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Fundamentals of Protein Interaction Network Mapping Authors: Jamie Snider, Max Kotlyar, Punit Saraon, Zhong Yao, Igor Jurisica and Igor Stagljar Published: December 2015, Molecular Systems Biology Presentation Outline • Why are Protein-Protein Interactions (PPIs) Important? • Methods for Detecting PPIs • Overview of 14 methods • Conclusions • Questions Why are Protein-Protein Interactions (PPIs) Important? • Proteins can act as: • • • • Molecular machines Sensors Transporters Structural elements • Proteins rarely act alone • Rely on interactions with other proteins to perform cellular functions https://www.youtube.com/watch?v=y-uuk4Pr2i8 In order to understand cellular functions, signaling, and disease states, we must understand these protein-protein interactions! Yeast Two Hybrid (Y2H) Method • Developed by Fields & Song in 19891 • Used to detect interactions between: • Two proteins • Proteins and nucleic acids • Small-molecule screens • One of the most popular PPI methods https://en.wikipedia.org/wiki/Two-hybrid_screening 1. Fields, Stanley, and Ok-kyu Song. "A novel genetic system to detect protein protein interactions." (1989): 245-246. Yeast Two Hybrid (Y2H) Method Legend AD – Activation domain BD – DNA-binding domain Prey – DNA sequence for prey protein Bait – DNA sequence for bait protein Genetically Modified Yeast Cell Yeast Two Hybrid (Y2H) Method Genetically Modified Yeast Cell “Prey” Protein “Bait” Protein DNA Binding Domain Yeast Two Hybrid (Y2H) Method Genetically Modified Yeast Cell “Prey” Protein “Bait” Protein DNA Binding Domain Promotor Region Reporter Gene (HIS3) DNA Segment Yeast Two Hybrid (Y2H) Method Genetically Modified Yeast Cell “Prey” Protein “Bait” Protein DNA Binding Domain Promotor Region Reporter Gene (HIS3) DNA Segment Yeast Two Hybrid (Y2H) Method Genetically Modified Yeast Cell “Prey” Protein “Bait” Protein DNA Binding Domain Promotor Region Reporter Gene (HIS3) DNA Segment Yeast Two Hybrid (Y2H) Method Yeast Cell Dies! Genetically Modified Yeast Cell “Prey” Protein “Bait” Protein DNA Binding Domain Promotor Region Reporter Gene (HIS3) DNA Segment Yeast Two Hybrid (Y2H) Method Advantages • • • • Simple Well established Low cost Scalable for large-scale screening studies • No need for cell lysis Limitations • Yeast host limits PPIs • No post translation modifications • No cofactors or other binding partners • Cannot be used on membrane bound proteins Membrane Yeast Two Hybrid (MYTH) Method • Developed by Staglijar et al, 1998 • Designed specifically for analysis of membrane proteins • Based on a splitubiquitin approach Thaminy, Safia, et al. "Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system." Genome research 13.7 (2003): 1744-1753. 1. Stagljar, Igor, et al. "A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo." Proceedings of the National Academy of Sciences 95.9 (1998): 5187-5192. Membrane Yeast Two Hybrid (MYTH) Method Transcription Factor Promotor Region Ubiquitone Reporter Gene (HIS3) DNA Segment Membrane Yeast Two Hybrid (MYTH) Method Advantages • Simple • Low cost • Scalable for both lowand high-throughput formats • Can detect PPIs between membrane bound proteins • At least one protein HAS to be membrane bound Limitations • Yeast host limits PPIs • No post translation modifications • No cofactors or other binding partners • Membrane proteins must have at least one terminus in the cytosol Luminescence-Based Mammalian Interactome Mapping (LUMIER) Method • Developed by BarriosRodiles et al, 2005 • Coimmunoprecipitationbased approach • Utilizes Renilla luciferase to determine proteinprotein interactions https://www.dkfz.de/gpcf/757.html 1. Barrios-Rodiles, Miriam, et al. "High-throughput mapping of a dynamic signaling network in mammalian cells." Science 307.5715 (2005): 1621-1625. Luminescence-Based Mammalian Interactome Mapping (LUMIER) Method Affinity Tag Antibody (anti-affinity tag) Luminescence-Based Mammalian Interactome Mapping (LUMIER) Method Advantages • Can be used in a highthroughput format • No specialized equipment needed • Approach can be used on different cell lines Limitations • Requires lysis of cells • Can disrupt weak/transient PPIs • Need careful controls to account for background fluorescence • Cannot study how PPIs change spatially or over time Mammalian Protein-Protein Interaction Trap (MAPPIT) Method • Developed by Eyckerman et al, 2001 • Based on a cytokine signal transduction mechanism (JAK/STAT Pathway) 1. Eyckerman, Sven, et al. "Design and application of a cytokine-receptor-based interaction trap." Nature cell biology 3.12 (2001): 1114-1119. Mammalian Protein-Protein Interaction Trap (MAPPIT) Method Mammalian Protein-Protein Interaction Trap (MAPPIT) Method Nucleus Transcription of reporter genes (Luciferase) Mammalian Protein-Protein Interaction Trap (MAPPIT) Method Advantages Limitations • Allows for direct PPI examination in mammalian cells • Suitable for highthroughput screening • Requires no specialized equipment • Method requires that PPI occur in the cytoplasmic sub-membrane region • Size of bait-tag may block certain PPIs due to steric hindrance • Not compatible with fulllength transmembrane proteins Kinase Substrate Sensor (KISS) Method • Developed by Tavernier et al in 2012 • Utilizes TYK2-gp130 coupling to activate STAT3 and regulate STAT3-dependent reporter systems (i.e. luciferase) 1. 