1. Genome Biology and Biotechnology 10. The proteome Prof. M. Zabeau Department of Plant Systems Biology Flanders Interuniversity Institute for Biotechnology (VIB) University of Gent International course 2005

2. Summary ¤Protein interactome –Yeast two-hybrid protein interaction mapping ¤Proteome –Isolation of protein complexes ¤Multilevel functional genomics –Combination of phenome analysis protein interaction mapping

3. Functional Maps or “-omes” proteins ORFeome Localizome Phenome Transcriptome Interactome Proteome Genes or proteins Genes Mutational phenotypes Expression profiles Protein interactions 1 2 3 4 5 n DNA InteractomeProtein-DNA interactions “Conditions” After: Vidal M., Cell, 104, 333 (2001)Vidal M., Cell, 104, 333 (2001) Cellular, tissue location

4. Basic Concept of the Yeast Two-hybrid System ¤Eukaryotic transcription factors –activate RNA polymerase II at promoters by binding to upstream activating DNA sequences (UAS) ¤Basic structure of eukaryotic transcription factors –The DNA binding and the activating functions are located in physically separable domains The DNA-binding domain (DB) The activation domain (AD) –The connection between DB and AD is structurally flexible ¤Protein-protein interactions can reconstitute a functional transcription factor –by bringing the DB domain and the AD domain into close physical proximity Reprinted from:Vidal M. and Legrain P., Nucleic Acids Res. 27: 919 (1999)

5. Yeast two-hybrid system ¤‘Architectural blueprint’ for a functional transcription factor –DB-X/AD-Y, where X and Y could be essentially any proteins from any organism UAS Upstream Activating Sequence Selectable marker gene Gal4 transcription-activation domain Gal4 DNA binding domain bait prey DB AD X Y

6. Yeast two-hybrid system ¤The yeast two-hybrid system allows –Genetic selection of genes encoding potential interacting proteins without the need for protein purification System is to isolate genes encoding proteins that potentially interact with DB-X (referred to as the ‘bait’) in complex AD-Y libraries (referred to as the ‘prey’) –Limitations of the system include False positives: clones with no biological relevance False negatives: Failure to identify knowm interactions –Stringent criteria must be used to evaluate both the specificity and the sensitivity of the assay Reprinted from:Vidal M. and Legrain P., Nucleic Acids Res. 27: 919 (1999)

7. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development ¤Landmark paper presents –First demonstration of large-scale two-hybrid analysis for protein interaction mapping in C. elegans starting with 27 proteins involved in vulval development in C. Elegans Walhout et al, Science 287: 116 (2000)

8. Experimental Approach ¤Start from known genes in vulval development –Used Recombinational cloning to introduce ORFs of 29 known genes involved in vulval development into two-hybrid vectors ¤Matrix two-hybrid experiment with 29 ORFs –Each DB-vORF/AD-vORF pairwise combination was tested for protein-protein interactions by scoring two-hybrid phenotypes ¤Exhaustive two-hybrid screen –using 27 vORF-DB fusion proteins as baits to select interactors from a AD-Y cDNA library sequenced the selected clones: interaction sequence tag (IST) Reprinted from:Walhout et al, Science 287: 116 (2000)

9. Construction of DB and AD Fusions by Recombinational Cloning Phage lambda excision: Integrase, IHF & Exisionase DNA binding domain Activation domain Reprinted from: Walhout et al, Science 287: 116 (2000)Walhout et al, Science 287: 116 (2000) DB-ORF fusionsAD-ORF fusions

10. Matrix of Two-hybrid Interactions Between the vORFs Reprinted from:Walhout et al, Science 287: 116 (2000)

11. Interaction Sequence Tag (IST) screening Reprinted from:Walhout et al, Science 287: 116 (2000)

12. Results ¤Matrix two-hybrid experiment with 29 ORFs –~ 50% (6 of 11) of the interactions reported were detected Two novel potential interactions were identified –Typically the yeast two-hybrid system will detect ~50% of the naturally occurring interactions ¤Two-hybrid screen –Identified 992 AD-Y encoding sequences –ISTs corresponded to a total 124 different interacting proteins 15 previously known –Provides a functional annotation for 109 predicted genes Reprinted from:Walhout et al, Science 287: 116 (2000)

