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Non-Invasive Imaging of Protein Interactions within Living Models


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Title: Non-Invasive Imaging of Protein Interactions within Living Models

Non-Invasive Imaging of Protein Interactions
within Living Models
  • Sean Courtney

Why study Protein Interactions?
  • Regulates many of the essential biological
  • Transcription
  • Translation
  • Metabolic pathways
  • Signal Transduction
  • Cell Cycle Progression
  • etc.
  • Yields information on possible roles of genes
    with as of yet unknown functions
  • Detects novel interactions between proteins of
    other various functions

Protein-Protein Interaction Techniques
  • Split Ubiquitin
  • Protein Fragment Complementation
  • Yeast Two-Hybrid

Split Ubiquitin
  • Fusions of Ubiquitin (Ub) and a target protein
    are recognized and cleaved by Ub-specific
    Proteases (UBPs), that recognize the folded
    conformation of Ub not the sequence
  • Ub can be expressed in yeast as an N-terminal
    half (Nub) and a C-terminal half (Cub), which
    have affinity for each other and spontaneously
    assemble forming the split ubiquitin
  • If Nub and Cub are coexpressed in a single cell,
    the reporter protein will be cleaved upon
  • The bait protein is fused to Cub followed by a
    reporter protein and a prey protein is fused to a
    mutated Nub (NubG, lost affinity)
  • If interaction occurs then the NubG and Cub are
    brought into proximity allowing reassembly and
    cleavage by the UBPs, thus releasing the reporter

Dualsystems Biotech
Protein Fragment Complementation
  • Uses an enzyme that usually produces a detectable
    product (colorimetric, fluorometric, or survival)
  • i.e. murine dihydrofolate reductase (mDHFR)
    reduces dihydrofolate into tetrahydrofolate
  • A bait protein is fused to one part of the
    enzyme and a prey protein is fused to the other
    part of the enzyme, both of which are
    transfected/transformed into cells
  • If the proteins interact, they bring the subunits
    of the enzyme within a close proximity, thus
    enabling their reassembly into an active enzyme,
    upon which the enzymes substrate is added and
    the product can then be detected

Michnick et al. 1998
Yeast Two-Hybrid
  • Gal4 is a yeast transcription factor
  • Each Gal4-responsive gene contains a target
    sequence, UAS
  • When Gal4 binds the UAS, transcription is
    activated from a downstream promoter
  • Bait gene fused to a GAL4 DNA-BD and a cDNA
    library (or another known protein) fused to a
  • GAL4 DNA-BD can bind the UAS alone but cannot
    activate transcription until bound with the GAL4
  • When the two domains interact, BD and AD are
    brought into proximity, thus activating
    transcription of a downstream reporter gene

Various Imaging Techniques
  • Magnetic Resonance Imaging (MRI)
  • Positron Emission Tomography (PET)
  • Single-Photon Emission Computed Tomography
  • Fluorescence Resonance Energy Transfer (FRET)
  • Fluorescence
  • Bioluminescence

Magnetic Resonance Imaging (MRI)
  • Used to visualize the inside of living organisms
  • Demonstrates pathological or other physiological
    alterations of living tissues (i.e. tumors)
  • Uses radio frequency signals to acquire images
  • Based on the relaxation properties of excited
    Hydrogen nuclei in water

MR Imaging Ability
  • http//

Positron Emission Tomography (PET)
  • A nuclear medicine imaging technique that
    produces a 3D image or map of functional
    processes in the body
  • Uses a short-lived radioactive tracer isotope
    which decays by emitting a positron (has been
    chemically incorporated into a metabolically
    active molecule) and is injected into the living
    animal, usually in the blood
  • A waiting period ensues while the metabolically
    active molecule (usually fluorodeoxyglucose, FDG)
    becomes concentrated in tissues of interest
  • Result of two simultaneous annihilation photons
    emitted back-to-back
  • The image produced is not the location of the
    radionucleoside but that of where the
    annihilation event occurs
  • Commonly used alongside CT scans or MRI scans,
    giving both anatomic and metabolic information

Single-Photon Emission Computed Tomography (SPECT)
  • A nuclear medicine tomographic imaging technique
    using gamma rays able to provide true 3D
  • A 2D view of the 3D distribution of a
    radionucleotide from multiple angles
  • A computer is used to apply a tomographic
    reconstruction algorithm to yield a 3D dataset
  • Can be manipulated to show thin slices along any
    chosen axis of the body

Fluorescence Resonance Energy Transfer (FRET)
  • Energy transfer mechanism between two fluorescent
  • Useful tool to quantify molecular dynamics in
    biophysics, such as protein-protein interactions,
    protein-DNA interactions, and protein
    conformational changes
  • Monitors the complex formation between two
    molecules, one is labeled with a donor and the
    other with an acceptor, which are then mixed
  • When they dissociate, the donor emission is
    detected upon the donor excitation, but when
    together, the acceptor emission is predominant

  • Production and emission of light by a living
    organism as the result of a chemical reaction
    during which chemical energy is converted to
    light energy
  • Uses an external light source with a low-pass
    filter to excite the fluorescent molecules
  • Green Fluorescent Protein, originally found in
    the Aequorea victoria species of jelly fish
  • Been biochemically modified to produce Green,
    Yellow, Blue, Cyan, and Red Fluorescent Proteins
    for use in various research techniques using a
  • Limited by tissue autofluorescence, as well as
    the light being able to first get into the living
    model and sensing the target fluorescent
    molecule, then having that fluorescence get back
    out of the model and to the detector (a lot of
    scattering occurs)

  • Luciferase, an enzyme found in Fireflies, is also
    commonly used as a reporter
  • Must be incorporated into the cell (i.e. tumor
  • Just before imaging, luciferin is added via IV or
    IP, which can then rapidly travel throughout the
    body and where ever it encounters luciferase,
    oxygen, and ATP it will be converted to
    oxyluciferin and produce a detectable light
  • Pros No external light source, no
    autofluorescent background noise from surrounding
    tissues, and depth of penetration is not as
    limiting compared to its fluorescent counterparts
  • Cons Limited to studying genetically modified
    cells, transgenic animal models, or infectious
  • Gives a very weak signal and requires a highly
    efficient Charge-coupled device (CCD) because it
    has a strong dependence of signal intensity on
    source depth

Cherry et al. 2004
How to use the Molecular Imaging Techniques to
View Protein Interactions in Living Models
  • Must first define your target of interest ?
    usually Cell-Surface Receptors or Enzymes
  • Once chosen, need to next choose the method to
    view it in the living model and therefore need to
    also choose the contrast agent
  • Dependent on spatial arrangement and the desired
    resolution, location and distribution of the
    target, concentration of the target, and the
    specificity of the target
  • Need an exogenous agent detectable by its
    physical or chemical properties
  • Common agents include radioactive atoms,
    fluorescent molecules, paramagnetic ions, or
    small molecules covalently linked to the target
    with similar properties as those listed

Various Contrast Agents
Cherry et al. 2004
Cell-Surface Receptor
  • Similar concept to pharmaceuticals in that it
    must find the target in the body and accumulate
  • Binds to the target, unbound portion must be
    cleared from that tissue to be able to
    distinguish the signal between specific and
  • Administration is usually intravenously (IV) into
    the bloodstream where it can rapidly travel
    through the body, sometimes injection is
    intraperitoneal (IP) within the abdominal cavity
  • The target must successfully trap the signal
    molecule within the cell or tissue and thus
    accumulate to provide an adequate signal

Cherry et al. 2004
  • Agents are designed to interact with the enzyme
  • Interaction of the signal agent with the enzyme
    causes a change in the agent (i.e. charge) so
    that it remains in that cell, trapped
  • Gadolinium (Gd3)
  • Activatable agent
  • Highly paramagnetic
  • Enclosed in a molecular case where it is unable
    to interact with water in the tissue
  • Interaction with the target enzyme breaks the
    linker (the lid) thus causing a structural
    change in the molecule where the Gd3 can now
    interact with the water, changing the relaxation

Cherry et al. 2004
Reporter Gene Method of Detection
  • Genetically modify the cell
  • Place the reporter gene under the control of the
    same promoter of the gene of interest
  • cells expressing the reporter also express the
    gene of interest in a ratio of approximately 11
  • This is limiting because the reporter must be
    introduced into the living model
  • Introduce the reporter gene into cancer cell
    lines to track cancer cells and their progeny via
    xenographs and tumor transplant models

Cherry et al. 2004
Various Reporter Gene Systems
Cherry et al. 2004
Noninvasive imaging of protein-protein
interactions in living animals
  • Gary D. Luker, Vijay Sharma, Christina M. Pica,
    Julie L. Dahlheimer, Wei Li, Joseph Ochesky,
    Christine E. Ryan, Helen Piwnica-Worms, and David
  • Molecular Imaging Center, Mallinckrodt Institute
    of Radiology and Departments of Molecular Biology
    and Pharmacology, Cell Biology and Physiology,
    and Internal Medicine, and Howard Hughes Medical
    Institute, Washington University School of
    Medicine, St. Louis, MO 63110
  • Proposal To develop a method for detecting
    protein-protein interactions in living mice by
    combining the yeast two-hybrid system with
    various reporter proteins sufficient for imaging

Construction of Reporter Proteins
  • HSV-1-TK ? Nucleoside analogs are actively
    transported into cells and are preferentially
    phosphorylated by the viral TK and not the
    mammalian TK
  • Previous mutagenesis studies by Black et al.
    showed a mutant HSV-1-TK with enhanced
    sensitivity to 8-18F-fluoropenciclovir (PCV) ?
  • Degreve et al. showed that a mutation in the NLS
    sequences of HSV-1-TK provided better uptake of
  • Disrupted one N-terminal NLS sequence of the
    HSV-1-TK NLS ? mNLS-sr39TK
  • Fused EGFP into both ?
  • HSV-1-sr39TK-EGFP
  • mNLS-sr39TK-EGFP