2. Tavernier, Jan, and Samuel Lievens. "Kinase substrate sensor." U.S. Patent Application No. 14/002,320. Lievens, Sam, et al. "Kinase Substrate Sensor (KISS), a mammalian in situ protein interaction sensor." Molecular & Cellular Proteomics 13.12 (2014): 33323342. Kinase Substrate Sensor (KISS) Method STAT3 Recruitment Sites TYK2 Nucleus Kinase Substrate Sensor (KISS) Method STAT3 Recruitment Sites TYK2 Nucleus Transcription of reporter genes (Luciferase) Kinase Substrate Sensor (KISS) Method Advantages Limitations • Effective for use with both membrane and cytosolic proteins • Allows for direct PPI examination in mammalian cells • Suitable for highthroughput screening • Requires no specialized equipment • No spatial analysis of PPIs • Does not allow for analysis of PPIs involved in STAT signaling Biomolecular Fluorescence Complementation (BiFC) Method • Developed by Hu et al in 2002 • Based on the division of a fluorescent protein into two nonfluorescent fragments • Most commonly used fluorescent protein is Yellow Fluorescent Protein (YFP) 1. Rizzuto R, Brini M, Pizzo P, Murgia M, Pozzan T. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 1995; 5 (6):635-42. / pmid: 7552174 Hu, Chang-Deng, Yurii Chinenov, and Tom K. Kerppola. "Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation." Molecular cell 9.4 (2002): 789-798. Biomolecular Fluorescence Complementation (BiFC) Method N-YFP C-YFP Biomolecular Fluorescence Complementation (BiFC) Method Advantages Limitations • Allows for direct visualization of PPIs in living cells! • Can be used for membrane bound, cytosolic, or organelle bound proteins • Different fluorescent proteins can be used in parallel for multiple PPI interactions • There is a delay in generation of fluorescence upon protein interaction • Fluorochrome formation is irreversible • Non-specific interactions can cause false-positives Fluorescence Resonance Energy Transfer (FRET) Method • Developed by Clegg et al in 1995 • Utilizes the concept of energy transfer to determine protein interaction • PPI detected by change in emission signal 1. Clegg, Robert M. "Fluorescence resonance energy transfer." Current opinion in biotechnology 6.1 (1995): 103-110. Fluorescence Resonance Energy Transfer (FRET) Method Excitation: 450nm Emission: 500nm No Interaction! Fluorescence Resonance Energy Transfer (FRET) Method Emission: 530nm Excitation: 450nm Detected Protein Interaction! Fluorescence Resonance Energy Transfer (FRET) Method Advantages Limitations • Monitor instantaneous, real-time PPIs • Direct detection in live cells • Complex interaction dynamics can be monitored • Protein fusions to appropriate fluorophores may be difficult • Strong background autofluorescence in cells • Photobleaching can occur over time Bioluminescence Resonance Energy Transfer (BRET) Method • Developed by Xu et al in 1999 • Similar to FRET but uses bioluminescence protein, eliminating need for excitation signal 1. Xu, Yao, David W. Piston, and Carl Hirschie Johnson. "A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins." Proceedings of the National Academy of Sciences 96.1 (1999): 151-156. Bioluminescence Resonance Energy Transfer (BRET) Method Light: 480nm No Interaction! Bioluminescence Resonance Energy Transfer (BRET) Method Light: 530nm Detected Protein Interaction! Bioluminescence Resonance Energy Transfer (BRET) Method Advantages • Does not require excitation illumination • Can be used on photosensitive cell types (i.e. retina) • No photobleaching as with FRET • Minimized background noise as compared to FRET Limitations • Protein fusions to appropriate fluorophores may be difficult • Signal tends to be significantly weaker as compared to FRET Mammalian membrane two hybrid(MaMTH) In vivo technology To analyze mammalian membrane PPIs MaMTH CubNub TF TF MaMTH MaMTH: Pros and Cons • Pros: • Analysis of full length mammalian membrane PPIs in cellular context. • Suitable for weak/transient and dynamic interactions. • Low cost, scalable, and no specialized equipment is needed. • Cons: • Works only for membrane associated bait. • Not suitable for spatial or real-time temporal analysis of PPIs. Affinity purification-mass spectrometry(AP-MS) • Affinity purification AP-MS