12/11/12. Dr. Sanjeeva Srivastava. IIT Bombay 2. Interactomics Yeast Two-Hybrid Immunoprecipitation Protein microarrays. Proteomics Course NPTEL

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1 Dr. Sanjeeva Srivastava IIT Bombay Interactomics Yeast Two-Hybrid Immunoprecipitation Protein microarrays IIT Bombay 2 1

2 IIT Bombay Tremendous effect of interactions IIT Bombay 4 2

3 Complex formation Protein modifications Protein transport Many more IIT Bombay 5 Function of uncharacterized proteins New roles for characterized proteins Mechanisms to regulate protein activity Networks of protein interactions IIT Bombay 6 3

4 Transient Weak Obligate Homo-oligomer Permanent Strong Non-obligate Hetero-oligomer IIT Bombay 7 Electrostatic Steric? Hydrophobic Hydrogen bonds IIT Bombay 8 4

5 IIT Bombay 9 Protein Interaction Networks IIT Bombay 10 5

6 Protein Inactivation Catalysis Altered kinetics New binding site formation Substrate binding IIT Bombay 11 DNA replication Signal transduction Transcription Translation Splicing Cell Cycle control Morphology Metabolism Motility Growth IIT Bombay

7 Dynamic, interact with a wide variety of biomolecules lipids, nucleic acids, small drug inhibitors, and many others Proteins also interact with one another form macromolecular complexes to regulate signal transduction & gene regulation IIT Bombay 13 IIT Bombay 7

8 Traditional approaches High throughput approaches Yeast two hybrids Affinity chromatography Immunoprecipitation Protein microarrays Label-free technologies IIT Bombay 15 IIT Bombay 8

9 IIT Bombay Purification of protein complexes by Immunoprecipitation (IP) or tandem affinity purification (TAP) Target protein and its interacting partner isolated from complex sample IIT Bombay 18 9

10 Antigen (known) Unknown ligands A Antibody Non-denaturing conditions Denaturing conditions IIT Bombay 19 Proteins in native state Interactions are natural Large order complexes can be observed IIT Bombay 20 10

11 Sticky proteins appear regularly Unclear whether interaction is direct or indirect Expensive Applications in other organisms challenging IIT Bombay 21 IIT Bombay 11

12 YTH system demonstrated by Fields and Song (1989) to detect protein interactions The bait-bd and prey-ad hybrid proteins are jointly expressed in yeast nucleus If protein-protein interaction is established results in activation of reporter gene transcription IIT Bombay 23 AD Bait Prey BD Lac Z Reporter IIT Bombay 24 12

13 AD Bait Prey BD Transcription Lac Z Reporter IIT Bombay 25 Used for protein-rna, protein-dna interactions, analysis of particular complexes, construction of protein interaction network Simple protocol No expensive equipment requirement Ability to screen large libraries IIT Bombay 26 13

14 High false positive/negative rates Proteins must localize and interact in nucleus Application in a non-yeast environment questionable Sensitive to toxic gene Limited to pair-wise interaction IIT Bombay 27 IIT Bombay 14

15 IIT Bombay Microscopic arrays comprising thousands of discrete proteins High throughput platform biomarker discovery protein-protein interactions functional characterization IIT Bombay 30 15

16 Antibody Array YY YYYYYYYY YY YYYYYYYY YY YYYYYYYY Microscopic array of antibodies Measure levels of proteins or other biomolecules in samples Protein Array Microscopic array of proteins Assay protein function, Protein interactions, Small molecule interactions, Identify substrates IIT Bombay 31 Direct Label-based Spotting purified proteins (E. coli) Limited number of proteins MacBeath, G., and Schreiber, S Science 289:1760 IIT Bombay 32 16

17 Direct Label-based 5800 yeast clones (His-tagged) Screening for known and novel Calmodulin & lipid binding partners Zhu et al Science, 293, 2101 IIT Bombay 33 IIT Bombay 17

