1. Network Clustering 7000 Yeast interactions among 3000 proteins

2. The Interactome: the Next ‘omic Step Interactome Proteome Transcriptome Genome

3. protein-gene interactions protein-protein interactions PROTEOME GENOME Citrate Cycle METABOLISM Bio-chemical reactions Bio-Map

4. A Parts List Approach to Bike Maintenance What are the shared parts (bolt, nut, washer, spring, bearing), unique parts (cogs, levers)? What are the common parts -- types of parts (nuts & washers)? How many roles can these play? How flexible and adaptable are they mechanically? Where are the parts located?

6. A Protein may interact with: –Other proteins –Nucleic Acids –Small molecules Protein Interactions

7. Genome: 30.000 genes Transcriptome: 40-100.000 mRNAs Proteome: 100-400.000 proteins >1.000.000 interactions Dimensions of Information Complexity Genomics vs. Post-Genomics Human Genome Human Proteome Transcripts Protein Interaction 10 5 10 6

9. Comprehensive Analysis of Complex Protein Structures in the Cell Total Protein Characterization Protein Identification: What’s there Post Translational Modifications: Regulation Quantification: Dynamics Multiprotein Complex/Organelle

10. Protein-Protein Interactions: The “Interactome” Experimental methods: Mass Spec, yeast 2-hybrid system, microarrays,… Computational techniques: phylogenic profiles, sequence analysis,… 2 challenges: - find which proteins interact (the partners) - find which residues participate in the interactions

14. Figure 1. General principle of far-Western analysis. The ProFound Far-Western Protein:Protein Interaction Kits follow the non-radiolabeled bait path.

15. Finding Protein Partners

16. Approaches

17. Predicting Protein-Protein Interactions Genome-based approach Proximity of genes on chromosome Genes that appear near each other on a chormosome are often expressed together. They may interact (need confirmation from biology, or annotation) Example: operons Gene 1 Gene 2 Gene 3

18. Global view of protein family interaction networks for 146 genomes

19. Structure DB Predicted Human Interactome 

21. Binding Domain Activation Domain Yeast 2-hybrid system Two hybrid proteins are generated with transcription factor domains Both fusions are expressed in a yeast cell that carries a reporter gene whose expression is under the control of binding sites for the DNA-binding domain

22. Binding Domain Activation Domain Binding Domain Prot1 Activation Domain Prot2 Yeast 2-hybrid system

23. Binding Domain Activation Domain Binding Domain Prot1 Activation Domain Prot2 Reporter Gene Promoter Region Binding Domain Prot1 Activation Domain Prot2 mRNA Yeast 2-hybrid system If Prot1 and Prot2 interact: Interaction of bait and prey proteins localizes the activation domain to the reporter gene, thus activating transcription. Since the reporter gene typically codes for a survival factor, yeast colonies will grow only when an interaction occurs.

24. Principle of the Yeast Two-Hybrid (Y2H) System Reporter Gene DNA Binding Domain Bait Activation Domain Prey ( No Reporter Gene Activity ) Scenario B: Proteins X and Z do not Interact Readout: No growth of yeast colonies Protein Z Protein X DNA Reporter Gene DNA Binding Domain Protein X Bait Protein Y Activation Domain Prey Scenario A: Proteins X and Y do Interact Readout: Yeast colonies grow DNA

25. Yeast 2-hybrid system In words: A transcription factor is split into 2 domains 2 hybrid proteins are designed, each containing one of the two proteins that are tested If the two proteins interact, the two domains from the transcription factor will interact, causing expression of a (detectable) reporter gene The reporter can be: - essential, in which case the yeast colony dies if the 2 proteins do not interact - reversely, the reporter gene can be attached to a green fluorescent protein Unfortunately, the rate of false positive is high (estimated > 45%)

27. Bait Design Fragments of proteins representing folded domains are often more effective than the full-length protein in identifying physiologically relevant interactions If the domain structure of a given bait protein was already established, the specific baits were designed to represent one or more folded domains. For cases in which domain structure was not available, a variety of secondary structure prediction algorithms were used to predict domains and thus direct bait design. Baits were designed to cover the entire protein, with several overlapping fragments, as not all baits will work effectively.

30. Caveats! Y2H data requires validation by secondary assay –Protein complementation –Pull-downs Didn’t observe source library in data analysis Didn’t analyze all bait and prey coordinates to map sites of interaction –Cant assume multi-protein complexes since proteins may be competing for same site of interaction –This level of analysis requires more sophisticated computational approach

32. Protein-Protein Interactions (1) Affinity Purification (2) Phage Display (3) Yeast 2-hybrid tag affinity support protease cleavage PAGE MS no interaction positive interaction transcription Protein Chips GST-fusion proteins GST-fusion proteins ORF-activation domain fusion cell lysate phage cDNA display library ORF-binding domain fusion wash unbound proteins wash unbound proteins nutritional selection elute bound proteins amplify phage particles grow up surviving yeast colonies MS repeat and/or sequence repeat and/or sequence

33. General Strategy for Protein Characterization Purification/Enrichment 1-DE2-DESolution Identification Identification Sequencing Sequencing PeptidesProteinor Measurement Analysis Mass Spectrometry

34. Comprehensive Analysis of Protein-Protein Interactions Co-immunoprecipitation ProteolysisLC/MS/MSLC/LC/MS/MS Identification of Protein Components Identification of Modifications Dynamics of components and modifications Multiprotein Complex Protein Interaction Chromatography Agarose Protein GST Agarose Ig-G Agarose C L TEV TAP-Tagged Proteins Cell Biology/ Genetics

