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Imaging Ligand-Driven PPAR Activities Using Single-Chain Bioluminescent Probes.

Sung-Bae Kim1, Tadaomi Furuta2, Genta Kamiya3

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Researchers developed a novel molecular imaging platform using bioluminescent probes to monitor nuclear receptor (NR) activities. This system, exemplified with PPARγ, shows promise for studying NR-related signaling pathways in metabolism and homeostasis.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Medical Imaging

Background:

  • Nuclear receptors (NRs) are critical regulators of metabolism and homeostasis, involved in diverse cell signaling pathways.
  • Monitoring NR activity is essential for understanding various physiological and pathological processes.
  • Existing methods for NR activity assessment have limitations in sensitivity and applicability.

Purpose of the Study:

  • To develop a broadly applicable molecular imaging platform for assessing nuclear receptor activities.
  • To design and validate a portfolio of single-chain bioluminescent probes (P1-P4) for NR imaging.
  • To demonstrate the platform's efficacy using the peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding domain (LBD).

Main Methods:

  • Engineering of four single-chain bioluminescent probes (P1-P4) based on protein-fragment complementation assay (PCA), molecular strain (MS), circular permutation (CP), and bioluminescence resonance energy transfer (BRET) systems.
  • Fusion of the PPARγ-LBD with luciferases and/or fluorescent proteins at optimized dissection sites.
  • In vitro and in vivo validation of probe performance using various ligands and furimazine (FMZ) substrate analogs (e.g., Ad-FMZ).

Main Results:

  • Probes P3 and P4 demonstrated strong bioluminescent (BL) signal intensities in response to a PPARγ agonist, achieving signal-to-background ratios up to 14-fold.
  • The developed probes sustained agonist-dependent BL signals for up to 24 hours in animal models.
  • Optimal dissection sites for the marine luciferase RLuc were identified for PCA systems.

Conclusions:

  • The developed molecular imaging platform offers a versatile tool for interrogating NR activities.
  • The probe portfolio can be adapted to study other NR-LBDs, broadening its application in NR-related research.
  • This platform has significant potential for advancing the study of NR-mediated cellular signaling in metabolism and homeostasis.