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Protein protein interactions

Protein protein interactions. Better understanding of protein b’s function. Introduction. Protein b unknown. Protein a known. Interaction. Detecting relationship between pathways. Detecting new pathways. Introduction. Protein a Function a. Protein b Function b. Interaction.

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Protein protein interactions

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  1. Protein protein interactions

  2. Better understanding of protein b’s function Introduction Protein b unknown Protein a known Interaction

  3. Detecting relationship between pathways Detecting new pathways Introduction Protein a Function a Protein b Function b Interaction

  4. OLD WORLD Discrete methods (1 X 1) NEW WORLD Comprehensive methods Old world vs. new world MAINLY IN THIS LECTURE

  5. AD = Activation Domain DBD = DNA Binding Domain AD DBD UAS gene upstream activating sequence Yeast two hybrid system Simple transcription Transcription Complex transcription

  6. AD bait DBD fish Yeast two hybrid system Hybrid proteins

  7. AD bait DBD fish UAS Reporter gene Yeast two hybrid system Hybrid proteins Transcription Complex transcription

  8. Plasmid A Plasmid B DBD AD bait fish promoter promoter Yeast Yeast two hybrid system plasmids

  9. Report of the gene only in case of interaction Between the two proteins Yeast two hybrid system plasmids Yeast

  10. Yeast array – producing the array AD ORF • Produce plasmids: each contains ORF + AD promoter • Transformation the plasmids into yeast cells Yeast A comprehensive analysis of protein-protein interactions in S. cerevisiae P. Uetz et al.. • ORF – Open Reading Frames • Produce the yeast’s 6000 ORFs • 2 colonies of each transformation are • inserted to the array

  11. ORF of protein x+ AD ORF of protein y+ AD Yeast array – producing the array

  12. Producing similar plasmids (DBD+protein) • Transformation the plasmids into yeast cells DBD ORF promoter Yeast Yeast array – using the array • Selection of 192 “easy” proteins MATING & creating diploids SELECTION OF LIVING COLONIES BASED ON HIS3 PRODUCTION DETECTING THE ARRAY’S PROTEIN ACCORDING TO ITS POSITION

  13. RNA15 RNA14 Yeast array -results BEFORE (Pcf11) AFTER

  14. Yeast array - Results over-view • 2 undependable assays were preformed for each of the 192 proteins. • 87 out of 192 proteins were detected as involved in protein-protein interactions (passed the 2 assays) • total of 281 interactions were detected

  15. Production of ORF+AD plasmids and transformants AD ORF promoter • Production of ORF+DBD plasmids and transformants Duplicates of a single DBD transformant DBD ORF promoter The AD library Activation Domain library • Production of an AD library MATING (haploids to diploids) Transferring to a selection plate Detecting the ORF’s using PCR

  16. Activation Domain library - results • 817 out of ~6000 proteins were detected as involved in protein-protein interactions • total of 692 interactions were detected

  17. Array vs. library - Comparison SENSETIVE, BETTER RESULTS QUICK, SIMPLER, CHEAPER

  18. Protein arrays – producing the array Protein chips: from concept to practice Young-Sam Lee et al.. • Producing the yeast’s 6000 ORF’s using plasmids • Attaching histidine anchors to every protein • Attaching the proteins to an array

  19. The poured Protein is labeled Using antibodies that detect the interaction’s product Sophisticated assays Detecting the interactions Protein arrays – using the array • Pouring a protein onto the array

  20. Production of chimeric tagged proteins using plasmids • The protein creates a complex of proteins • The complex is separated using gel • electrophoresis Mass Spectrometry of purified complexes • The complex is isolated using the tag • Each protein is identified by Mass Spectrometry

  21. BENEFITS Identifying complex interactions Reliability can be checked DRAWBACKS Needs specific conditions Can lose loosely associated components Tagging might disturb the “complexing” Mass Spectrometry of purified complexes

  22. Examine 2 genes , “viable” and mutants GENES PR’ LEVELS LIVES LIVES THE CREATURE LIVES DEAD Synthetic Lethal Mutations What’s lethal mutation? • Examining the creature carrying them

  23. Create a yeast array, every yeast contains a different mutation Synthetic Lethal Mutations Hypothesis : these proteins are in interactions METHOD # 1: Synthetic Lethal Mutations • Create an artificial DNA containing 2 genes with conditional mutations • Change the conditions and detecting dead creatures METHOD # 2: Synthetic Lethal Mutations Arrays • Pour different yeasts carrying different mutations MATING & creating diploids STIMULATING THE CREATION OF SPORES + SELECTION FINDING THE DEAD

  24. IN VIVO, EXAMINE DIFFERENT CELL’S CONDITIONS NOT SO ACCURATE Computational methods • Mentioned in this seminar, mainly for understanding proteins’ • Functions and using to detect interactions Correlated mRNA expression • Measuring mRNA levels under a variety of cellular conditions • Grouping the genes that have similar transcriptional responses

  25. CREATURE A ORTHOLOGS CREATURE B Computational methods Genome analysis – IN SILICO Prokaryotic's operons • Genes that are consistently in the same operon, in the same order but in different and distanced creatures

  26. CREATURE A 1 polypeptid HOMOLOGY CREATURE B 3 polypeptids Computational methods Genome analysis Phylogenetic profile • Interacting proteins have a tendency to be either present ot absent • together from fully sequenced genomes Gene fusion (ROSETTA STONE) • One gene in creature A = some genes in creature B

  27. Inexpensive, fast, “widened” with the genomes DB Computational methods Genome analysis Otology relationships are not so clear, not always reliable

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