Protein-Protein Interactions.pdf

Protein-Protein Interactions BY ARLEEN YOUSUF  PROTEIN-PROTEIN INTERACTIONS 1 2 3 Proteins, Almost all of the cellular Proteins can carry out Biomolecules or processes require that...

Protein-Protein Interactions BY ARLEEN YOUSUF  PROTEIN-PROTEIN INTERACTIONS 1 2 3 Proteins, Almost all of the cellular Proteins can carry out Biomolecules or processes require that their roles by interacting macromolecules, proteins specifically with other molecules, performs a wide range recognize a multitude of including DNA, RNA, of functions in different interaction Proteins, and small organisms. partners. molecules.  PROTEIN-PROTEIN INTERACTIONS protein-protein interactions (PPIs) refer to intentional physical contacts established between two or more proteins as a result of biochemical events and/or electrostatic forces. Human genome: 25-30,000 genes ~500,000 different proteins ~10,000 proteins expressed in a cell ~80% of proteins operates in oligomeric states  Biological Effects of Protein-Protein Interactions PPIs play important roles in various biological processes, including cell- to-cell interactions, cell cycle progression, signal transduction, and metabolic pathways. 1. PPIs can alter the kinetic properties of enzymes, which may lead to subtle changes in substrate binding or allosteric effects. 2. PPIs can act as a general mechanism to allow for substrate channeling by moving a substrate between domains or subunits. 3. PPIs can create a novel binding site for small effectors molecules. 4. PPI can inactivate or suppress a protein. 5. PPI can change the specificity of a protein for its substrate by interacting with different binding parameters. 6. In a upstream or a downstream event, PPIs can work as a regulatory role. Types of the Protein-Protein Interactions Composition Affinity Lifetime  Types of the Protein-Protein Interactions Homo- PPI occurs between oligomer identical chains COMPOSITIONS Hetero- oligomer PPI occurs between non- identical chains GroEL consists of 14 identical subunits of 57 kDa each forming two heptameric rings arranged back-to-back. A chaperonin protein is formed by seven GroEL proteins associating as a homo-heptamer. Several enzymes, carrier proteins, scaffolding proteins, and transcriptional regulatory factors carry out their functions as homo-oligomers. Types of the Protein-Protein Interactions Constituents (promoters, Obligate monomers) of a complex are unstable on their own in vivo Affinity Non- The components of non-obligate obligate interactions can exist independently Ku protein, which are involved in DNA repair, are shown to bind DNA as obligate homodimers.  Types of the Protein-Protein Interactions The components of transient Transient interaction associate and dissociate temporarily in vivo Lifetime Permanent Permanent interactions are usually very stable and irreversible Types of the Protein-Protein Interactions Components are stable only in complex form. Association r dissociation takes place under certain triggers. Complexes are broken and formed continuously and Kd is in a micromolar range. The domain-peptide complexes are mostly transient Types of the Protein-Protein Interactions  Protein-Protein Interactions in Drug Development Targeted modulation of PPIs with small molecules is one of the most promising approaches with more than 50 PPIs that have successfully been targeted with small molecules. Most of these are inhibitors of PPIs, but stabilization of PPIs is also a strategy for targeted PPI modulation. Protein-Protein Interactions in Drug Development Techniques to Detect Protein- Protein Interaction Detection Techniques: Co-Immunoprecipitation Yeast Two-Hybrid System Fluorescence Resonance Energy Transfer Biomolecular Fluorescence Complementation Tandem Affinity Purification GST Pull-Down Far-Western Blot CO-IMMUNOPRECIPITATION Application of Co-Immunoprecipitation Co-immunoprecipitation can be used to: Determine whether two target proteins are bound in the body. Identify a new role for a particular protein Isolate and obtain the interaction protein complex in its natural state Co-immunoprecipitation Co-immunoprecipitation YEAST TWO-HYBRID SYSTEM  Yeast Two-Hybrid System Quickly and directly analyze the interactions between known proteins Search for and isolate ligands that interact with known proteins Discover new proteins and discover new functions of proteins Screening drug action sites and effects of drugs on protein interactions Create a genomic protein linkage map  Yeast Two-Hybrid System The principle of yeast two-hybrid Two target proteins (proteins X and Y) are constructed into fusion plasmids with BD and AD domains. Constructed plasmids were transferred to homozygous yeast cells for expression. If No interaction between X and Y: the downstream reporter genes would not be transcribed for expression. If Yes interaction: BD and AD are spatially close and the downstream reporter gene is transcribed. Interaction is determined by judging the expression of the reporter gene.  Yeast Two-Hybrid System How to determine whether there is an Interaction? Reporter genes are commonly used as nutritional markers such as HIS3, URA3, LacZ, and ADE2. The corresponding host bacteria are labeled defective cells that must be grown in a medium containing the nutrient marker. Thus, when interacting proteins are present, reporter gene expression is activated so that they can be grown in cultures without nutrient markers to verify the presence of interactions. FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) Detection principle of FRET The target proteins X and Y are coupled with D and A fluorescent proteins, respectively. D and A: fluorescent substances, or donor and acceptor, respectively. When X-fusion protein is excited with 430nm of purple light, it can produce blue fluorescence at 490nm. Similarly, when using 490nm blue light to excite the Y fusion protein, yellow fluorescence at 530nm is produced.  FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) The principle of FRET No interaction between proteins X and Y (spatial distance between the two was >10nm); fusion proteins X and Y are detected by corresponding fluorescence. Yes, interaction between proteins X and Y (space distance