Experimental Bioinformatics Lab Institute for Research in Biomedicine Barcelona Barcelona Supercomputing Center
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- Jemimah Young
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1 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions Montse Soler-López Experimental Bioinformatics Lab Institute for Research in Biomedicine Barcelona Barcelona Supercomputing Center 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 1
2 High-throughput experimental approach to validate predicted protein-protein interactions: Alzheimer s disease PPI network Soler-Lopez et al. Genome Res th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 2
3 Tools for the analysis of biomolecular interactions Charbonnier et al. Biotechnol Annu Rev th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 3
4 Yeast two-hybrid: what is it? 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 4
5 Y2H: what is it? A genetic assay for detecting physical protein-protein interactions in yeast developed by Stan Fields in early 90s (Fields, S. Proteomics 2009 Dec;9(23): ) not a perfect method, but allows high throughput screening of protein interactions used primarily for initial identification of interacting proteins 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 5
6 Y2H: what is it? most of available PPI data in databases still come from Y2H two hybrid pooling approach tandem affinity purification anti tag coimmunoprecipitation two hybrid pull down dihydrofolate reductase reconstruction two hybrid array coimmunoprecipitation peptide array protein array fluorescence microscopy ubiquitin reconstruction x-ray crystallography surface plasmon resonance imaging technique affinity chromatography technology number of evidences Extracted from MINT database: 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 6
7 Y2H: how does it work? 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 7
8 Y2H: how does it work? Suppose you have two proteins Protein X Protein Y and you want to know if they interact 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 8
9 Y2H: how does it work? 1) Fusion of our protein of interest X with a DNA Bindingdomain bait DB 18kD Protein X UAS Gene promoter Reporter gene Based on modular organization of transcription factors (TF) for gene regulation in yeast 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 9
10 Y2H: how does it work? 2) Fusion of our protein of interest Y with Activationdomain bait DB Protein X 18kD prey AD Protein Y 14kD UAS Gene promoter Reporter gene Based on modular organization of transcription factors (TF) for gene regulation in yeast 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 10
11 Y2H: how does it work? 3) If X and Y interact, a functional Transcription Factor is formed X DB bait Y AD prey UAS Gene promoter Reporter gene Based on modular organization of transcription factors (TF) for gene regulation in yeast Reporter gene 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 11
12 Y2H: how does it work? 4) TF binds to UAS activating transcription of reporter gene X DB Y AD RNA POL Expression of reporter protein UAS Upstream activation seq Gene promoter Reporter gene Transcription machinery ON Based on modular organization of transcription factors (TF) for gene regulation in yeast 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 12
13 Y2H: variants Transcription factors: Reporter genes: Yeast GAL4-based E.coli LexA+VP16-based HIS3 URA3 LacZ ADE2 LYS2 enzyme involved in biosynthesis of histidine in biosynthesis of uracil in activation of b-galactosidase in biosynthesis of adenine in biosynthesis of lysine 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 13
14 Considerations in designing Y2H 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 14
15 Considerations in designing Y2H Prior to beginning a two-hybrid screen: Protein family determine as much information regarding the protein of interest members of large protein families may interact at varying degrees Self-activation Spurious activation of reporter genes Possible solutions: Screen with fragments that lack these activities Swap the DBD and AD domains of the 2 proteins 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 15
16 Considerations in designing Y2H Interaction Artifacts Interactions might not occur in vivo under native conditions Often only segments of the protein are analyzed, revealing (or masking) domains DBD or AD fusion proteins may have little resemblance to native folding Post-translational modifications are absent (or incorrectly modified) in yeast Interactions can be mediated non-specifically (large hydrophobic regions) Interactions might be biologically irrelevant (e.g., the proteins exist in different cell types, compartments or at different times during development or cell cycle) 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 16
17 Y2H: features 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 17
18 Two-Hybrid System with Gateway cloning (ProQuest Invitrogen) Uses low-copy-number (ARS/CEN) vectors Incorporation of the Gateway Technology to allow rapid and easy generation of bait and prey constructs Contains three different reporter genes with independent promoters: HIS3, URA3, LacZ Reporter gene (URA3) allows both positive and negative selection 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 18
19 Y2H cloning vectors Bait vector Prey vector 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 19
20 Y2H screen: approaches 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 20
21 Y2H screen: approaches Matrix: array, bait x prey Baits Preys Library: bait x pool of preys 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 21
22 Y2H screen: matrix approach 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 22
23 Y2H screen: matrix (I) Co-transformation in yeast diploid strain (MaV203) agar plate with yeast media w/o Leu nor Trp 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 23
24 Y2H screen: matrix (I) Mating yeast haploid strains (bait/matα x prey/mata) Yeast media without Leu nor Trp 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 24
25 Testing reporter genes HIS induction 3AT Very weak weak medium strong most sensitive reporter gene yeast media w/o histidine enables modulation of the stringency estimation of the interaction affinity 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 25
26 Testing reporter genes URA induction 5FOA No binding Strong binding yeast media with uracil enables negative selection by dose-dependent 5FOA least sensitive reporter gene strong promoter that supresses transcriptional background 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 26
27 Testing reporter genes lacz induction colorimetric assay 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 27
