Brief Introduction of Yeast Two-hybrid System

1. Brief Introduction of Yeast Two-hybrid System Xiaoqin Lai Dec. 2, 2004

3. Dimensions of Information ComplexityGenomics vs. Post-Genomics Protein Interaction 106 Human Proteome 105 Transcripts Human Genome Genome: 30.000 genes Transcriptome:40-100.000 mRNAs Proteome: 100-400.000 proteins >1.000.000 interactions

4. What is proteomics? • Proteome: Expressed protein complement of a cell • Proteomics: – “The systematic study of the many and diverse properties of proteins in a parallel manner with the aim of providing detailed descriptions of the structure, function and control of biological systems in health and disease” Patterson & Aebersold Nat Genetics 33:S311 (2003) – Just about everything that has something to do with proteins and is “high-throughput” in some way

5. Proteomics tasks a. Interaction mapping b. Expression profiling c. Activity profiling d. Modification profiling e. Localization profiling

6. Protein-Protein Interaction • Protein-protein interactions are one of the most important regulatory mechanisms in cells. • Most of the cellular processes are coordinated by specific protein interactions. • By figuring the functions of non-annotated proteins, biologist will better understand molecular mechanism of biological events

7. Yeast-two hybrid Ito et al. PNAS (2000, 2001), Uetz et al. Nature (2000) (yeast) Giot et al. Science (2004) (Drosophila) Li S., et al (2004) Science (C. Elegans) Biochemical purifications Yeast Gavin et al. (2002) Nature Ho et al. (2002) (HMS-PCI) Nature Krogan et al. (2004) Mol Cell Protein Chip In silico predictions Methods Co-occurrence (Phylogenetic profiling) Neighborhood (Operon) Fusion (Rosetta) See review by Osterman and Overbeek in Curr Opin Chem Biol. (2003) mRNA-co-expression Eisen et al., PNAS (1998) Marcotte Nature (1999) Synthetic lethals Tong (2004) Others Pull down, Far western,Co-IP Phage display Fluorescence resonance energy transfer Surface plasma resonance transfer Protein interaction analysis methods

8. Interactions MIPS DIP YPD Intact (EBI) BIND/ Blueprint GRID MINT Prediction server Predictome (Boston U) Plex (UTexas) STRING (EMBL) Protein complexes MIPS YPD Reference databases

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

16. Yeast Two Hybrid Assay Standard genetic screen for physical protein-protein interactions • Benefits: – Simple – Inexpensive – Scalable and automatable

17. Comparison of protein-protein interaction screens Differences between individual methods and reference sets

18. energy production aminoacid metabolism other metabolism translation transcription transcriptional control protein fate cellular organization transport and sensing stress and defense genome maintenance cellular fate/organization uncharacterized Interaction density E G 0 10 M Interaction pro 1000 possible P T B F O A R D C U E G M P T B F O A R D C U Interaction density

19. Functional biases

20. Comparison

21. Conclusions • The overlap between the individual methods is surprisingly small • Different methods complement each other • Individual methods are not exhausted • Single experimental methods can be as reliable as combined sets • Integration [ Bader, G. and Hogue, C. (2002) Nat. Biot.] [Kemmeren H., et al. (2002) Mol. Cell] [Von Mering C., Krause, R., et al. (2002) Nature] [Edwards et al. (2002) Trends Genet. ]

22. Number of citations in Medline that describe use of the yeast two-hybrid system in 1989-1999 is growing exponentially. Total number of citations to date approaches 3000. Year

27. 酵母双杂交系统 常用的DNA结合结构域: GAL4（1-147） LexA（E.coli 转录抑制因子） 常用的转录激活结构: GAL4（768-881） B42（ E.coli） VP16（疱疹病毒）

28. 工作原理 • 酵母或大肠杆菌的转录因子(Gal4、LexA)的DNA结构域可以将一个与其融合的蛋白质分子X(诱饵)带至报告基因的上游激活序列(UAS)，并与之结合； • 与转录因子的转录激活结构域(来自Gal4或VP16)融合的蛋白质分子Y(靶蛋白)，可通过其与X蛋白质的相互作用，将激活结构域带至报告基因的调控区； • DNA结合结构域和转录激活结构域在空间上的靠近，重建了转录因子的功能，激活了下游报告基因的表达，表现为酵母可以在特定的缺省培养基上生长，或在有底物X-gal时，形成蓝色菌落

29. 三个基本组成部分 • 表达诱饵蛋白的载体，诱饵即我们感兴趣的蛋白， 它和DNA结合结构域融合。 • 表达靶蛋白的载体，靶蛋白可以是一个已知的蛋白，也可以是cDNA或基因组文库编码的蛋白。靶蛋白和转录激活结构域融合。 • 一个或多个报告基因(如控制氨基酸合成的基因、大肠杆菌的lacZ基因等)，位于DNA结合结构域识别的调控区的下游。

35. Applications of YTH • 高灵敏度地检测蛋白－蛋白的相互作用 • 确定蛋白相互作用的结构域或重要活性位点 • 寻找与靶蛋白相互作用的新蛋白 • 寻找具有药物治疗作用的小分子肽 • 寻找控制蛋白相互作用的化合物 • 蛋白相互作用图谱的绘制

36. Sources of yeast two-hybrid system

37. Matchmaker Yeast Two-hybrid System 3 DNA-BD : Amino acids 1-147 of the yeast Gal4 protein binding to the Gal UAS upsream of the report genes AD : Amino acids 768-881 of the Gal4 protein a transcriptional activator

38. Reporter constructs in yeast strains AH109 and Y187

39. Sequence of the Gal4 DNA-BD recognition sites

40. MATCHMAKER Yeast Two-hybrid System 3 Vectors

41. Overview of performing a yeast two-hybrid screen

42. Interactions can be screened for strength 3-AT: Competitive Inhibitor of HIS3 protein

43. Yeast Two Hybrid Assay • Y2H screens are notorious for a high level of false positives! • Some baits activate transcription alone • Some interactions occur by chance or due to incorrectly folded proteins • Some proteins are just ‘sticky’ Uetz, 2001

44. Protein interactions indicated by a YTH screen False positives should be eliminated Step 1: double screens avoid false positives Step 2: independent validation of interaction Pull-down assays (affinity isolation) Co-localization of protein expression

45. Verification of putative positive clones

46. Verification of putative positive clones (con’d)

47. Yeast mating to verify protein interactions

48. Ito et al, 2001 Genome Scale Yeast Two Hybrid Assay Red: positive screen 1 Green: positive screen 2 Yellow: positive for both Uetz, 2001

49. Interactions in the yeast proteome

50. EXAMPLE