. TFIIH Nucleolar proteins U3 snoRNA Ribosomal Leucine synthesis pathway