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Protein protein interactions
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- 2. Contents.... Introduction Examples of PPIs Typesof PPIs Protein domains methods to investigate PPIs Protein Interactions Database (PIDs) Applications of PPIs
- 3. gene expression nutrient uptake, intercellularcommunication motility apoptosis cell growth proliferation morphology
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- 5. PPIs refer tointentional physical contacts established between two or more proteins as a result of biochemical events and/or electrostatic forces
- 6. Signal transduction The activityof the cell is regulated by extracellular signals Transport across membranes A protein may be carrying another protein. Cell metabolism In many biosynthetic processes enzymes interact with each other to produce small compounds or other macromolecules. Muscle contraction Myosin filaments act as molecular motors and by binding to actin enables filament sliding. Examples of protein–protein interactions
- 7. TYPES OF PROTEIN–PROTEIN INTERACTIONS Homo-oligomers Hetero-oligomersNon-covalent : Covalent : Transient Stable
- 8. Homo-oligomers are macromolecularcomplexes constituted by only one type of protein subunit Several enzymes, carrier proteins and transcriptional regulatory factors carry out their functions as homo-oligomers. Homo-oligomers Joel Edt and Shoshana Wodak, 2002 Protein subunits assembly is guided by the establishment of non-covalent Interactions in the quaternary structure of the protein E.g. : PPIs in Muscle Contraction Homo-oligomers complex
- 9. Distinct protein subunitsinteract in hetero-oligomers, which are essential to control several cellular functions Hetero-oligomers Heterologous proteins - cell signaling events E.g. : PPI between Cytochrome Oxidase and TRPC3 (Transient receptor potential cat ion channels) Hetero-oligomers complex Eg: Hemoglobin Hb or Hgb
- 10. Covalent : Strongest association- disulphide bonds or electron sharing - Post translational modifications E.g.: ubiquitination and SUMOylation Non-covalent : Established during transient interactions by the combination of weaker bonds Hydrogen bonds, Ionic interactions, Van der waals forces, or Hydrophobic bonds
- 11. Ubiquitination Plays arole in the degradation of defective and superfluous proteins , single-chain polypeptid Ubiquitination (or ubiquitylation) is an enzymatic post- translational modification in which a ubiquitin protein is attached to a substrate protein Steps: activation, conjugation, and ligation, By: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s)
- 12. Transient Interactions : Interactionsthat last a short period of time reversible manner E.g.: G protein-coupled receptors only transiently bind to Gᵢ/ₒ proteins when they are activated by extracellular ligands Stable Interactions: Proteins - interact for a long time, taking part of permanent complexes as subunits -carry out Functional or Structural roles e.g. Cytochrome c
- 13. cytochrome c –**CcO complex Eg: Stable Interactions stabilized by a few electrostatic interactions between long side chains within a small contact surface. In contrast to other Cyt.c complexes, numerous water molecules are found in the long inter‐molecular span between Cyt.c and CcO.. **Cytochrome c oxidase
- 15. Protein Domains • Interactionsonly possible due to structural domains within the proteins • A protein domain is a conserved part of a given protein sequence and (tertiary) structure that can evolve, function, and exist independently of the rest of the protein chain • Proteins hold structural domains that allow their interaction with and bind to specific sequences on other proteins
- 16. 1. phosphotyrosine-containing motifs, -Examples for protein who carry this motif: activated receptors for growth factors, cytokines and antigens. - Recognizing protein protein interaction domain: a. SH2 domains b. PTB domains, also binds unphosphorylated peptides 2. phosphoserine/threonine motifs, - Recognizing protein protein interaction domain: a. 14-3-3 proteins b. FHA domains c. WW domains, also binds unphosphorylated peptides, Proline-rich d. WD40-repeat domains
- 17. 3. acetylation oflysine residues - Proteins who carry the motif: histones - Recognizing proteins: creates binding sites for the Bromo domain 4. methylation of lysine residues - Proteins who carry the motif: histones - Recognizing proteins: creates binding sites for the Chromo domains,
- 18. Other protein-protein interactiondomains Apoptosis DD - death domain DED - Death Effector Domain CARD- caspase recruitment domain BH1-4 - Bcl-2 Homology Chromatin CSD - Cold-shock domain Proteolysis F-box Hect - homologous to the E6AP carboxyl terminus RING - really interesting new gene Dimerization • SAM - Sterile α Motif Vessicle Traffic • GYF • Snare • VHS Undefined • ANK • ARM • WD40 • LIM
- 19. Src homology 2(SH2) domain Role - cellular communication Structure - contains 2 alpha helices and 7 beta strands It has a high affinity to phosphorylated tyrosine residues It is known to identify a sequence of 3-6 amino acids within a peptide motif Represent the largest class of known pTyr-recognition domains.
