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Molecular imaging of homodimeric protein–protein interactions in living subjects

TARIK F. MASSOUD^*,,, RAMASAMY PAULMURUGAN^|| and SANJIV S. GAMBHIR^*,,,||,1

^* The Crump Institute for Molecular Imaging
Department of Molecular & Medical Pharmacology and Department of Biomathematics
UCLA-Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA;
Departments of Radiology and Oncology, University of Cambridge School of Clinical Medicine, Cambridge, UK;
Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA; and
^|| Department of Radiology and the Bio-X Program, Stanford University School of Medicine, Stanford, California, USA

¹Correspondence: Stanford University School of Medicine, James H. Clark Center, 318 Campus Dr., East Wing, 1st Floor, Stanford, CA 94305-5427, USA. E-mail: sgambhir{at}stanford.edu

SPECIFIC AIMS

Homodimeric protein interactions are potent regulators of cellular functions, but are challenging to study in living subjects. Our aims were to 1) quantify and image homodimeric protein-protein interactions in mammalian cells and in living mice using a split synthetic renilla luciferase (hRLUC) complementation-based bioluminescence assay and the herpes simplex virus type 1 thymidine kinase (TK) as a test protein and 2) explore the topological factors that affect the interaction of chimeras made of split hRLUC reporter fragments fused to proteins taking part in protein-protein interactions.

PRINCIPAL FINDINGS

1. In vitro luminometry and in vivo bioluminescence imaging of TK homodimerization
We previously identified suitable split sites in the molecule of hRLUC that generated inactive amino-terminal 229-residue fragment (N-hRLUC) and carboxyl-terminal 82-residue fragment (C-hRLUC) of the reporter protein that, together, were able to produce significant recovered activity through assisted complementation. We performed in vitro luminometry of cell lysates and in vivo whole-body bioluminescence imaging of intact cells for assaying hRLUC activity after transient cotransfection of two plasmids in different sets of 293T cells, each plasmid containing one of four vectors in a pcDNA3.1 (+) backbone. Cotransfection strategies were performed such that opposing split hRLUC domains would be expressed and made available to complement when spatially positioned in either a tail-to-head (N-hRLUC upstream and C-hRLUC downstream of the two TK monomers, respectively), head-to-tail (downstream and upstream, respectively), tail-to-tail, or head-to-head configuration with respect to the two TK monomers. Accordingly, we used four different combinations of vector constructs in these cotransfection experiments.

We obtained the greatest degree of in vitro recovered activity for hRLUC (68-fold increase [P<0.01] above background levels of light emission expressed in relative light units/min, normalized transfection by cotransfecting and assaying for firefly luciferase activity and normalized to micrograms of total protein) (Fig. 1 a), and the highest in vivo imaging signal (2.7-fold increase [P<0.01] above background light emission in maximum photons per second/cm²/steradian) (Fig. 1b ) after interaction of the two TK chimeras in the tail-to-head configuration (tail-to-head homodimer).

View larger version (31K): [in this window] [in a new window]   Figure 1. a) Homodimerization-mediated fragment-assisted complementation of the split hRLUC system in transiently transfected 293T cells. Graph shows luminometry results on cell lysates 24 h after cotransfection of constructs described in text. Coexpression of the tail-to-head combination of chimeric proteins resulted in successful homodimerization of the TK monomers, which in turn resulted in significant recovered activity of hRLUC compared with other vector combinations including hRLUC without TK monomers. Signals from the head-to-tail, tail-to-tail, and head-to-head combinations were only minimally above those of mock-transfected cells (not show, <10 RLU/µm protein/min). Error bar is the SEM for 3 samples. Results are normalized for transfection efficiency. b) Optical CCD camera imaging of living mice engrafted with transiently transfected 293T cells for in vivo assessment of TK homodimerization-mediated complementation of split hRLUC. The image of a representative mouse is shown as a visible light image superimposed on the CCD bioluminescence image with a scale in photons/sec/cm2/steradian. Mice were imaged in the prone position after tail vein injection of coelenterazine. Each mouse was imaged several minutes after s.c. implantation of 10x106 293T cells transiently cotransfected 24 h previously with the tail-to-head combination of monomers (site A) or with mock transfected cells (site B). There was significantly higher (P<0.01) recovered activity of hRLUC from site A than with site B (graph). Error bar is the SEM for 3 samples.

2. Monitoring the lack of association of TK monomers
By contrast, we recorded very minimal levels of recovered hRLUC activity in vitro for the remaining three combinations of protein chimera configurations, varying from 1.5% to 3.2% of the activity seen in the tail-to-head homodimers (Fig. 1a ). These combinations gave insufficient bioluminescence signal for in vivo imaging (data not shown); unlike the tail-to-head homodimer, these combinations were unsuitable for detecting TK homodimers whether present or not. The least favorable complementation was seen when N-hRLUC was in the downstream position of TK. This was made even worse when combined with a chimera carrying an opposing C-hRLUC in the downstream position. We found that a mutation of arginine to cysteine at position 318 of TK, previously reported to inhibit dimerization, also led to a significant decrease in complementation signal for the split reporter assay.