28 HT screening: replica plating Baits Preys Master plate 96 pin replicator HIS3 induction URA3 induction URA3 induction β-gal induction Replica plates 1) -L-W-H + 10mM 3AT 5) -L-W-U 6) -L-W % 5FOA 9) -L-W + membrane 2) -L-W-H + 25mM 3AT 3) -L-W-H + 50mM 3AT 4) -L-W-H + 100mM 3AT 7) -L-W + 0.1% 5FOA 8) -L-W + 0.2% 5FOA 10) -L-W-H-U + 25mM 3AT 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 28
29 Y2H matrix: evaluating phenotypes 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 29
30 Y2H: evaluating phenotypes URA & HIS inductions: -L-W-U-H HIS3 induction sensitivity: HIS3 induction sensitivity: -L-W-H + 25mM 3AT 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 30
31 Y2H: evaluating phenotypes HIS3 induction sensitivity: -L-W-H + 100mM 3AT 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 31
32 Y2H: evaluating phenotypes URA3 induction: -L-W-H % 5FOA 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 32
33 Y2H: evaluating phenotypes URA3 induction: -L-W-H + 0.2% 5FOA 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 33
34 Y2H: evaluating phenotypes lacz induction: X-gal assay Strong interactors show blue color within 1 hour Weak interactors show blue color within 24 h, but can remain very faint blue 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 34
35 Y2H screen: library approach 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 35
36 Y2H screen: library Prey library: Pool of cdnas corresponding to the transcriptome of a cell type/tissue Total mrna cdna Digested into ~1.5kb fragments inserted into prey vector Normalized copy number 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 36
37 Y2H screen: library ProQuest pre-made cdna Libraries 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 37
38 Y2H screen: library 1 bait/mav203 x 1 prey library Transformation yeast media w/o Leu,Trp Transformation efficiency ~ th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 38
39 Y2H screen: library Yeast media without Leu, Trp, Ura Pick up single colony Isolate plasmid DNA Transform E.coli Isolate plasmid DNA DNA sequencing Gene identification by BLAST 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 39
40 Y2H: scoring interactions 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 40
41 Y2H: scoring interactions Scoring criteria to reduce false positives: Select interactions that activate at least 2 reporter genes (medium, strong interactions) Select interactions that are observed at least in two independent screens (weak interactions) Select interactions that activate at least 2 reporter genes and are observed in more than 1 screen (very strong interactions) Select interactions that show positive binding in downstream assays: co-immunoprecipitation, pull-down, colocalization 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 41
42 Y2H: scoring interactions Library approach: Confirm the identified interactions by matrix Y2H Discard identified genes that show frameshift with the AD Select interactions that show positive binding in downstream assays: co-immunoprecipitation, pull-down, colocalization 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 42
43 Y2H: applications 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 43
44 Y2H applications: Pathway generation Alzheimer s interactome Soler-Lopez et al. Genome Res th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 44
45 Y2H applications: Interaction domain mapping Novel interactors and domain mapping of Hd3a Taoka et al. Nature : th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 45
46 Y2H applications: Characterization of interactions by in frame-mutations Taoka et al. Nature : th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 46
47 Y2H applications: ligand/inhibitor screening Three-hybrid for ligand-receptor screening Medium: 1uM FK506 10uM FK506 Licitra et al. Proc.Natl.Acad.Sci.U.S.A : th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 47
48 Y2H applications: ligand/inhibitor screening Anthrax inhibitor screening Kim et al. Biochem Biophys Res Commun (1): th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 48
49 Y2H PPI verification: downstream assays 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 49
50 In vitro verification of Y2H PPIs in mammalian cells APP CDC37 DYNC1H1 ECSIT GCDH PDCD4 RNF32 ST13 ECSIT FARSA FBXL12 NOS3 RNF32 ST13 APOE APOE FARSA APP FBXL12 FARSA IFIT3 FBXL12 NOS3 IFIT3 PRAM1 NOS3 PRDX2 LC set PSEN1 Co-IP Pull down PSEN2 STAMBPL1 Detected Not detected Detected Not detected Not tested HC set GST pull-downs Co-immunoprecipitations Soler-Lopez et al. Genome Res th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 50
51 In vivo verification of Y2H PPIs in neuronal cells Double immunofluorescence and confocal microscopy Soler-Lopez et al. Genome Res th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 51
52 Y2H: conclusions 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 52
53 Why should you use Y2H? Very high numbers of genes/constructs assayed in a relatively simple experiment Physical interactions detected and characterized following one single commonly used protocol In vivo assay No need for protein purification Eukaryotic system It can be used in various stages of a structural biology project 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 53
54 Why should you not use Y2H? Transcription factors (self-activators) Membrane proteins (insufficient folding and/or stability of a fusion protein) Toxic proteins in yeast If post-translational modifications are required for the interaction No time/space constraint 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 54
55 Alternative Y2H systems 18 th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 55
56 Alternative Y2H : MAPPIT Mammalian protein-protein interaction trap Bait: hybrid erythropoietein(epo)-leptin receptor; Prey: gp130 When bait-prey interact, JAKs phosphorylate gp130 Phosphorylated gp130 attracts STAT which activates transcription of reporter (luciferase) Generates bioluminescent readout Operates in mammalian background enabling posttranslational modification Ulrichts et al. Methods Mol Biol : th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 56
57 Alternative Y2H : MYTH Split-ubiquitin based membrane yeast two-hybrid Prey: ubiquitin N-terminal fragment (Nub); bait: C-terminal fragment (Cub) if an bait-prey interact, a pseudoubiquitin is formed This pseudo-ubiquitin is recognized by cytosolic deubiquitinating enzymes which cleave off the transcription factor, allowing it to enter the nucleus and activate a reporter system consisting of the HIS3, ADE2 and lacz genes Snider et al. Nat Protoc (7): th October 2011 EMBO Practical Course on Modern Biophysical Methods for protein-ligand interactions 57