13. Validation of Potential Interactions ¤Conservation of interactions in other organisms –If X' and Y' are orthologs of X and Y, respectively X/Y conserved interactions are referred to as "interologs" Reprinted from:Walhout et al, Science 287: 116 (2000)

14. Validation of Potential Interactions ¤Systematic clustering analysis –closed loop connections between vORF- encoded proteins X interacts with Y, Y interacts with Z, Z interacts with W, and so on (X/Y/Z/W/...) Reprinted from:Walhout et al, Science 287: 116 (2000) Mutations with Similar phenotypes

15. Conclusions ¤Demonstrated the feasibility of generating a genome- wide protein interaction maps –Two-hybrid screens are Simple sensitive amenable to high-throughput –Feasible using the C. elegans ORFeome ¤Y2H detects approximately 50% of the interactions –provides a useful coverage of biologically important interactions Reprinted from:Walhout et al, Science 287: 116 (2000)

16. A Comprehensive Analysis of Protein–protein Interactions in Saccharomyces Cerevisiae ¤Landmark paper presents –The first Large scale high throughput mapping of protein-protein interactions between ORFs predicted in S. cerevisiae using –Two complementary yeast two-hybrid screening strategies Two-hybrid array of 6.000 hybrid proteins High-throughput library screen Uetz et al., Nature 403: 623 (2000)

17. The two-hybrid array screening ¤Two-hybrid array of 6.000 hybrid proteins comprises –Haploid yeast colonies derived from ~6,000 yeast ORFs fused to the Gal4 activation domain (AD) –The two-hybrid array contained on 16 plates of 384 colonies ¤Matrix screen for interactions –192 different Gal4 DB ORF hybrids were mated to the two-hybrid array –192 two-hybrid array screens were performed in duplicate Each yielded 1–30 positives But only ~ 20% were reproduced in the duplicate screen ¤Putative interacting partners identified –87/192 DB hybrids yielded putative protein–protein interactions –Identified 281 interacting protein pairs Reprinted from: Uetz et al., Nature 403: 623 (2000)

18. The two-hybrid array screening Reprinted from: Uetz et al., Nature 403: 623 (2000) Positive control: 6,000 haploid yeast Gal4 activation domain - ORF fusions Two-hybrid positives from a mating with a Gal4 DNA-binding domain - ORF fusion 16 microassay plates

19. High-Throughput Library Screen ¤Used a library Made by pooling ORF-AD fusions –Each ORFs was fused separately to a gal4 activation domain –ORF-AD fusions were pooled to form an activation-domain library Advantage over traditional cDNA libraries is the uniform presentation of each ORF ¤Protein interactions were screened by –mating the 6.000 DNA-binding domain hybrids in duplicate to the activation domain library –817 yeast ORFs (15%) yielded protein–protein interactions –Identified 692 interacting protein pairs 68% of the interactions were identified multiple times Reprinted from: Uetz et al., Nature 403: 623 (2000)

20. Results of the Systematic Two-Hybrid Screens ¤The matrix array screens –gave more interactors 45% of the 192 proteins in the array screens yielded interactions –are much more labour- and material-intensive limits the number of screens that can be performed Full matrix would require testing 6.000 * 6.000 = 36.000.000 interactions! ¤The library screens gave –fewer interactors 8% of the proteins tested in the library screens yielded interactions –a much higher throughput Reprinted from: Uetz et al., Nature 403: 623 (2000)

21. Analysis of the protein-protein interactions ¤The analysis reveals –Interactions that place unknown proteins into a biological context –Novel interactions between proteins involved in the same biological function –Novel interactions that connect biological functions into larger cellular processes

