Energy status regulates levels of the RAR/RXR ligand 9-cis-retinoic acid in mammalian tissues: Glucose reduces its synthesis in ß-cells.

9-cis-retinoic acid (9cRA) binds retinoic acid receptors (RAR) and retinoid X receptors (RXR) with nanomolar affinities, in contrast to all-trans-retinoic acid (atRA), which binds only RAR with nanomolar affinities. RXR heterodimerize with type II nuclear receptors, including RAR, to regulate a vast gene array. Despite much effort, 9cRA has not been identified as an endogenous retinoid, other than in pancreas. By revising tissue analysis methods, 9cRA quantification by liquid chromatography-tandem mass spectrometry becomes possible in all mouse tissues analyzed. 9cRA occurs in concentrations similar to or greater than atRA. Fasting increases 9cRA in white and brown adipose, brain and pancreas, while increasing atRA in white adipose, liver and pancreas. 9cRA supports FoxO1 actions in pancreas ß-cells and counteracts glucose actions that lead to glucotoxicity; in part by inducing Atg7 mRNA, which encodes the key enzyme essential for autophagy. Glucose suppresses 9cRA biosynthesis in the ß-cell lines 832/13 and MIN6. Glucose reduces 9cRA biosynthesis in 832/13 cells by inhibiting Rdh5 transcription, unconnected to insulin, through cAMP and Akt, and inhibiting FoxO1. Through adapting tissue specifically to fasting, 9cRA would act independent of atRA. Widespread occurrence of 9cRA in vivo, and its self-sufficient adaptation to energy status, provides new perspectives into regulation of energy balance, attenuation of insulin and glucose actions, regulation of type II nuclear receptors, and retinoid biology.

Microsecond MD Simulations to Explore the Structural and Energetic Differences between the Human RXRα-PPARγ vs. RXRα-PPARγ-DNA.

The heterodimeric complex between retinoic X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) is one of the most important and predominant regulatory systems, controlling lipid metabolism by binding to specific DNA promoter regions. X-ray and molecular dynamics (MD) simulations have revealed the average conformation adopted by the RXRα-PPARγ heterodimer bound to DNA, providing information about how multiple domains communicate to regulate receptor properties. However, knowledge of the energetic basis of the protein-ligand and protein-protein interactions is still lacking. Here we explore the structural and energetic mechanism of RXRα-PPARγ heterodimer bound or unbound to DNA and forming complex with co-crystallized ligands (rosiglitazone and 9-cis-retinoic acid) through microsecond MD simulations, molecular mechanics generalized Born surface area binding free energy calculations, principal component analysis, the free energy landscape, and correlated motion analysis. Our results suggest that DNA binding alters correlated motions and conformational mobility within RXRα-PPARγ system that impact the dimerization and the binding affinity on both receptors. Intradomain correlated motions denotes a stronger correlation map for RXRα-PPARγ-DNA than RXRα-PPARγ, involving residues at the ligand binding site. In addition, our results also corroborated the greater role of PPARγ in regulation of the free and bound DNA state.

Intranasal delivery of 9-cis retinoic acid reduces beta-amyloid deposition via inhibiting astrocyte-mediated inflammation.

Alzheimer's disease (AD) is associated with the accumulation and deposition of a beta-amyloid (Αß) peptide in the brain, resulting in increased neuroinflammation and synaptic dysfunction. Intranasal delivery of targeted drugs to the brain represents a noninvasive pathway that bypasses the blood-brain barrier and minimizes systemic exposure. The aim of this study was to evaluate the therapeutic effect of intranasally delivered 9-cis retinoic acid (RA) on the neuropathology of an AD mouse model. Herein, we observed dramatically decreased Αß deposition in the brains of amyloid precursor protein (APP) and presenilin 1 (PS1) double-transgenic mice (APP/PS1) treated intranasally with 9-cis RA for 4 weeks compared to that in the brains of vehicle-treated mice. Importantly, intranasal delivery of 9-cis RA suppressed Αß-associated astrocyte activation and neuroinflammation and ultimately restored synaptic deficits in APP/PS1 transgenic mice. These results support the critical roles of Αß-associated neuroinflammation responses to synaptic deficits, particularly during the deposition of Αß. Our findings provide strong evidence that intranasally delivered 9-cis RA attenuates neuronal dysfunction in an AD mouse model and is a promising therapeutic strategy for the prevention and treatment of AD.

