Optimizing Luciferase Protein Fragment Complementation for Bioluminescent Imaging of Protein–Protein Interactions in Live Cells and Animals
Introduction
Protein–protein interactions regulate a variety of cellular functions, including cell cycle progression, signal transduction, and metabolic pathways. On a whole organism scale, protein–protein interactions regulate signals that affect overall homeostasis, patterns of development, normal physiology, and disease in living animals.1, 2, 3 In addition, protein–protein interactions have considerable potential as therapeutic targets.4, 5 Evidence is accumulating that pathways of protein interactions in specific tissues produce regional effects that cannot be investigated fully with in vitro systems, and thus there is considerable interest in evaluating protein interactions in living animals.
Fundamentally, the detection of physical interaction among two or more proteins can be assisted if association between the interactive partners leads to the production of a readily observed biological or physical readout.6 Compared with studies of protein interactions in cultured cells, strategies to interrogate protein–protein interactions in living organisms impose even further constraints on reporter systems and mechanisms of detection. This Chapter summarizes briefly various strategies for detecting protein-binding partners using conventional cell biology assays with the intent of identifying properties that might be exploited for imaging applications.
Section snippets
Detecting Protein Interactions in Intact Cells
Most strategies for detecting protein–protein interactions in intact cells are based on fusion of the pair of interacting molecules to defined protein elements to reconstitute a biological or biochemical function. Examples of reconstituted activities include activation of transcription, repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity.6 A variety of these techniques have been developed to investigate protein–protein
Detecting Protein–Protein Interactions in Living Animals
Of the available strategies, complementation of firefly and Renilla luciferases is readily amenable to near real-time applications in living animals,18, 28 but the available fragments suffer from considerable constitutive activity of the N terminus fragments, thereby precluding general use. Thus, no enzyme fragment pair has yet been found that satisfies all criteria for noninvasive analysis of protein–protein interactions and enables interrogation in cell lysates, intact cells, and living
Optimizing Luciferase Fragments for Complementation
To identify an optimal pair of firefly luciferase fragments that reconstitutes an active (bioluminescent) heterodimer only upon association, one can construct and screen a comprehensive combinatorial incremental truncation library as shown in Fig. 2.29 This library employs a well-characterized protein interaction system: rapamycin-mediated association of the FRB domain of human mTOR (residues 2024–2113) with FKBP-12.16, 19, 30 Initial fusions of FRB and FKBP with N- and C-terminal fragments of
Selection of Protein Pairs for LCI
LCI can theoretically be used to study any process that alters the association state of a pair of proteins, including conformational changes, compartmentation changes, posttranslational modifications such as phosphorylation, or protein association mediated by small molecules. However, the practical utility of LCI to study a particular pair of interacting proteins should be considered carefully. While any reporter strategy may perturb the system under investigation, the expression of certain
Conclusions
The detection of protein interactions in living animals can provide useful information about the molecular basis of physiologic and pathophysiologic events, as well as the molecular response to therapeutic agents. Luciferase complementation imaging of protein interactions in cells and small animal models has been developed to permit the rapid and repetitive measurement of protein pairs of interest. Optimized luciferase complementation, quantified by imaging with a CCD camera equipped with
Acknowledgements
We thank colleagues of the molecular imaging center for insightful discussions and excellent technical assistance. Work reviewed herein was supported by a grant from the National Institutes of Health (P50 CA94056).
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