Targeting Cellular Retinoic Acid Binding Protein 1 to Modulate Non-Canonical Retinoic Acid Signaling

Abstract

All-trans-retinoic acid (atRA) is the principle active metabolite of vitamin A and is essential for almost all biological functions. Canonically, atRA exerts its actions through retinoic acid receptors (RARs) located in the nucleus to regulate gene transcription. atRA also possesses “non-canonical activities” that modulate cell signaling, and is defined by (1) RAR-independence, (2) a rapid time-scale and (3) cytosolic localization. The primary mediator of this non-canonical activity is the highly conserved cellular retinoic acid binding protein 1 (CRABP1). CRABP1 was previously thought to only function in the binding and sequestration of atRA to regulate cellular bio-availability. However, studies of two non-canonical pathways have established CRABP1 as a mediator of non-canonical atRA activity. The first is CRABP1-mediated regulation of the mitogen activated protein kinase (MAPK) pathway with physiological and disease relevance in stem cell proliferation, cancer, immune regulation, and obesity. The second is CRABP1-mediated regulation of calcium (Ca2+)-calmodulin dependent kinase II (CaMKII) activation with physiological and disease relevance in cardiac dysfunction and motor neuron degenerative diseases such as amyotrophic lateral sclerosis (ALS). Nuclear Magnetic Resonance (NMR) spectroscopy and molecular studies determined the structural and molecular mechanism underlying CRABP1-mediated regulation of CaMKII activation. Mechanistically, CRABP1 preferentially complexes with the inactive form of CaMKII to ultimately dampen CaMKII activation. Alanine mutagenesis studies have determined that CRABP1 residues within a proposed CaMKII interaction surface and an allosteric site maintain this preference. Mutation of these residues can shift CRABP1 preference towards the active form of CaMKII. Two novel CRABP1 ligands (C4 and C32) were also characterized as potential therapeutic agents that may be developed to target the CRABP1-CaMKII pathway in motor neuron (MN) diseases. In a reconstituted MN culture model, C4 and C32 can dampen CaMKII activation in a CRABP1-dependent manner. In an immortalized MN cell line (MN1) C4 and C32 can protect against excitotoxic-mediated MN death induced by ionomycin treatment. The primary sequence of CRABP1 is extremely conserved among animal species, with only one substitution observed at amino acid position 86, suggesting important functional constraints placed upon CRABP1 sequence during evolution. Data mining of reported human studies was performed to determine the relevance of CRABP1 in human health and disease. Associations of CRABP1 with various human diseases were identified, including altered human CRABP1 gene expression and the presence of variants in cancers, ALS, and several rare diseases. The studies within this dissertation elucidate the structural and molecular mechanism of CRABP1-mediated regulation of cell signaling, specifically in CaMKII activation. The results suggest a potential therapeutic approach in targeting the CRABP1-CaMKII pathway with CRABP1-selective ligands to manage MN diseases. These results expand our understanding of CRABP1 in mediating the non-canonical activity of atRA hormone, particularly in modulating various cell signaling pathways to maintain health. The results also uncover complex mechanisms through which CRABP1-selective, atRA-like compounds may be further developed in therapeutic applications.