Molecular imaging of homodimeric protein–protein interactions in living subjects

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Abstract
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 [PP3H 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.
Funding Information
  • National Institutes of Health (2 RO1 CA82214‐05)
  • Health Foundation
  • Royal College of Radiologists
  • U.S. Department of Energy (DE‐FG02‐03ER63687)