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    Department of Biotechnology
    Speaker - HinaOjha
  • 2. Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells.
  • 3. Functional proteomics
    Critical aspects required to understand the function of a protein include:
    • Protein sequence and structure–used to discover motifs that predict protein function
    • 4. Expression profile–reveals cell-type specificity and how expression is regulated
    • 5. Post-translational modifications (e.g., phosphorylation, acylation, glycosylation, ubiquitination)–suggests localization, activation and/or function
    • 6. Intracellular localization–may allude to the function of the protein
    • 7. Interactions with other proteins-function may be extrapolated by knowing the function of binding partners.
  • Why we are studying protein-protein interactions?
    • Until the late 1990's, protein function analyses mainly focused on single proteins.
    • 8. But because the majority of proteins interact with other proteins for proper function, they should be studied in the context of their interacting partners to fully understand their function.
    • 9. With the development of the field of proteomics, understanding how proteins interact with each other and identifying biological networks is vital to understanding how proteins function within the cell.
  • Biological effects of protein-protein interactions
    • Alter the kinetic properties of enzymes, which may be the result of subtle changes in substrate binding or allosteric effects
    • 10. Allow for substrate channeling by moving a substrate between domains or subunits, resulting ultimately in an intended end product
    • 11. Create a new binding site, typically for small effector molecules
    • 12. Inactivate or destroy a protein
    • 13. Change the specificity of a protein for its substrate through the interaction with different binding partners; e.g., demonstrate a new function that neither protein can exhibit alone
    • 14. Serve a regulatory role in either an upstream or a downstream event
  • R0 = 4.9 nm
    Common Methods to Analyze Protein-Protein Interactions
    Biochemical and biophysical approaches
    • Affinity chromatography
    • 15. Coimmunoprecipitation
    • 16. Fluorescence Resonace Energy Trasfer (FRET)
    • 17. Surface Plasma Resonance
    • 18. Atomic Force Microscopy (AFM)
    • 19. X-ray Diffraction
    Molecular genetic approaches
    • Yeast Two-hybrid
  • Why Yeast Two-Hybrid system
    The yeast two hybrid system has a clear advantage over classicalbiochemical or genetic methods
    • It is an in vivo technique that uses the yeast cell as a living test-tube.
    • 20. It bears a greater resemblance to higher eukaryotic systems than a system based on a bacterial host.
    • 21. With regards to classical biochemical approaches, which can require high quantities of purified proteins or good quality anti-bodies, the two hybrid system has minimal requirements to initiate screening, since only the cDNA of the gene of interest is needed.
    • In signaling cascades, weak and transient interactions are often very important. Such interactions are more readily detected with the two hybrid system since the reporter gene response often leads to significant amplification.
    • 22. The two hybrid assay is also useful for analysis of known interactions, which can be achieved by modifying important residues or modules and observing this effect on binding.
    • 23. Interactions can be measured semi-quantitatively using the two hybrid system, allowing discrimination between high, intermediate, and low-affinity bindings, the power of which correlates with that of in vitro approaches
    • Two hybrid screens are sometimes termed "functional screens", since if at least one of the proteins screened has a known function in a well-defined pathway, it might provide a functional hint in the current interaction.
    • 24. Although there are certain disadvantages involving the two hybrid assay, the most convincing argument for its use is the speed and ease by which the molecular mechanisms of many signaling cascades have been defined using this technique.
  • An Introduction to Yeast
    Saccharomycescerevisiae is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much likeE.coli as the model prokaryote.
    Saccharomycescerevisiae cells are round to ovoid, 5–10 micrometers in diameter. It reproduces by a division process known as budding
    S. cerevisiae was the first eukaryoticgenome that was completely sequenced. (April 1996)
    The genome is composed of about 13,000,000bp and 6,275 genes, although only about 5,800 of these are believed to be true functional genes.
  • 25. The classical yeast two-hybrid system
    The early yeast two-hybrid systems were based on the finding that many eukaryotic transcription factors have separable DNA-binding and transcription activation
  • 26.
    • The protein of interest, the “bait”, is fused to a DNA-binding domain.
    • 27. Proteins that bind to bait, the “fish” or “prey”, are fused to a transcription activation domain.
    • Proteins that do not bind to the bait will not activate the transcription of the reporter gene (HIS in this case)
    • 28. Any protein that binds to the bait will activate the transcription of the reporter gene
    (a) No binding
    (b) Binding between protein and bait
  • 29. The first step is to construct a bait plasmid and a library. Each type of plasmid contains a selectable marker such as an essential amino acid.
  • 30.
  • 31.
  • 32.
  • 33. Overall summary of Yeast two-hybrid experiment
    • Yeast two-hybrid experiments yield information on protein protein interactions
    • 34. GAL4 Binding Domain
    • 35. GAL4 Activation Domain
    • 36. X and Y are two proteins of interest
    • 37. If X & Y interact then reporter gene is expressed
  • Major applications of classical system
    Used to determine whether two known proteins interact with one another
    Used to identify unknown proteins, encoded by a cDNA library, that interact with a protein of interest
    Powerful tool for investigating the network of interactions that form between proteins involved in particular biological processes
  • 38. Selection of host strain
    The yeast strains used for two-hybrid experiments carry mutations in a number of genes required for amino acid biosynthesis, such as TRP1, LEU2, HIS3 and URA3.
    If these amino acids are omitted from the growth medium the yeast strain will fail to grow.
    Many of the two-hybrid plasmids carry genes that complement these mutations and allow for selection of the transformant yeast.
  • 39. Reporter Genes
    • LacZ reporter - Blue/White Screening
    • 40. HIS3 reporter - Screen on His+ media (usually need to add 3AT to increase selectivity)
    • 41. LEU2 reporter - Screen on Leu+ media
    • 42. ADE2 reporter - Screen on Ade+ media
    • 43. URA3 reporter - Screen on Ura+ media (can do negative selection by adding FOA)
  • Modifications of the Yeast Two-Hybrid system
    The hSos recruitment system
    Three protein system
    The dual-bait system
    The reverse two-hybrid system
  • 44. The hSos recruitment system
    • Plasmid encoding X fused in-frame to (Ras GEF) hSos
    • 45. Plasmid encoding Y fused in-frame with a v-Srcmyristylation signal
  • Procedure
    Two plasmids are constructed, one encoding protein X fused in-frame to the human Rasguanyl nucleotide exchange factor (Ras GEF) hSos, the second encoding protein Y fused in-frame with a v-Srcmyristylation signal.
    The plasmids are co-transformed into a temperature-sensitive yeast mutant containing a lesion in the Cdc25 gene which encodes a yeast Ras GEF.
    The protein Y fusion is targeted to the yeast plasma-membrane. Interaction between proteins X and Y recruits hSos to the yeast plasma membrane where it complements the cdc25 mutation by activating the Rassignalling cascade.
    Interactions are detected through growth of yeast cells at the restrictive temperature (37 ◦C).
  • 46. Applications
    Cytoplasm-based yeast two-hybrid system
    Do not rely on a transcriptional readout
    Transcriptional repressors
    Auto-activation of reporter genes by bait constructs
    The problem of certain proteins not localizing to the yeast cell nucleus
  • 47. Three protein system
  • 48. Procedure
    This system is based on the classical yeast two-hybrid system. Proteins X and Y are expressed in-frame with a transcription factor DNA-binding domain and transcription activation domain, respectively.
    A third protein, Z, is expressed with a nuclear localization signal, without any added domains, in the yeast nucleus.
    Protein Y may only interact with X in the presence of Z.
    (i) A domain formed through the interaction between X and Z may provide an interaction interface for protein Y.
    (ii) Alternatively, protein Z may act as a bridge between proteins X and Y
  • 49. The dual-bait system
    X1 fused to DNA-binding domain LexA
    X2 fused to DNA-binding domain λcI
  • 50. Procedure
    Two test proteins (X1 and X2) are fused to two different DNA-binding domains (LexA and λcI, respectively).
    The two fusion constructs are co-expressed in the same yeast cell and tested for interaction with proteins fused to the B42 transcription activation domain.
    Interaction with X1 induces expression of LexA-dependent reporter genes (lacZ and LEU2).
    Interaction with X2 induces expression of λcI-dependent reporter genes (LYS2 and gusA)
  • 51. Applications
    Two test proteins can be analyzed for protein-protein interaction partners in a single library screening
    To test the specificity of a protein-protein interaction amongst evolutionarily conserved proteins
    To identify domains or residues required for interaction with one partner but not another
  • 52. The reverse two-hybrid system
  • 53. Procedure
    This system is based on the classical two-hybrid system except that expression of the reporter gene is toxic to the yeast cells under certain conditions.
    In this example, the reporter gene URA3 allows for selection of protein-protein interactions between X and Y on media minus uracil and counter-selection of disrupted protein-protein interaction between X and Y on media containing 5FOA.
  • 54. Applications
    This system can be used to identify residues required for protein-protein interaction by making use of a mutagenised copy of the cDNA encoding one of the proteins.
    cDNAs encoding proteins no longer able to interact can be sequenced to reveal amino acids essential for interaction.
  • 55. What is the yeast two-hybrid system used for?
    • Identifies novel protein-protein interactions
    • 56. Can identify protein cascades
    • 57. Identifies mutations that affect protein-protein binding
    • 58. Can identify interfering proteins in known interactions (Reverse Two-Hybrid System)
  • Advantages
    • Immediate availability of the cloned gene of the interacting protein
    • 59. Only a single plasmid construction is required
    • 60. Interactions are detected in vivo
    • 61. Weak, transient interactions can be detected
    • 62. Can accumulate a weak signal over time
    • 63. Protein purification not necessary
    • 64. No antibodies requries
    Two hybrid systems - > to uncover unanticipated interactions
  • 65. Disadvantages
    • False positives are the largest problem with the yeast two-hybrid system. Can be caused by the ability of bait to induce transcription without interaction with the bait
    • 66. Possible incorrect protein folding in yeast
    • 67. gene encoding target protein must be available
    • 68. failed to detect some know interactions
    Elimination of False Positives
    • Sequence Analysis
    • 69. Retransformation of both strain with bait plasmid and strain without bait plasmid
    • 70. Test for interaction with an unrelated protein as bait
  • Examples of Uses of the Yeast Two-Hybrid System
    • Identification of caspase substrates
    • 71. Interaction of Calmodulin and L-IsoaspartylMethyltransferase
    • 72. Genetic characterization of mutations in E2F1
    • 73. Peptide hormone-receptor interactions
    • 74. Pha-4 interactions in C. elegans
  • WAOOW !! This is my favorite protein
    My bad day!
    When my protein will come?
    I don't like that at all.