Breakdown Bait is immobilized with solid Support. Bait captures a prey from Soluble target through affinity purification Captured protein is “cut” into pieces, Ionized and detected using MS AP-MS Pros and Cons • Pros: • Library independent method with true genomewide HT capability • Proteins are purified in their natural form, eliminating need of interrogating multiple isoforms • Epitope tagging allows study of proteins for which native antibodies are not available • Cons: • Needs to perform cell lysis and affinity purification barring detection of spatial or temporal PPIs • Data anaylsis is more difficult, and expertise is needed for MS Proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS) BioID-MS Breakdown Biotin is vitamin H Proximal proteins are biotinylated by BirA. Then they can be captured for detection, Immobilization or affinity purification. (avidin-biotin affinity interaction) Bait is fused to biotin-ligase Molecule BirA. Preys are Captured by the bait. Biotin attached molecules can Be immobilized with avidin. These Captured proteins are identified Using MS> BioID-MS Pros and Cons • Pros: • PPIs are detected in their natural cellular context • Instability coming from cell lysis is avoided • Can detect weak/transient interactions • More effective at detecting low abundance proteins • Cons: • BirA can compromise bait’s targeting or function • Low expression levels of PPI partners can lead to false negative • Requires expertise in specific bioinformatics tools and MS Proximity ligation assay (PLA) In situ detection of PPIs in fixed cells And tissues Proximity ligation assay (PLA) Two DNA strands interact through Addition of two additional cycle forming Strands. Proximity probe: Antibodies + DNA oligonucleotide s A cycle is formed and amplified resulting in Long DNA sequence. Long sequence is bound by fluorophoreLabeled probes allowing visualization. PLA: Pros and Cons • Pros: • Ability to detect and localize single molecule resolution and objectively quantify them in cells and tissues • Can monitor transient or weak interactions • Cons: • Dependence on enzyme makes the method expensive • Use of antibodies makes it costly and they are not always available for all proteins • Not suited for high throughput screening Ligand-receptor capturetrifunctional chemoproteomics reagents (LRC-TriCEPS) ligand Biotin streptavidin receptor LRC-TriCEPS Breakdown Cell is lysed (membrane is broken down) Cell is enzymatically digested w/ trypsin Peptides are purified via biotin tag Peptides are freed and identified using MS LRC-TriCEPS: Pros and Cons • Pros: • Ability to detect ligand-receptor interactions w/o genetic manipulation • Effective for detecting transient surface interactions • Can identify various cell-surface binding partners • Cons: • Only useful for N-glycoprotein receptors • Coupling of ligands with TriCEPS might interfere with functions/binding Avidity-based extracellular interaction screen (AVEXIS) Avidity: “the accumulated strength of multiple affinities of individual non-covalent binding interactions” This technique is used to detect extra-cellular Receptor-ligand pairs in cellular recognition Process. AVEXIS • Step 1: collect secreted protein using expression of recombinant proteins AVEXIS • Step 2: Strengthening Prey and Capturing Baits on Solid phase • Step 3: Apply different preys to the affixed baits AVEXIS: Pros and Cons • Pros: • Can detect very poor PPI associated with membraneembedded receptor proteins. • Has been adapted for use on a higher-throughput scale. • Cons: • Limited to the study of membrane proteins with self contained extracellular domains. • Selecting, preparing and validating is a lengthy process. Conclusions • Understanding protein-protein interaction is critical to understanding cellular functions • Due to this need, many variations of determining PPIs have been developed • Every PPI method has its advantages and limitations • Much choose your PPI method carefully! Questions? FRET Em/Ex Profiles Mammalian Membrane Two Hybrid (MaMTH) Method • Developed by Petschnigg et al. in 2014 • Similar to MYTH but utilized for mammalian systems • Uses luciferase as a reporter gene 1. Petschnigg, Julia, et al. "The mammalian-membrane two-hybrid assay (MaMTH) for probing membrane-protein interactions in human cells." Nature methods 11.5 (2014): 585-592. Mammalian Membrane Two Hybrid (MYTH) Method Ubiquitone Transcription Factor Promotor Region Reporter Gene (Luciferase) DNA Segment Mammalian Membrane Two Hybrid (MaMTH) Method Advantages • Simple • Low cost • Scalable for both lowand high-throughput formats • Can detect PPIs between membrane bound proteins • Can be used in multiple cell lines Limitations • Membrane proteins must have at least one terminus in the cytosol • Not suitable for spatial or real-time analysis