Protein Interaction Maps and Model Organisms Guang-Chao Chen IBC, Academia Sinica

Genome sizes of humans and other organisms Organism estimated size estimated gene average gene chromosome number density number Homo sapiens (human) 2900 million bases ~30,000 1 gene per 100,000 bases 46 Rattus norvegicus (rat) 2750 million bases ~30,000 1 gene per 100,000 bases 42 Mus musculus (mouse) 2500 million bases ~30,000 1 gene per 100,000 bases 40 Drosophila melanogaster 180 million bases 13,600 1 gene per 9,000 bases 8 (fruit fly) Arabidopsis thaliana 125 million bases 25,500 1 gene per 4000 bases 10 (plant) Caenorhabditis elegans 97 million bases 19,100 1 gene per 5000 bases 12 (roundworm) Saccharomyces cerevisia 12 million bases 6300 1 gene per 2000 bases 32 (yeast) Escherichia coli (bacteria) 4.7 million bases 3200 1 gene per 1400 bases 1

Network Biology

Conventional approaches: one or a few proteins Functional genomic approaches: tens of thousands of proteins -microarrays (gene expression profile) -large scale gene knockout (RNA-mediated interference) -large scale protein localization (GFP) -protein interaction maps

Protein interactions are crucial in many aspects of biological function.

Genetic vs physical interaction maps

Two directions for proteomics in protein interaction mapping. Gene (DNA) Transcription (RNA) Protein Genome Transcriptome Proteome Forward genetics Reverse genetics Reverse proteomics Classical proteomics

The life cycle of Saccharomyces cerevisiae

The Nobel Prize in Physiology or Medicine 2001 "for their discoveries of key regulators of the cell cycle" Leland H. Hartwell R. Timothy Hunt Sir Paul M. Nurse

Yeast mutants with cell-cycle defects Wild-type Haploid meiosis/ pat1 cdc2 cdc28 Nat Rev Genet. 2001

Suppressor mechanisms Nat Rev Genet. 2001

Cloning suppressors ts- ts+ Nat Rev Genet. 2001

A novel genetic system to detect protein–protein interactions Nature 340, 245 - 246 (20 July 1989); doi:10.1038/340245a0     A novel genetic system to detect protein–protein interactions Stanley Fields & Ok-kyu Song

High-throughput two hybrid screen and interactome mapping

Stringent Y2H screening strategy

False-positives and false-negatives in Y2H

I. Validation of Y2H by orthogonal assays Classification and validation of potential interactions. I. Validation of Y2H by orthogonal assays II. Interologues III. Lirature

(EGF) (Ras) Y2H

Clustering analysis suggests the existence of a multiprotein complex The synthetic multivulva (synMuv) genes in C. elegans

Boulton et al. (2002) Science

The Nobel Prize in Physiology or Medicine 1995 "for their discoveries concerning the genetic control of early embryonic development" Edward B. Lewis Christiane Eric F. Wieschaus Nüsslein-Volhard

Second-site enhancers and suppressors

GAL4-dependent ectopic expression of a gene (the GAL4-UAS system) expression of engrailed (en) Brand and Perrimon 1993 Development

Clonal (mosaic) analysis Nat Rev Genet. 2002

Global views of the protein-interaction map

Global views of the protein-interaction map

Local pathway views

Quality control in high-throughput protein interaction networks - Existing protein complex Reproducibility of interactions Evolutionary conservation - Functional classification - Subcellular localization - Gene expression

The awesome power of comparative interactomics

Limitations of the yeast two-hybrid system interaction in the nucleus not suitable for membrane proteins and large-size proteins may not undergo posttranslational modification

Analysis of protein-protein interactions in vivo B2H Integral membrane protein adenylate cyclase - galactosidase lactamase GFP luciferase Miller (2005) PNAS Karimova (2005) J Bacteriol.

Posttranslational modification Cytoplasmic interaction - effective phosphorylation - cytotoxicity Prey fused with a myristylation sequence Guo (2004) Nature Biotechnol Aronheim (2004) Mol Cell Biol

Epitope phage display

Protein-peptide interactions with random phage display

Zebrafish embryonic development Haffter et al. 1996 Development

The zebrafish toolbox Forward genetics Reverse genetics - Chemical mutagenesis - Insertional mutagenesis Reverse genetics - Morpholinos - Tilling (Targeting Induced Local Lesions IN Genomes) Expression profilling - Whole-embryo in situ hybridization - Gene chip - Spotted microarrays

Chemical screens in whole, mutant zebrafish gridlock Small molecules that suppress gridlock circulation defects Nat Rev Drug Discov. 2005

Zebrafish as a system for small-molecule screens small molecular library 200-300 embryos/female 96 or 384-well assay plates examine phenotypes (automated readout) search library database Nat Rev Drug Discov. 2005

RNA interference (RNAi) as a platform for dissecting the function of independent genes High-throughput genetics: large-scale RNAi libraries and large number of cell-based assays

High-throughput RNAi screens by cell imaging. Kiger et al. 2003 J Biol.

Possible fates of mammalian cells in vitro Hela neuronal cells 293T epithelial cells

Short hairpin RNA (shRNA)

Genome-wide screens in mammalian cells (RNAi)

Direct loss-of –function versus modifier screens Nat Rev Genet. 2006