22. Interactions involving unknown proteins Reprinted from: Uetz et al., Nature 403: 623 (2000)

23. Interactions Between Proteins in the RNA Splicing Complex Reprinted from: Uetz et al., Nature 403: 623 (2000) Interactions are consistent with the crystallographic data

24. Interaction Connecting two different Complexes Reprinted from: Uetz et al., Nature 403: 623 (2000) spindle checkpoint complexmicrotubule checkpoint complex

25. Analysis of Interologs Reprinted from: Uetz et al., Nature 403: 623 (2000) Yeast Human

26. Conclusions ¤The two-hybrid array approach is feasible –for systematic genome-wide analysis of protein interactions ¤The large scale mapping of protein-protein interactions reveals –many new interactions between proteins –that protein interactions should be viewed as potential interactions that must be confirmed independently –This conclusion is supported by the fact that the results of different screens only partially overlap Reprinted from: Uetz et al., Nature 403: 623 (2000)

27. A Map of the Interactome Network of the Metazoan C. elegans ¤Paper presents –Large scale mapping of protein-protein interaction in C. elegans using yeast two-hybrid screens with a subset of metazoan- specific proteins identified > 4000 interactions –Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome map contains 5500 interactions Li et. al., Science, 303, 540-543 (2004)

28. Worm Interactome map Reprinted from: Li et. al., Science, 303, 540-543 (2004) Phylogenetic classes Eukaryotic Multi cellular Worm

29. A Protein Interaction Map of Drosophila melanogaster ¤Paper presents –a two-hybrid–based protein-interaction map of the fly proteome by screening 10,623 ORFs against cDNA libraries to produce a draft map of 7048 proteins and 20,405 interactions. Computational rating of interaction confidence produced –a high confidence interaction network of 4679 proteins and 4780 interactions showing two levels of organization a short-range organization, presumably corresponding to multiprotein complexes a more global organization, presumably corresponding to intercomplex connections Giot et. al., Science, 302, 1727-1736 (2003)

30. The fly protein- interaction map: Protein family/human disease orthologs Reprinted from: Giot et. al., Science, 302, 1727-1736 (2003)

31. The fly protein- interaction map: Subcellular localization Reprinted from: Giot et. al., Science, 302, 1727-1736 (2003)

32. Towards a proteome-scale map of the human protein–protein interaction network ¤Paper presents –First step towards a systematic and comprehensive analysis of the human interactome using stringent, high-throughput yeast two-hybrid system to test pairwise interactions among the products of 8,100 currently available Gateway-cloned open reading frames Rual et. al., Nature 424: 1173-1178 (2005)

33. Reprinted from: Rual et. al., Nature 424: 1173-1178 (2005) High-throughput yeast two-hybrid pipeline ¤Stringent test –Second test using GAL1::HIS3 and GAL1::lacZ –Reduces the number of false positives ¤Detected 2,800 interactions

34. Reprinted from: Rual et. al., Nature 424: 1173-1178 (2005) Overlap of CCSB-HI1 with literature data ¤Compared the overlap between –Observed interactions –Interactions reported in the literature ¤Conclude that the CCSB- HI1 data set contains 1% of the human interactome –Human interactome is estimated at 200.000 to 300.000 interactions.

35. Reprinted from: Rual et. al., Nature 424: 1173-1178 (2005) Interaction network of disease-associated CCSB-HI1 proteins ¤The human interactome will further –the understanding of human health and disease ¤Illustrated by –The network of disease- associated proteins (green nodes) EWS protein

36. Functional Maps or “-omes” proteins ORFeome Localizome Phenome Transcriptome Interactome Proteome Genes or proteins Genes Mutational phenotypes Expression profiles Protein interactions 1 2 3 4 5 n DNA InteractomeProtein-DNA interactions “Conditions” After: Vidal M., Cell, 104, 333 (2001)Vidal M., Cell, 104, 333 (2001) Cellular, tissue location

