Molecular Drivers of FABP-mediated Endocannabinoid Signaling for Appetite Regulation - Stark, Ruth E. Molecular Drivers of FABP-mediated Endocannabinoid Signaling for Appetite Regulation
Metabolic signaling by endogenous cannabinoids (ECs) is essential to the regulation of human
appetite, pain, and neuroprotection. Fatty acid-binding proteins (FABPs) can either sequester the
hydrophobic ECs or transport them to hydrolytic enzymes; ECs routed to the nucleus also activate the
peroxisome proliferator-activated receptors (PPARs). EC levels have been correlated with obesity in
knockout mice for liver (L) FABP that is co-expressed with intestinal (I) FABP in enterocytes and also for
PPARa knockouts, underscoring the regulatory roles of these proteins. We will probe poorly understood
EC-FABP, EC-PPAR, and FABP-EC-PPAR complexes in vitro at near-physiological concentrations,
determining affinities, metabolic fates, molecular binding interfaces, and conformational changes to test
mechanistic hypotheses regarding EC signaling. Trainees at multiple career stages, including those
recruited from underrepresented groups in STEM, will be integrally involved in this research program.
Specific questions to be addressed are as follows: (1) How do ECs choose between FABP
chaperones to produce obese or lean outcomes? The possibility that ECs are delivered by LFABP to
hydrolytic enzymes for metabolic breakdown rather than sequestered by IFABP in the enterocyte will be
tested enzymatically, whereas the rationale for LFABP’s diminished affinity will be explored using high-
pressure solution-state NMR to identify energetically favored candidate sites for binding. (2) Could
transcriptional activity be driven by EC binding preferences for LFABP vs. PPARa? The PPARa
ligand-binding domain (PPARa_LBD) will be purified, rigorously delipidated, and tested in vitro for EC-
modulated transcriptional activity. EC binding affinities will be compared for PPARa_LBD and FABPs.
(3) Could transcriptional activity be driven by EC-modulated FABP-PPAR collisions that cause
conformational changes? To determine the interactions involved in the proposed FABP-mediated EC
activation of PPARa transcriptional function, we will first use surface plasmon resonance to measure the
binding affinity of LFABP-PPARa_LBD protein complexes, on their own and in the presence of ligands
with a range of known activation efficacies. The site-specific collision-associated impact on the LFABP
partner will be probed by solution-state NMR spectroscopy of the [U-15N]-enriched protein, using chemical
shift perturbations of each backbone NH resonance to define the binding interface with PPARa_LBD, any
allosteric structural changes that occur upon complex formation, and their modulation by EC ligands. Taken
together, these experiments will advance our understanding of (macro)molecular networks that function to
achieve metabolic signaling by ECs involved in appetite and pain regulation, thereby advancing our
understanding of health risks related to obesity and inflammation. This understanding can guide the design
of drugs that modulate these biomedically important ligand-protein and protein-protein interactions.
NIH SuRE Grant Proposal
