Abstract
Human soluble epoxide hydrolase (hsEH) is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity. Here, we uncover the molecular bases of hsEH inhibition mediated by the endogenous 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Our data reveal a dual inhibitory mechanism, whereby hsEH can be inhibited by reversible docking of 15d-PGJ2 in the catalytic pocket, as well as by covalent locking of the same compound onto cysteine residues C423 and C522, remote to the active site. Biophysical characterisations allied with in silico investigations indicate that the covalent modification of the reactive cysteines may be part of a hitherto undiscovered allosteric regulatory mechanism of the enzyme. This study provides insights into the molecular modes of inhibition of hsEH epoxy-hydrolytic activity and paves the way for the development of new allosteric inhibitors.
Highlights
Human soluble epoxide hydrolase is an enzyme responsible for the inactivation of bioactive epoxy fatty acids, and its inhibition is emerging as a promising therapeutical strategy to target hypertension, cardiovascular disease, pain and insulin sensitivity
This indicates that the Michael addition of 15d-PGJ2 to both C423 and C522 induces conformational changes in the enzyme that could account for the impairment of hydrolytic activity
We revealed that 15d-PGJ2 exerts inhibition of the human sEH ortholog by binding both non-covalently in the catalytic pocket and covalently to reactive cysteine residues, one of which, namely C423, was not characterised in earlier work
Summary
15d-PGJ2 covalently modifies two cysteines in hsEH CTD. To investigate whether human sEH C-terminal Domain (hsEH CTD) was covalently modified by the endogenous electrophilic lipid 15d-PGJ2, as reported for the murine ortholog[30], electrospray ionisation mass spectrometry (ESI-MS) experiments were performed. UV spectra revealed a three-peak absorbance profile, with the expected aromatic amino acids absorbance at 280 nm and two additional peaks at 250 and 330 nm, which matched the UV spectrum of 15d-PGJ2 (Fig. 2b, Supplementary Fig. 3a) Did this indicate a successful separation of prostaglandinmodified proteins from the apoproteins, and suggested that in the covalently adducted species the active site of the enzyme is somewhat altered, becoming unable to bind to the benzylmercaptan compound. Deconvolution analyses of CD data indicate that thiol modification by 15dPGJ2 is accompanied by a decrease in α-helical and an increase of β-strand content, and that double adduction of both cysteine residues results in a somewhat cumulative effect (Table 1) This indicates that the Michael addition of 15d-PGJ2 to both C423 and C522 induces conformational changes in the enzyme that could account for the impairment of hydrolytic activity.
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