- 20. PPIs Identification Methods •Yeast two-hybrid system • split ubiquitin system • split lactamase / split galactosidase system • split yellow fluorescent protein (YFP) system Experimental ((In vivo • Co-immunoprecipitation • Tagged Fusion Proteins • X-ray Diffraction • Biacore • Phage display Experimental ((In vitro • BIND • DIP • MINT • IntAct Computational (In silico)
- 21. Methods to InvestigatePPIs Immuno-precipitation, Protein microarrays, Analytical ultracentrifugation, Light scattering, Fluorescence spectroscopy, Resonance-energy transfer systems, Surface Plasmon resonance, protein-fragment complementation assay, and Calorimetry etc… The two most prominent methods used for investigating PPIs are: Yeast two-hybrid screening and Affinity purification coupled to mass spectrometry Xue-Wen Chen andMei Liu
- 22. Yeast two-hybrid • Bait– The protein fused to the DBD is referred to as the ‘bait’ (yeast transcription factor, like Gal4) • Prey- The protein fused to the AD • Reporter gene: LacZ reporter - Blue/White Screening • Testing for physical interactions between two proteins • first proven using Saccharomyces cerevisiae as biological model by Fields and Song
- 23. Co-immunopercipitation • Co-IP isa classic technology widely used for protein-protein interaction identification and validation • New binding partners, binding affinities, the kinetics of binding and the function of the target protein Principle of co-Immunoprecipitation
- 24. The advantage ofthis technology includes: • Both the bait and prey proteins are in their native conformation in the co-IP assay • The interaction between the bait and prey proteins happens in vivo with little to no external influence The limitation of this technology lies in • Low affinity or transient interaction between proteins may not be detected.
- 25. Yeast two-hybrid Saccharomyces cerevisiaeas biological model by Fields and Song One technique that can be used to study protein-protein interactions is the "yeast two hybrid" system transcription requires both the DNA-binding domain (BD) and the activation domain (AD) of a transcriptional activator (TA) Normal Transcription
- 26. If protein Xand protein Y interact, then their DNA-binding domain and activation domain will combine to form a functional transcriptional activator (TA). The TA will then proceed to transcribe the reporter gene that is paired with its promoter Basic principle
- 27. The yeast two-hybridassay uses two plasmid constructs Bait plasmid, Hunter plasmid The bait plasmid, which is the protein of interest fused to a GAL4 binding domain, and the hunter plasmid, which is the potential binding partner fused to a GAL4 activation domain Selection genes encoding for amino acids, such as histidine, leucine and tryptophan Solmaz Sobhanifar (2005)
- 28. Plasmid construction The'bait' DNA is isolated and inserted into a plasmid adjacent to the GAL4 BD DNA. When this DNA is transcribed, the 'bait' protein will now contain the GAL4 DNA- binding domain as well. The ‘Prey‘/ Hunter fusion protein contains the GAL4 AD Solmaz Sobhanifar (2005)
- 29. Transfection : The'bait' and 'hunter' plasmids are introduced into yeast cells by transfection. cells containing both plasmids are selected for by growing cells on minimal media. Only cells containing both plasmids have both genes encoding for missing nutrients, and consequently, are the only cells that will survive. Solmaz Sobhanifar (2005)
- 30. Transcription of reportergene No transcription of reporter gene The reporter gene most commonly used in the Gal4 system is LacZ, an E. coli gene whose transcription causes cells to turn blue4 LacZ gene is inserted in the yeast DNA immediately after the Gal4 promoter Solmaz Sobhanifar (2005)
- 31. Identify novel protein-proteininteractions Characterize interactions already known to occur protein domains Conditions of interactions manipulating protein-protein interactions in an attempt to understand its biological relevance To know how mutation affects a protein's interaction with other proteins Applications
- 32. Genome-wide protein-protein interactionnetworks in different organisms Bang et al 2011