Characterization of the Two-Hybrid System
  • Treatment with Doxycycline activates a reverse
    tetracycline-responsive transactivator inducing
    bi-directional transcription
  • p53 and TAg are known to interact
  • BD binds the promoter and upon interaction
    between p53 and TAg, AD is now in place to
    promote transcription of the downstream reporters
  • mNLS-sr39TK can be used for microPET
  • EGFP can be used for Fluorescence Microscopy

Development of a Reporter Cell Line
  • Stably transfected HeLa cells with
    Gal4-mNLS-sr39TK-EGFP to develop a reporter cell
  • Only cells expressing p53 and TAg showed cellular
    accumulation of PCV
  • Stably transfected HeLa-Gal4 cells with the
    various constructs and treated with doxycycline
  • Only expressed upon antibiotic treatment?tightly
    regulated expression
  • Measured the activity of the nucleoside analog
  • Only saw activity when cells expressed both p53
    and TAg, with treatment
  • GFP expression only in the presence of doxycycline

Time course of Reporter Gene Induction
  • To determine the peak expression time of the
    reporter, cells either expressing p53/CP or
    p53/TAg were treated with doxycycline for the
    displayed times and PCV accumulation was then
  • Peak expression time was at 48 h and then began
    to decrease thereafter

Imaging in Vivo Protein Interactions
  • Produced xenograft tumors of TAg and CP cells of
    nude mice
  • Once tumors grew to 5 mm, mice were treated and
    imaged 1h after tail injection of 18F-FHGB
  • Fluorescent microscopy of excised tumors
    displayed expression of GFP
  • Treated TAg tumor mice with doxycycline for 12,
    24, and 48 h to determine if microPET could
    quantify hybrid protein levels
  • Also looked at the biodistribution of 18F-FHGB
  • The intensity of the reporter was proportional to
    the amount of interacting proteins in vivo

  • Binding of p53/TAg in living mice was detected by
    microPET imaging with 18F-FHGB
  • An approximate 6-fold increase in mNLS-sr39TK
    activity occurred in response to the interaction
    of p53 and TAg in vivo
  • Function of the reporter protein was enhanced
    when increasing the amounts of interacting
    proteins were used
  • In vivo microPET imaging could be used to
    determine relative affinity differences between
    interacting proteins previously shown in vitro
  • GFP fluorescent imaging in vivo could provide a
    rapid screening assay for detecting the presence
    or absence of protein-protein interactions
  • Xenograft models may be useful in the initial
    characterization of drugs targeted to specific
    protein interactions
  • Transgenic mice with reporter genes could allow
    the interaction of proteins to be monitored in
    their native environment
  • Potential to advance our understanding of how
    protein interactions affect our normal
    physiology, development, disease progression and
    response to therapy

  • Cherry, S. R. (2004). "In vivo molecular and
    genomic imaging new challenges for imaging
    physics." Phys Med Biol 49(3) R13-48.
  • Gross, S. and D. Piwnica-Worms (2005). "Spying on
    cancer molecular imaging in vivo with
    genetically encoded reporters." Cancer Cell 7(1)
  • Gross, S. and D. Piwnica-Worms (2006). "Molecular
    imaging strategies for drug discovery and
    development." Curr Opin Chem Biol 10(4) 334-42.
  • Haberkorn, U. and A. Altmann (2003). "Noninvasive
    imaging of protein-protein interactions in
    living organisms." Trends Biotechnol 21(6)
  • Luker, G. D., J. P. Bardill, et al. (2002).
    "Noninvasive bioluminescence imaging of herpes
    simplex virus type 1 infection and therapy in
    living mice." J Virol 76(23) 12149-61.
  • Luker, G. D., V. Sharma, et al. (2002).
    "Noninvasive imaging of protein-protein
    interactions in living animals." Proc Natl Acad
    Sci U S A 99(10) 6961-6.
  • Luker, G. D., V. Sharma, et al. (2003).
    "Molecular imaging of protein-protein
    interactions controlled expression of p53 and
    large T-antigen fusion proteins in vivo." Cancer
    Res 63(8) 1780-8.
  • Luker, G. D., V. Sharma, et al. (2003).
    "Visualizing protein-protein interactions in
    living animals." Methods 29(1) 110-22.
  • Luker, K. E., M. C. Smith, et al. (2004).
    "Kinetics of regulated protein-protein
    interactions revealed with firefly luciferase
    complementation imaging in cells and living
    animals." Proc Natl Acad Sci U S A 101(33)
  • Winnard, P., Jr. and V. Raman (2003). "Real time
    non-invasive imaging of receptor- ligand
    interactions in vivo." J Cell Biochem 90(3)