AlloFinder: a strategy for allosteric modulator discovery and allosterome analyses.

Allostery tweaks innumerable biological processes and plays a fundamental role in human disease and drug discovery. Exploration of allostery has thus been regarded as a crucial requirement for research on biological mechanisms and the development of novel therapeutics. Here, based on our previously developed allosteric data and methods, we present an interactive platform called AlloFinder that identifies potential endogenous or exogenous allosteric modulators and their involvement in human allosterome. AlloFinder automatically amalgamates allosteric site identification, allosteric screening and allosteric scoring evaluation of modulator-protein complexes to identify allosteric modulators, followed by allosterome mapping analyses of predicted allosteric sites and modulators in human proteome. This web server exhibits prominent performance in the reemergence of allosteric metabolites and exogenous allosteric modulators in known allosteric proteins. Specifically, AlloFinder enables identification of allosteric metabolites for metabolic enzymes and screening of potential allosteric compounds for disease-related targets. Significantly, the feasibility of AlloFinder to discover allosteric modulators was tested in a real case of signal transduction and activation of transcription 3 (STAT3) and validated by mutagenesis and functional experiments. Collectively, AlloFinder is expected to contribute to exploration of the mechanisms of allosteric regulation between metabolites and metabolic enzymes, and to accelerate allosteric drug discovery. The AlloFinder web server is freely available to all users at http://mdl.shsmu.edu.cn/ALF/.

Conservation of DNA and ligand binding properties of retinoid X receptor from the placozoan Trichoplax adhaerens to human.

Nuclear receptors are a superfamily of transcription factors restricted to animals. These transcription factors regulate a wide variety of genes with diverse roles in cellular homeostasis, development, and physiology. The origin and specificity of ligand binding within lineages of nuclear receptors (e.g., subfamilies) continues to be a focus of investigation geared toward understanding how the functions of these proteins were shaped over evolutionary history. Among early-diverging animal lineages, the retinoid X receptor (RXR) is first detected in the placozoan, Trichoplax adhaerens. To gain insight into RXR evolution, we characterized ligand- and DNA-binding activity of the RXR from T. adhaerens (TaRXR). Like bilaterian RXRs, TaRXR specifically bound 9-cis-retinoic acid, which is consistent with a recently published result and supports a conclusion that the ancestral RXR bound ligand. DNA binding site specificity of TaRXR was determined through protein binding microarrays (PBMs) and compared with human RXRɑ. The binding sites for these two RXR proteins were broadly conserved (∼85% shared high-affinity sequences within a targeted array), suggesting evolutionary constraint for the regulation of downstream genes. We searched for predicted binding motifs of the T. adhaerens genome within 1000 bases of annotated genes to identify potential regulatory targets. We identified 648 unique protein coding regions with predicted TaRXR binding sites that had diverse predicted functions, with enriched processes related to intracellular signal transduction and protein transport. Together, our data support hypotheses that the original RXR protein in animals bound a ligand with structural similarity to 9-cis-retinoic acid; the DNA motif recognized by RXR has changed little in more than 1 billion years of evolution; and the suite of processes regulated by this transcription factor diversified early in animal evolution.

9-cis retinoic acid induces neurorepair in stroke brain.

The purpose of this study was to examine the neurorestorative effect of delayed 9 cis retinoic acid (9cRA) treatment for stroke. Adult male rats received a 90-min right distal middle cerebral artery occlusion (dMCAo). Animals were separated into two groups with similar infarction sizes, based on magnetic resonance imaging on day 2 after dMCAo. 9cRA or vehicle was given via an intranasal route daily starting from day 3. Stroke rats receiving 9cRA post-treatment showed an increase in brain 9cRA levels and greater recovery in motor function. 9cRA enhanced the proliferation of bromodeoxyuridine (+) cells in the subventricular zone (SVZ) and lesioned cortex in the stroke brain. Using subventricular neurosphere and matrigel cultures, we demonstrated that proliferation and migration of SVZ neuroprogenitor cells were enhanced by 9cRA. Our data support a delayed and non-invasive drug therapy for stroke. Intranasal 9cRA can facilitate the functional recovery and endogenous repair in the ischemic brain.