- 33. Fang et al.,2002 List of rice genes used as baits for YTH screening
- 34. Fang et al.,2002 List of interacting proteins found for eight bait proteins
- 35. Protein interactions database Protein interactions are collected together in specialized biological databases Databases can be subdivided into primary databases, meta-databases, and prediction databases Primary databases - published PPIs proven to exist via small-scale large- scale experimental methods. Eg: DIP, Biomolecular Interaction Network, BIND, BioGRID), HPRD Meta-database – Primary and original data Eg: APID, The Microbial, MPIDB, and PINA , and GPS-Prot etc. Prediction Databases – predicted using several techniques Eg: Human Protein–Protein Interaction Prediction Database (PIPs), I2D, STRING, and Unified Human Interactive (UniHI).
- 36. BIND (Biomolecular Interaction NetworkDatabase) • http://bind.ca • A free, open-source database for archiving and exchanging molecular assembly information. • The database contains - Interactions - Molecular complexes - Pathways
- 37. PPI methodologies havebeen developed in yeast-methods are sometimes not suitable for plant systems Proteomic approaches still challenging International Plant Proteomics Organization (www.inppo.com), global initiative to develop and improve connections between plant proteomics researchers and related fields Conclusions
Editor's Notes
- #5 Protein–protein interactions occur when two or more proteins bind together In fact, proteins are vital macromolecules, at both cellular and systemic levels, but they rarely act alone identification of interacting proteins can help to elucidate their function Aberrant PPIs are the basis of multiple diseases, such as Creutzfeld-Jacob, Alzheimer's disease, and cancer.
- #6 Protein–protein interactions occur when two or more proteins bind together In fact, proteins are vital macromolecules, at both cellular and systemic levels, but they rarely act alone identification of interacting proteins can help to elucidate their function Aberrant PPIs are the basis of multiple diseases, such as Creutzfeld-Jacob, Alzheimer's disease, and cancer.
- #7 Physiology of muscle contraction involves several interactions.
- #11 Strongest association formed by the - disulphide bonds or electron sharing. sUMO small ubiquitin-like modifiers
- #13 These comprise of interactions that last for a long duration
- #14 The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between long side chains within a small contact surface. In contrast to other Cyt.c complexes, numerous water molecules are found in the long inter‐molecular span between Cyt.c and CcO. This new interaction mode likely contributes to the rapid association/dissociation of the Cyt.c–CcO complex.
- #18 A bromodomain is an approximately 110 amino acid protein domain that recognizes acetylated lysine residues
- #20 SH2 domains play a vital role in cellular communication. Its length is approximately 100 amino acids long and it is found within 111 human proteins.[5] Regarding its structure, it contains 2 alpha helices and 7 beta strands. Research has shown that it has a high affinity to phosphorylated tyrosine residues and it is known to identify a sequence of 3-6 amino acids within a peptide motif.
- #22 There are many methods to investigate Protein-Protein Interactions namely
- #23 The most common screening approach.
- #24 analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein Antibodies that are specific for a particular protein (or group of proteins) are immobilized on a solid-phase substrate such as agarose beads. The beads with bound antibodies are then added to the protein mixture, and the proteins that are targeted by the antibodies are captured onto the beads via the antibodies; in other words, they become immunoprecipitated.
- #28 Yeast two-hybrid assays typically use selection genes encoding for amino acids, such as histidine, leucine and tryptophan Hunter - Constructed as a library (several potential interacting proteins)
- #32 determining which protein domains are responsible for the interaction what conditions interactions take place, by altering the intracellular environment