Non-Invasive Imaging of Protein Interactions within Living Models

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Non-Invasive Imaging of Protein Interactions
within Living Models

• Sean Courtney

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Why study Protein Interactions?

• Regulates many of the essential biological
  processes
• 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

http//www.wellesley.edu/Chemistry/chem227/nucleic
function/transcription/lac20operon/06eukaryotes.j
pg
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Protein-Protein Interaction Techniques

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

http//www.ittc.ku.edu/xwchen/Project_files/image
007.jpg
http//www.wesleyan.edu/mbb/faculty/imukerji/hbs3.
jpg
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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
  reassembly
• 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
  protein

Dualsystems Biotech
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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
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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-AD
• GAL4 DNA-BD can bind the UAS alone but cannot
  activate transcription until bound with the GAL4
  DNA-AD
• When the two domains interact, BD and AD are
  brought into proximity, thus activating
  transcription of a downstream reporter gene

Clontech
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Various Imaging Techniques

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

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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

http//en.wikipedia.org/wiki/ImageUser-FastFissio
n-brain.gif
http//en.wikipedia.org/wiki/Image3Dbrain.gif
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MR Imaging Ability

• http//video.google.com/videoplay?docid3477454458
  695092843qMRIhlen

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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

http//en.wikipedia.org/wiki/ImagePET-MIPS-anim.g
if
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Single-Photon Emission Computed Tomography (SPECT)

• A nuclear medicine tomographic imaging technique
  using gamma rays able to provide true 3D
  information
• 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
  molecules
• 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

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Fluorescence

• 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
  reporter
• 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)

http//wwwchem.leidenuniv.nl/metprot/armand/images
/029l.jpg
http//en.wikipedia.org/wiki/ImageAequorea_victor
ia.jpg
http//www.upenn.edu/pennnews/photos/704/mice.jpg
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Bioluminescence

• Luciferase, an enzyme found in Fireflies, is also
  commonly used as a reporter
• Must be incorporated into the cell (i.e. tumor
  xenograft)
• 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
  agents
• 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
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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

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Various Contrast Agents
Cherry et al. 2004
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Cell-Surface Receptor

• Similar concept to pharmaceuticals in that it
  must find the target in the body and accumulate
  there
• Binds to the target, unbound portion must be
  cleared from that tissue to be able to
  distinguish the signal between specific and
  non-specific
• 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
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Enzymes

• Agents are designed to interact with the enzyme
  target
• 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
  state

Cherry et al. 2004
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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
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Various Reporter Gene Systems
Cherry et al. 2004
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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
  Piwnica-Worms
• 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

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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) ?
  HSV-1-sr39TK
• Degreve et al. showed that a mutation in the NLS
  sequences of HSV-1-TK provided better uptake of
  124I-FIAU
• 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

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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

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Development of a Reporter Cell Line

• Stably transfected HeLa cells with
  Gal4-mNLS-sr39TK-EGFP to develop a reporter cell
  line?HeLa-Gal4
• 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
  reporter
• Only saw activity when cells expressed both p53
  and TAg, with treatment
• GFP expression only in the presence of doxycycline

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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
  measured
• Peak expression time was at 48 h and then began
  to decrease thereafter

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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

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Conclusions

• 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

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References

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