18 Direct Label-based IIT Bombay 35 Direct Label-based Sandwich IIT Bombay 36 18

19 Reverse-phase IIT Bombay 37 Chemical linkage IIT Bombay 38 19

20 Chemical linkage Peptide fusion IIT Bombay 39 Direct Label-based NAPPA IIT Bombay 40 20

21 MIST IIT Bombay 41 Abundance based Function based Direct Label-based Sandwich Reverse-phase Peptide fusion NAPPA MIST IIT Bombay 42 21

22 Achievements Challenges High density Small volume Multifunctional assays Protein purification Protein functionality IIT Bombay 43 Interactomics Yeast Two-Hybrid Immunoprecipitation Protein microarrays IIT Bombay 44 22

23 Niroshan Ramachandran, Dale N. Larson, Peter R. H. Stark, Eugenie Hainsworth, Joshua LaBaer Emerging tools for real-time label-free detection of interactions on functional protein microarrays. FEBS Journal. Volume 272, Issue 21, pages , November 2005 Harini Chandra, Panga Jaipal Reddy and Sanjeeva Srivastava Expert Review of Proteomics. Protein microarrays and novel detection platforms. February 2011, Vol. 8, No. 1, Pages /epr Sandipan Ray, Gunjan Mehta, Sanjeeva Srivastava, Label-free detection techniques for protein microarrays: Prospects, merits and challenges. PROTEOMICS. Volume 10, Issue 4, pages , No. 4 February 2010 Gerard Drewes, Tewis Bouwmeester Global approaches to protein protein interactions. Current Opinion in Cell Biology. Volume 15, Issue 2, April 2003, Pages Harini Chandra, Sanjeeva Srivastava Cell-free synthesis-based protein microarrays and their applications. PROTEOMICS. Volume 10, Issue 4, pages , No. 4 February 2010 IIT Bombay 45 Luis Berrade, Angie E. Garcia, Julio A. Camarero Protein Microarrays: Novel Developments and Applications. Pharmaceutical Research. July 2011, Volume 28, Issue 7, pp Oda Stoevesandt, Michael J Taussig and Mingyue He Protein microarrays: high-throughput tools for proteomics. Expert Review of Proteomics. April 2009, Vol. 6, No. 2, Pages Mingyue Hea, Ming-Wei Wangb Arraying proteins by cell-free synthesis. Biomolecular Engineering. Volume 24, Issue 4, October 2007, Pages Thorsten Stellberger, Roman Häuser, Peter Uetz, Albrecht von Brunn Functional Genomics. Yeast Two-Hybrid Screens: Improvement of Array-Based Screening Results by N- and C-terminally Tagged Fusion Proteins. Methods in Molecular Biology Volume 815, 2012, pp IIT Bombay 46 23

24 Bernhard Suter, Saranya Kittanakom, Igor Stagljar Current Opinion in Biotechnology. Protein technologies / Systems biology. Two-hybrid technologies in proteomics research. Volume 19, Issue 4, August 2008, Pages Andrea Fiebitz, Dominique Vanhecke. Protein Microarray for Disease Analysis. High-Throughput Mammalian Two-Hybrid Screening for Protein Protein Interactions Using Transfected Cell Arrays (CAPPIA). Methods in Molecular Biology Volume 723, 2011, pp Barry Causier Studying the interactome with the yeast two-hybrid system and mass spectrometry. Volume 23, Issue 5, pages , September/ October Joshua LaBaer, Niroshan Ramachandran Protein microarrays as tools for functional proteomics. Current Opinion in Chemical Biology. Volume 9, Issue 1, February 2005, Pages Bernhard Suter, Saranya Kittanakom, Igor Stagljar Two-hybrid technologies in proteomics research. Current Opinion in Biotechnology. Volume 19, Issue 4, August 2008, Pages IIT Bombay 47 24

Enhancers mutations that make the original mutant phenotype more extreme. Suppressors mutations that make the original mutant phenotype less extreme

Enhancers mutations that make the original mutant phenotype more extreme. Suppressors mutations that make the original mutant phenotype less extreme Interactomics and Proteomics 1. Interactomics The field of interactomics is concerned with interactions between genes or proteins. They can be genetic interactions, in which two genes are involved in the