Figure 3. Characterization of interaction of HMB with PPARα at in silico and molecular levels.

(A) A rigid body in silico docked pose of the PPARα LBD with HMB was derived from the SwissDock online server and then displayed in UCSF Chimera software. HMB was found to be docked in the ligand-binding pocket formed by Ser280, Y314, and H440.

(B) Magnified view of (A) is displayed.

(C) The most stable docked pose of Y314D (mutated) PPARα and HMB was derived from the SwissDock online server. HMB was found to be posed far (>5 Å) from the ligand-binding pocket of Y314D PPARα.

(D) A time-resolved fluorescence resonance energy transfer (TR-FRET) analysis was performed to examine the interaction between PPARα and HMB. The curve was plotted as a 520/490 nm ratio of response with increasing doses of HMB. Curve fit was done in GraphPad Prism software. The analysis generated EC50 (4.02 nM) and Hill slope (0.7949).

(E) TR-FRET was also performed to study the interaction between gemfibrozil (a known ligand of PPARα) and PPARα for comparison.

(F) Thermal shift assay of full-length PPARα was conducted with 10 μM HMB. The melting of PPARα was monitored using an SYBR Green real-time melting strategy.

(G) Thermal shift assay of mutated Y314D-PPARα was also conducted with 10 μM HMB. Results were analyzed and confirmed after three independent experiments.

(H and I) PPARα^−/− astrocytes were transduced with either lenti-FL-PPARα or lenti-Y314D-PPARα, and after 48 h of transduction, cells were stimulated wih 10 μM HMB (H). After 6 h, cells were immunostained for PPARα with GFP auto-labeling (I) followed by quantification of MFI of PPARα in six images per group from a total of three independent experiments using the NIH ImageJ. One-way ANOVA followed by Tukey’s multiple comparison test was used for statistical analysis.

***p < 0.001; NS, not significant.