37. Proteome Analysis ¤Large scale and comprehensive analysis of the proteome has so far not been feasible –Lack of suitable and sensitive protein fractionation methods 2-D gels are limited to a few 1000 proteins only – the most abundant –Protein characterization is slow and laborious Despite enormous improvements in mass spectrometry, the characterization of individual proteins remains the bottleneck –Level of proteome characterization to date is in the order of a few 1000 proteins at best Represents 5% to 25% of the proteome ¤Tandem affinity purification (TAP) technology constitutes an important breakthrough –Fast and reliable method of protein purification

38. A generic protein purification method for protein complex characterization ¤Paper presents –a generic procedure to purify protein complexes under native conditions using tandem affinity purification (TAP) tag procedure –Using a combination of high-affinity tags for purification Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999)

39. Reprinted from: Kumar A. and Snyder M., Nature 415, 123(2002) Tag-based Characterization of protein complexes

40. High-affinity Tags ¤High-affinity protein tags –Must allow efficient recovery of proteins present at low concentrations ProtA tag: two IgG-binding units of protein A of S. aureus –released from matrix-bound IgG under denaturing conditions CBP tag: calmodulin-binding peptide –released from the affinity column under mild conditions ¤Tandem affinity purification (TAP) tag –A fusion cassette encoding both the ProtA tag and the CBP tag Separated by a specific TEV protease recognition sequence which allows proteolytic release of the bound material under native conditions Reprinted from: Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999)

41. Tandem affinity purification (TAP) tag Reprinted from: Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999) ProtA CBP

42. The TAP Purification Procedure Reprinted from: Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999) ProtA affinity purification step CBP affinity purification step TEV protease cleavage step

43. Advantage of the Two-step Procedure ¤Purification of U1 snRNP –Single-step affinity purification yields a high level of contaminating proteins –Tow-step affinity purification yields highly specific purification with very low background Reprinted from: Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999)

44. Functional organization of the yeast proteome by systematic analysis of protein complexes ¤Landmark paper presents –Large-scale application of the TAP technology for a systematic analysis of multiprotein complexes from yeast Generated gene-specific TAP tag cassettes by PCR Insert TAP cassettes by homologous recombination at the 3' end of the genes to generate fusion proteins in their native location Purified protein assemblies from cellular lysates by TAP –Separate purified assemblies by denaturing gel electrophoresis –Digest individual bands by trypsin Analyze peptides by MALDI–TOF MS to identify the proteins using database search algorithms Gavin et. al., Nature 415, 141 (2002)

45. Reprinted from: Gavin et. al., Nature 415, 141 (2002) The Gene Targeting Procedure TAP tag gene-specific cassette

46. Large-scale Analysis of Protein Complexes ¤Experimental outline –Started with a selection of 1,739 genes 1,143 genes representing eukaryotic orthologues 596 genes nonorthologous set –Generated 1,167 strains expressing tagged proteins to detectable levels –Analyzed 589 protein complexes Comprising 418 different orthologues –Generated 20,946 samples for mass spectrometry Identified 16,830 proteins –Characterized a total of 232 protein complexes Comprising 1,440 distinct proteins ~ 25% of the ORFs in the genome Reprinted from: Gavin et. al., Nature 415, 141 (2002)

47. Purification and Identification of TAP Complexes Reprinted from: Gavin et. al., Nature 415, 141 (2002)

48. Sensitivity and Specificity of the Approach ¤Very efficient large-scale purification and identification of protein complexes –78% of the 589 purified complexes have associated proteins –The remaining 22% showing no interacting proteins May not form stable or soluble complexes The TAP tag may interfere with complex assembly or function ¤Complexes are stable and show the same composition when purified with different entry points –Example: the polyadenylation machinery, responsible for eukaryotic messenger RNA cleavage and polyadenylation Identified 12 of the 13 known components Identified 7 new components

49. Reprinted from: Gavin et. al., Nature 415, 141 (2002) The Polyadenylation Protein Complex new components of the polyadenylation complex

