Discovery Research via in vivo Evolution Huang Lei, Tian He, Wen Ya, and Zhang Yi Peking University, and National Institute of Biological Sciences, Beijing 2008
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Discovery Research via in vivo Evolution Huang Lei, Tian He, Wen Ya, and Zhang Yi Peking University, and National Institute of Biological Sciences, Beijing 2008 03 02 Discovery Research in Biology • To answer the question: ‘what’/‘whether’ • Example 1: what activates receptor X? – Whether drug alpha activates X? • Example 2: what suppresses gene Y? – Whether gene beta suppresses Y? • Example 3: what maintains stem cell state? – Whether kinase gamma maintains stem cell state? Strategies for Discovery Research • Two strategies towards the goal: • Guess: answering whether, intelligent but very few novel insight • Screen: answering what, laboring but can give anti-intuition insight • Hereinafter we concentrate on screening Complexity Theory for Screen • You always have it in your first 100 lines or you never have it -- Seymour Benzer on flies • Complexity theory: when dimension grows, for serial screening, complexity grows in geometrical metrics • Monte Carlo method complexity • Simulated annealing: decelerating Monte Carlo method Example of Simulated Annealing in Biological System • Adaptive Immunity Molecular components of adaptive immunity • Somatic hypermutation Molecular components of adaptive immunity • DNA break and repair AID at the center of adaptive immunity How does AID works? C •AID converts C to U, causing U:G mispairs. •The mispairs are repaired through the base excision repair (BER) or the mismatch repair (MMR) pathways •Mutations are introduced through the intervention of translesion DNA polymerases. U UNG regulates transition/transversion ratio Limiting AID function • Transcription rate of the target gene: AID only targets ssDNA • AID promoters and enhancers • Epigenetic insulators • Specific sequence bias -Hotspots: DGYW/WRCH (R = A/G, Y = T/C, W = A/T, D = A/G/T). A Problem: How to restrict AID function within the targeted sequence? The genomic damage must be avoided! Possible solution: Mimic the Immunoglobin structure? in vivo evolution application based on adaptive immunity Problems (and solutions?) • Mammalian cells grow slow – Bacteria/yeast grow fast • Mammalian cells are expensive – Bacteria/yeast are cheap • Eukaryote protein has to be correctly folded and glycosylated – Yeast better than bacteria? AID can work in yeast An Example Class of drug target Species Number of molecular targets Targets of approved drugs Pathogen and human 324 Human genome targets of approved drugs Human 266 Targets of approved small-molecule drugs Pathogen and human 248 Targets of approved small-molecule drugs Human 207 Targets of approved oral small-molecule drugs Pathogen and human 227 Targets of approved oral small-molecule drugs Human 186 Targets of approved therapeutic antibodies Human 15 GPCR, deorphanization and drug discovery • GPCR: G protein coupled receptors • A huge gene family • Important pharmacological target Sexual Reproduction in yeast -- a GPCR signaling pathway How to get it done in yeast? GPCR signaling mating pathway expression of heterologous GPCRs Four modification for heterogolous GPCRs Introducing heterologous GPCRs add a cleavable leader sequence to aid transport to the plasma membrane remove regions not required for interacting with the ligand or G protein. Modifying the G protein develop chimeric G alpha subunits to incorporate receptor binding properties of mammalian subunits into a Gpa1 subunit that retains efficient interaction with the yeast G beta gamma Four modification for heterogolous GPCRs Knockout some native genes and incorporating reporter genes knock out Ste2, Sst2, Far1 combine reporter genes behind PRE Autocrine system establish an autocrine system combine the ligand to a factor or alpha factor facilitating its secreting but restrict on the membrane What can we do with it ?? Our Plan … Protocol… Ade2 Peptide-alpha factor IRES His3 PRE Ade2 lacO IRES lacZ lacI hAID The whole system One example using GPCR protocol for artificial evolution No binding between peptide and GPCR x Signalling Initially……………….. Peptide-alpha factor Ade2 x His3 PRE Ade2 IRES lacO IRES lacZ lacI hAID No GPCR signalling: hAID is expressed to mutate peptide ligand Binding between peptide and GPCR Signalling Until the peptide become an agonist of GPCR…. Ade2 Peptide-alpha factor Fus1 IRES His3 PRE IRES lacZ lacI x Ade2 lacO hAID His3 GPCR is activated, AID is silenced… lacZ lacZ readout with fluorescence ……… or visual detection directly Positive and negative selections • Positive selection: – his3 mediated histidine- survival – High lacZ activity • Negative selection: – Raise in complete medium (let it grow!) – Low or no lacZ activity Applications for drug discovery • Peptidergic ligand for specific GPCR • Optimizing peptidergic ligand hits • Finding conserved motif for agonist/antagonist Assay procedure: it is easy! • Transform GPCR to ready-knockout lines • Assay for constitutive activity • Transform the peptide-encoding vector library into a nice coupled GPCR line • Grow the transformant in large vial with evolution medium (His-, 3AT+) • After sometime, collect the solution and plate for colonies • Sequence individual colony for hits Further development on compound structure GPCR other than ligand Taking the complexity of the GPCR pathway into account We can first use the simple yeast twoor three- hybrid systems for a test. Yeast two-hybrid system Peptide-Gal4-AD Ade2 IRES His3 UAS Ade2 lacO IRES lacZ lacI hAID For example, the core circuit could be adopted into Y2H The methods in two-hybrid systems Generally, the cDNA encoding the DBD-X fusion protein and the cDNA encoding the AD-Y fusion protein are inserted into two plasmids, respectively, and then both transformed into the yeast cells. Sexual Reproduction in yeast Interaction mating methods can also be used in two-hybrid systems. The AD and DBD fusion proteins begin in two different haploid yeast strains with opposite mating types, a and α, respectively. To test for interaction,the hybrid proteins are brought together by mating, a process in which two haploid cells fuse to form a single diploid cell. Further, Yeast three-hybrid system Application of Y3H • RNA aptamer screen • … or: RNAinteracting protein? Applications other than GPCR • • • • Nuclear receptor ligand screen Protein interaction screen Novel bacterial transcriptional biosensors? Whatever you can think about! :) Summary • We present a simple core genetic circuit which can evolve any desired target in vivo • We present a unified, inexpensive solution for both academical and industrial needs • In vivo evolution brings greater capacity and flexibility to screening • Further assay development based on mammalian systems such as immune cell lines Acknowledgements • • • • • Wang Yiping Youri Pavlov Rao lab members iGEM 2007 members :) PKU iGEM 2008 society :)