35. Fishing for Partners Recomb. Protein Histidine-Tag Biotinyl-Tag GST-Protein Flag-Protein Specific Ab Ni +2 Streptavidin Glutathione Specific mAB Protein Bait Ligand

37. GST “X” “Y” GST pull-down assay Sepharose GSH Sepharose GSH GST “Y”

38. GST pull-down assay Sepharose GSH GST “X” “Y” Run Western blot Input GST-X GST anti-Y

39. GST pull-down assay mix and incubate express GST-fusion protein in E.coli pGEX GST gene X prepare protein extract from brain

40. GST pull-down assay GST-fusion protein GST alone

41. Protein Bait Specific bait ligand on beads Bait linked to the support Specific Interactions Cellular extract incubated in batch with the immobilised bait Unbound proteins Elution of bait and partner(s) SDS-PAGE separation Fishing for Partners Strategy MS Identification

42. |BamH1 | |EcoR1 ||SmaI |SalI | XhoI | NotI |... ATC GAA GGT CGT GGG ATC CCC AGG AAT TCC CGG GTC GAC TCG AGC GGC CGC...... TAG CTT CCA GCA CCC TAG GGG TCC TTA AGG GCC CAG CTG AGC TCG CCG GCG...... Ile Glu Gly Arg Gly Ile Pro Arg Asn Ser Arg Val Asp Ser Ser Gly Arg... | Factor Xa | Engineered protease site allows removal of fusion partner

43. His 6 Tag add 6 consecutive His to either end binds metals Addition of a few residues should have minimal effect on recombinant protein Epitope Tag 6-12 amino acids mAb for detection or purification

44. Immunoprecipitation affinity purification based on isolation of Ag-Ab complexes analyze by gel electrophoresis initially based on centrifugation of large supramolecular complexes [high] and  equal amounts isolation of Ag-Ab complexes protein A-agarose protein G-agarose Bacterial proteins that bind IgG (Fc): protein A (Staphylococcus aureus) protein G (Streptococcus) binds more species and subclasses

45. Typical IP Protocol 1. Solubilize antigen usually non-denaturing SDS + excess of TX100 2. Mix extract and Ab 3. Add protein G-agarose, etc 4. Extensively wash 5. Elute with sample buffer 6. SDS-PAGE 7. Detection protein stain radioactivity G agarose

46. Protein identification In situ digestion Peptide extraction MALDI-MS

47. MS-MS of Peptide Mixtures LC MS MS/MS

48. 766.4868 836.4362 904.4685 997.5691 1209.5710 1221.7473 1570.6782 1697.8175 1800.9144 1890.9643 2061.1366 0 10000 20000 30000 40000 Counts 800 800 1000 1000 1200 1200 1400 1400 1600 1600 1800 1800 2000 2000 Mass (m/z) 1406.7220 … PPGTGKTLLAK AVANESGANFISVK FYVINGPEIM... Molecular Weight Fragmentation

49. Leu-Gly-Val-Asp-Glu-

50. 1410.6 Database IIGHFYDDWCPLK SPAFDSIMAETLK AFDSLPDDIHEK GGILAQSPFLIIK

51. What can Biacore do for you? SPR (surface plasmon resonance) technology enables real-time detection and monitoring of biomolecular events and provides quantitative information on: 1.Specificity – how specific is the binding between two moelcules? 2.Concentration – how much of a given molecule is present and active? 3.Kinetics – what is the rate of association/dissociation? 4.Affinity – how strong is the binding? 5.Binding partners - provide identification of binding targets by linking SPR to MS

52. The BIAcore uses an optical method (surface plasmon resonance) to measure changes in refractive index. Macromolecules binding to a sensor surface leads to an increase in refractive index near the surface. How the BIAcore works

53. A BIAcore sensorgram

54. MicroArray Analysis of thousands of proteins at one time. Many different types –Antibody arrayed - detect many proteins –Proteins arrayed - detect interacting proteins –Proteins arrayed - detect interacting small molecules –Etc.

55. Protein:protein interactions

57. Examples of protein-ligand combinations used in protein microarrays

59. Gene name Interaction Knowledge from proteomics studies is limited by our inability to analyze efficiently large data sets Proteomics studies highlight the extreme complexity of interactions in a genomic scale. Proteomics is facing the challenge of analyzing large and highly complex and very noisy data sets. Bioinformatics is integrated in proteomics projects to mine data and is becoming more and more important.

60. CD19 Actin Cytoskeleton PDK1 PI3K  (p110) Btk WISH CAP Dbl CamK II NdkB cdc42 CD22 Fyn SOS2

61. CD19 Btk Actin Cytoskeleton SOS2 WISH CAP Dbl CamK II NdkB cdc42 PDK1 CD22 Fyn PI3K  (p110) 19070197 6755399 6671538 4633514 8567325 13542677 20894430 26326968 3064262 Sam68 Protein 4.1G AIP Cbl-b

65. Databases of experimental protein interaction data –SPIN-PP: http://honiglab.cpmc.columbia.edu/SPIN/main.html http://honiglab.cpmc.columbia.edu/SPIN/main.html (existing protein-protein interfaces in the PDB) –MIPS: http://mips.gsf.de/proj/yeast/CYGD/interaction/ http://mips.gsf.de/proj/yeast/CYGD/interaction/ (protein-protein interactions in saccharomyces cerevisae) –InterAct: http://www.ebi.ac.uk/intact/index.htmlhttp://www.ebi.ac.uk/intact/index.html (protein interactions from literature curation) –DIP: http://dip.doe-mbi.ucla.edu/http://dip.doe-mbi.ucla.edu/ –BIND: http://bind.ca/http://bind.ca/ Protein Interactions

66. Protein Interactomics