3. In vitro luminometry and in vivo bioluminescence imaging of simulated inhibition of TK homodimerization
We designed a strategy for and assessed the effects of simulated homodimerization inhibition using in vitro luminometry and in vivo imaging. We constructed two variant protein chimeras: one with an N-hRLUC domain attached at either end of the same TK molecule and the other with C-hRLUC domains sandwiching the TK molecule. The double N-hRLUC chimera was combined with the chimera containing C-hRLUC downstream of TK in cotransfection strategies. The double C-hRLUC chimera was combined with that containing N-hRLUC upstream of TK. The first combination represented our tail-to-head TK homodimer with the addition of another N-hRLUC in the head position. Given our finding that the presence of an N-hRLUC domain in the head position leads to ineffective homodimerization, we reasoned that this new tail-to-double-head combination would inhibit the demonstrated homodimerization of the tail-to-head homodimer. The second combination represented our tail-to-head TK homodimer with the addition of another C-hRLUC in the tail position. It was reasoned that the presence of an additional smaller C-hRLUC, albeit in the tail position, might lead to a lesser degree of homodimerization inhibition than for the first combination tested. Finally, a third combination used both new chimeras in a double-tail-to-double-head combination.

We compared these new combinations with the tail-to-head homodimer and demonstrated that the extra N-hRLUC in the tail-to-double-head combination of monomers resulted in complete inhibition of hRLUC complementation (Fig. 2 a). The double-tail-to-head combination resulted in an intermediate loss of complementation, observed on both luminometry and whole-body imaging, down to 56% and 64%, respectively, of that seen for the tail-to-head homodimer (Fig. 2a, b ). We hypothesized that this smaller C-hRLUC might have induced more modest deleterious conformational movements at the TK dimerization interface. As expected, the double-tail-to-double-head combination inhibited completely the association of TK monomers. We monitored the dynamic temporal changes of these simulated dimerization inhibitors for 48 h after transient transfection.

View larger version (33K): [in this window] [in a new window]   Figure 2. a) Inhibition of homodimerization-mediated fragment-assisted complementation of the split hRLUC system in transiently transfected 293T cells. Graph shows luminometry results on cell lysates 24 h after cotransfection of constructs described in text. The recovered activity of hRLUC after successful homodimerization of the tail-to-head TK monomer combination (lilac) was mostly eliminated by the presence of extra N-hRLUC "spacers" in the downstream position of the tail-to-double-head and double-tail-to-double-head monomer combinations. The double-tail-to-head combination resulted in an intermediate inhibition of TK homodimerization and a corresponding decrease in the light signal. Positions of the spacers are shown in the diagram (N-hRLUC in red and C-hRLUC in blue). Signals from the tail-to-double-head and double-tail-to-double-head combinations were only minimally above those of mock-transfected cells (not shown, <15 RLU/µm protein/min). Error bar is the SEM for 3 samples. Results are normalized for transfection efficiency. b) Optical CCD camera imaging of living mice engrafted with transiently transfected 293T cells for in vivo assessment of inhibition of TK homodimerization-mediated complementation of split hRLUC. The image of one representative mouse is shown as a visible light image superimposed on the CCD bioluminescence image with a scale in photons/sec/cm2/steradian. Mice were imaged in the prone position after tail vein injection of coelenterazine. Each mouse was imaged after s.c. implantation of 10x106 293T cells transiently cotransfected 24 h before with the tail-to-head combination of monomers (site A), the double-tail-to-head combination of monomers (site B), and the tail-to-double-head combination of monomers (site C, shoulder region). There was a significant decrease (P<0.05) in recovered activity of hRLUC from sites B and C compared with site A, as shown in the graph. Error bar is the SEM for 3 samples.

CONCLUSIONS AND SIGNIFICANCE

We have demonstrated the use of an in vivo protein fragment-assisted complementation assay, based on a split hRLUC reporter technology, for bioluminescence imaging of homodimeric protein-protein interactions in intact living subjects (Fig. 3 ). We imaged in living mice the homodimerization of TK monomers tagged with split hRLUC reporter proteins. This technique depends on various factors, such as the size, orientation, and position of the split hRLUC reporter domains in relation to the test proteins. Our detailed characterization of this system should help broaden its application and make it a versatile methodology for the study of homodimeric protein-protein interactions. The high sensitivity of this assay for detecting, locating, and quantifying transient homodimerization of proteins, combined with the advantages of doing so in a living subject environment, should make it of potential value in 1) investigating the reasons for protein homodimerization. For many transient homodimers, the biological rationale for the physiological monomer/dimer equilibrium is unknown, unclear, or speculative; 2) Studying factors that drive the association or dissociation of homodimer subunits: linking the unique information gained from in vivo imaging with knowledge of biophysical phenomena, governed by the shape, chemical complementarities, and flexibility of the molecules involved, potentially offers a greater understanding of the conditions under which homodimeric subunits interact; 3) Assessment of known interactions within their intracellular physiological context. This is likely to be where the greatest value of this technique lies, rather than in screening proteomes for unknown interactions. Therefore, the protein fragment-assisted complementation assay we present is likely to complement and work in concert with other techniques (e.g., FRET, BRET, etc.) for studying homodimeric interactions in living environments; 4) The development of novel drugs based on inhibition of active protein assemblies: implementation of molecular imaging in the drug discovery process offers the strong advantage of being able to study a potential drug labeled for imaging in an animal model, often before phenotypic changes become obvious, and then quickly move into human studies. Ultimately, we foresee innovative molecular imaging tools enhancing our appreciation of entire biological pathway systems and their pharmacological regulation, and accelerating the achievement of a "systems biology" understanding of biological complexity.

View larger version (35K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the protein fragment-assisted complementation strategy using split synthetic renilla luciferase (hRLUC) to monitor the thymidine kinase (TK) homodimeric protein-protein interaction. The amino-terminal portion of hRLUC is attached to one TK monomer through a peptide linker, and the carboxyl-terminal portion of hRLUC is similarly attached to another TK monomer. Dimerization of the two TK monomers restores hRLUC activity through protein complementation and produces light in the presence of the substrate coelenterazine.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1128fje

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