Molecular imaging of homodimeric protein-protein interactions in living subjects.
Homodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation-based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N-terminal (N-hRLUC) or C-terminal (C-hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail-to-head homodimer). This was monitored using luminometry (68-fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7-fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant-TK to generate N-hRLUC mutant TK and mutant TK-C-hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo-dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8-3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N-hRLUC or C-hRLUC 'spacers') to monitor incremental lack of association of the tail-to-head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein-protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects.
Massoud
Tarik F
TF
The Crump Institute for Molecular Imaging, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095, USA.
Paulmurugan
Ramasamy
R
Gambhir
Sanjiv S
SS
D000818:Animals; D002460:Cell Line / Q000637:transplantation; D019281:Dimerization; D007668:Kidney; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics; D049410:Luciferases, Renilla / Q000737:chemistry* / Q000235:genetics; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008819:Mice, Nude; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012815:Signal Processing, Computer-Assisted; D013937:Thymidine Kinase / Q000737:chemistry* / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; D013937:Thymidine Kinase