Download Fundamentals of Protein Interaction Network Mapping

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