Protein interaction Computational (inferred) Experimental (observed)

Inference of Protein function

Guilt by association Five methods for inferring functional interactions: Complexes, Pathways Rosetta stone Phylogenetic profiles Expression profiles Gene neighbors Operon method

Five Known Rosetta Stone Links: Marcotte et al., Science, 285, pp (1999)

Pathways Detected by the Domain Fusion (Rosetta stone) Method AroHYDIBAroK PurF AroFAroE AroL AroGPur2 AroA AroB AroBPurTPur3 B AroDPurL AroEPur7 Pur5 Pur2 PurU AroKPurE Pur3 PurE C AroL Pur5 PurK AroAPurK PurTPur7 AroCGuaA GuaBPurB PurB A D GuaAPurHPurA Marcotte et al., Science, 285, pp (1999)

PHYLOGENETIC PROFILE METHOD

YC083W homology to thioredoxin MRPL2 MRPL6 MRPL7 MRPL10 known MRPL16 ribosomalpredicted to YGR021WMRPL23 proteins target member ofMRPS9 mitochondria highlyMRPS28 conservedMRF1 peptide chain release factor protein protein familyYJR113C homology to ribosomal protein S7 synthesis of unknownMSY1 ttrosyl-tRNA synthetase functionYGL068W probable ribosomal protein L12 MGE1 heat shock protein/chaperone YDR116C homology to bacterial ribosomal L1 protein YHR189W homology to peptidyl-tRNA hydrolase SIS1/XDJ homology to DraJ heat shock protein PDR13/SSE1/LHS1 homolgoy to Hsp70 RIB2 DRAP deaminase YDL036C homology to Rib2/pseudouridine synthase MIS1 C1-THF synthase ADE3 C1-THF synthase TPI1 tiose phosphate isomerase YGL236C homology to conserved gidA family, unknown function YOL060C homology to hypothetical C. elegans protein M02F4.4 Phylogenetic Profile: e.g. for a yeast protein

Microarray Co-expression Analysis Conclusion: P2 and P7 are functionally linked P3 and P6 are functionally linked Expression Profiles Rich med Starve High T P P P P P P P Profile Clusters P P P P P P P

Inferring Functional Linkages from the Gene Neighbor Method genome 1... genome 2genome 3 genome 4 A A A A B B B B C C C C A B C A statistically significant correlation is observed between the positions of proteins A and B across multiple genomes. A functional relationship is inferred between proteins A and B, but not between the other pairs of proteins:

gene A gene B gene C OPERON method of inferring functional linkages in the genome of Mycobacterium tuberculosis

Examples of Inferred Protein Networks

Features of Inferred Networks Linkages are between non-homologs The function of each protein is defined in its cellular context by its linkages Each linkage is assigned a probability Need to separate and visualize each module of functional proteins

Prions Can spontaneously change shape (10 -5 ) Can induce shape change in other proteins Contagious, inheritable Sup35 –Translation termination factor –Prion: reads through stop codons

Sup 35 Interactions

A Network of Proteins Related to Flagella fliA

Flagella-Related Transcription Factors

fliA Flagella-Specific ATP Synthase

fliA Chemotaxis-Related Proteins

Quantitative Assessment of Inferred Protein Linkages

Assessing Functional Linkages by method of Keyword Recovery X A Y Z Is the linkage inferred between A and Y valid ? Compare the keyword annotations common to A and Y, with the number expected at random = “signal-to-noise”

Assessing Inference of Linkages by Recovery of Keywords YEAST GENOME (Marcotte et al. Nature, 1999) Inferrence Method # functional Signal links to noise Individual Methods: Experiment (2-hybrid) Phylogenetic Profiles20,749 5 Rosetta Stone50,459 3 Correlated mRNA expression 26,013 2 Combined methods: Links by ≥ 2 methods 1,271 8

Observed Functional Linkages

Database of Interacting Proteins Experimentally detected interactions from the scientific literature

The DIP Database DOE-MBI LSBMM, UCLA

  Paul D. Boyer Nature (1999) XRay structure Stock et al. Science (1999) Two-hybrid assay Moritani C et al. Biochim Biophys (1996) ATP Synthase ATP Synthase in DIP HOW PROTEIN INTERACTIONS ARE REPRESENTED IN DIP

Number of proteins per network Number of clusters DIP contains 22,000 interactions from 2,200 articles, representing hundreds of networks

b Assessing DIP reliability with mRNA expression data (ERP)

Coexpression Measure Euclidean distance Protein A Protein B expression level log(e/e ref )

Extracting the Expression Profile (EPR) Index d2d2 p x 100 Model: non Y2H all vs all 40% of Y2H is highly reliable Overall: 50% of 8000 yeast DIP interactions are reliable = 4000 Interacting Experimental (Y2H) Random fitted  =0.4

Many proteins interacting in a Y2H may never meet each other in the cell!

Expression Profile Data Assesses Interaction Data (EPR Index; Deane et al. Molec & Cell Proteomics, 2002) DOE-MBI LSBMM, UCLA Uetz, P. et al.; Nature 403: (2000) Ito, T. et al.; PNAS 97: (2000) Ito, T. et al.; PNAS 98: (2001) Ito, T. et al.; PNAS 97: (2000) Ito, T. et al.; PNAS 98: (2001) Ho, Y. et al.; Nature 415: (2002) Gavin, A.C. et al.; Nature 415: (2002)  7.8+/-3.7%  19.5+/-3.7%  48.7+/-6.9%  89.5+/-6.6%

Measured vs Inferred Functional Interactions

EPR Assessment of Inferred Interactions vs. Y2Hybrid & MS MethodLinkagesEPR index Rosetta S +Phylo P 24, Yeast 2 hybrid ~4, Mass spec ~4, DIP yeast overall 6,1440.3

Some Conclusions A X Y Z B V C A protein’s function is defined by the cellular context of its linkages Many functional linkages are revealed from genomic data Validity of functional linkages can be assessed computationally by keyword recovery or experimentally by expression data Functional complexes can be discovered from genomic data