50. Composition of the Polyadenylation Complex Reprinted from: Gavin et. al., Nature 415, 141 (2002) protein tagged for affinity purification <

51. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Reliability of the TAP Method ¤High sensitivity –identify proteins present at 15 copies per cell ¤High reproducibility –70% of the proteins are detected in independent purifications ¤Low background –The background comprises highly expressed proteins Identified 17 contaminant proteins (heat-shock and ribosomal proteins) ¤Limitations –18% of the tagged essential genes gave no viable strains The carboxy-terminal tagging can impair protein function

52. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Organization of the purified assemblies into complexes ¤589 purified complexes characterized –245 complexes corresponded to 98 known multiprotein complexes in yeast –242 complexes correspond to 134 new complexes ¤In total 232 annotated TAP complexes are identified –102 proteins showed no detectable association with other proteins

53. Number Of Proteins Per Complex Reprinted from: Gavin et. al., Nature 415, 141 (2002) Average of 12 proteins per complex

54. Functional Classification Of The Complexes Reprinted from: Gavin et. al., Nature 415, 141 (2002) wide functional distribution of complexes

55. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Protein Complexes are Dynamic ¤Complexes are not necessarily of invariable composition –Using distinct tagged proteins as entry points to purify a complex Core components can be identified as invariably present Regulatory components may be present differentially ¤Dynamic complexes: e.g. signaling complexes –The interactions of a signalling enzyme may be sufficiently strong to allow the detection of distinct cellular complexes They may be diagnostic for the role of these enzymes in different cellular activities

56. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Higher-order Organization of The Proteome Map ¤Most complexes are linked together –Complexes belonging to the same functional class often share components mRNA metabolism, cell cycle, protein synthesis and turnover, intermediate and energy metabolism ¤Shared components linking complexes into a network –The network connections reflect physical interaction of complexes common architecture, localization or regulation –Relationships between complexes suggests integration and coordination of cellular functions –The more connected a complex, the more central its position in the network

57. Reprinted from: Gavin et. al., Nature 415, 141 (2002) cell cycle signalling Transcription DNA maintenance chromatin structure RNA metabolism protein synthesis and turnover cell polarity and structure intermediate and energy metabolism membrane biogenesis and traffic The Yeast Protein Complex Network protein and RNA transport

58. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Protein Complexes Have a Similar Composition in Yeast and Human

59. Reprinted from: Gavin et. al., Nature 415, 141 (2002) Conclusions ¤The paper clearly demonstrates the merits of the TAP technology for –characterizing protein complexes from different compartments, including low-abundance and large complexes –TAP data and yeast two-hybrid assay data show only a very small overlap The two methodologies address different aspects of protein interaction and are complementary ¤The TAP analysis provides an outline of the eukaryotic proteome as a network of protein complexes –The human–yeast orthologous proteome represents core functions for the eukaryotic cell Orthologous proteins are often responsible for essential functions

60. Recommended reading ¤Yeast two-hybrid interaction mapping –The yeast two-hybrid system Vidal M. and Legrain P., Nucleic Acids Res. 27: 919 (1999) –Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development –Walhout et al, Science 287: 116 (2000)Walhout et al, Science 287: 116 (2000) ¤Purification of protein complexes –Gavin et. al., Nature 415, 141 (2002)Gavin et. al., Nature 415, 141 (2002)

61. Further reading ¤Protein Interaction Mapping –Interaction map of yeast Uetz et al., Nature 403: 623 (2000) –Interaction map C. elegans Li et. al., Science, 303, 540-543 (2004)Li et. al., Science, 303, 540-543 (2004) –Interaction map Drosphila Giot et. al., Science, 302, 1727-1736 (2003)Giot et. al., Science, 302, 1727-1736 (2003) ¤Purification of protein complexes –Tandem affinity purification (TAP) tag method Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999)Rigaut et. al., Nat. Biotechnol. 17, 1030 (1999)