Discovery of a protein-metabolite interaction between unsaturated fatty acids and the nuclear receptor Nur77 using a metabolomics approach

Abstract

Neuron-derived clone 77 (Nur77) is an orphan nuclear receptor with currently no known natural ligands. Here we applied a metabolomics platform for detecting protein-metabolite interactions (PMIs) to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriches unsaturated fatty acids (UFAs) in tissue lipid mixtures. The interaction of Nur77 with arachidonic acid and docosahexaenoic acid was subsequently characterized using a number of biophysical and biochemical assays. Together these data indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. UFAs are the only endogenous lipids reported to bind to Nur77, which highlights the use of metabolomics in the discovery of novel PMIs.

Figure 1

A metabolomics strategy for identification of potential Nur77 ligands. (a) Metabolomics workflow for the discovery of PMIs with Nur77LBD. (b) Plotting each metabolite ion based on its statistical significance and fold enrichment value identifies significantly enriched ions. Negative- and positive-mode MS ions are represented as green circles and blue diamonds, respectively, with the corresponding unsaturated fatty acids (UFAs) ions highlighted in red circles. (c) UFAs were enriched by His6-Nur77LBD when compared to no-protein control in samples incubated with lipid extracts from brain and testes. The experiment identified UFAs as potential Nur77 ligands. Fold changes (Nur77/control) represent statistically significant differences between His6-Nur77LBD and no-protein control samples (Student’s t-test, *, p-value < 0.05, **, p-value < 0.01; N = 3). FFA, free fatty acid; ND, not detectable.

Figure 2

ANS displacement assay showed the displacement of 800 μM ANS from 0.5 μM His6-Nur77LBD by increasing concentrations of AA (a) or DHA (d), but not by PA (b) or AEA (c).

Figure 3

(a, b) CD spectra showed changes in conformation of His6-Nur77LBD when it was treated with 10 molar equivalents of AA or DHA, but not of PA or AEA.

Figure 4

Determination of oligomeric states of His6-Nur77LBD in the presence or absence of lipids by size exclusion chromatography. (a) Standard curve was plotted as a function of the logarithm of the molecular weight (MW) and elution volume (V_e). The MW of the proteins used as MW markers were indicated along the plot in blue. The data were fitted to a linear regression model (black line) and subsequently employed to calculate experimental (Exp.) MW of His6-Nur77LBD in various samples (in red and in the table). (b, c, d) UV traces at 280 nm of His6-Nur77LBD samples showed AA-induced oligomerization of His6-Nur77LBD from a monomer (1) to a trimer (3) and tetramer (4), whereas no change in oligomeric states of the protein was detected in the no-lipid control (b) or in PA-treated (d) samples.