Noninvasive quantitative imaging of protein-protein interactions in living subjects.
We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.
Ray
P
P
The Crump Institute for Molecular Imaging, University of California Los Angeles School of Medicine, Los Angeles, CA 90095, USA.
Pimenta
H
H
Paulmurugan
R
R
Berger
F
F
Phelps
M E
ME
Iyer
M
M
Gambhir
S S
SS
D000818:Animals; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1; D008156:Luciferases / Q000235:genetics; D051379:Mice; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics; D011401:Promoter Regions, Genetic; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; C497453:ID1 protein, human; C497454:ID1 protein, rat; C497455:Idb1 protein, mouse; D051798:Inhibitor of Differentiation Protein 1; D017570:MyoD Protein; D016328:NF-kappa B; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases