Year: 2004
Type: article
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 [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.
Source: The FASEB Journal
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
Awards: Center of Cancer Nanotechnology Excellence Focused on Therapy Response, In Vivo Cellular and Molecular Imaging Center@Stanford, MOLECULAR IMAGING OF STEM CELL SURVIVAL, APOPTOSIS, AND DIFFERENTIATION IN MYOCARDIUM, G222768804, G5693587585, G9000109420, Reporter Gene Technologies for Integrated Cancer Diagnostics
OpenAlex ID: W2158866962