Abstract 165: Proposed Mechanism of Inhibition of Paraoxonase 1 Activity by Isolevuglandins

Abstract

Paraoxonase 1 (PON1) is a High Density Lipoprotein (HDL) associated enzyme which binds to ApoAI and demonstrates lactonase, organophosphatase and arylesterase activity. Association of PON1 with ApoAI, results in a hyperactive state where PON1 activity increases 3-fold. Reduced PON1 activity has been consistently linked to increased atherosclerosis, but the mechanisms underlying this inhibition remain unclear. PON1 activity is found to be reduced in the settings of inflammation where myeloperoxidase (MPO) interacts with both ApoAI and PON1. Association of MPO with HDL generates substantial amounts of isolevuglandins (IsoLGs), lipid peroxidation products that irreversibly modify lysyl residues of proteins. We therefore hypothesized that association of MPO with HDL leads to IsoLG modifications of PON1 that reduce its activity. We found that incubating IsoLG with dextran purified HDL crosslinked PON1 and inhibited its PON1 activity (IC 50 7.5 μM). We then used purified recombinant PON1 to further elucidate the mechanism of inhibition. We considered four models for inhibition of PON1 activity by IsoLG: a) IsoLG directly modifies PON1 to render it inactive; b) IsoLG modifies PON1 near the ApoAI binding site so it cannot associate with ApoAI to become hyperactivated, but PON1 retains its basal activity; c) IsoLG modifies ApoAI near the PON1 binding site, blocking PON1 association and subsequent hyperactivation; d) IsoLG crosslinks PON1 associated with ApoAI to render it inactive. We found that incubation of IsoLG with PON1 inhibited both its basal activity and its hyperactivation when modified PON1 was subsequently incubated with HDL. However incubation of HDL with IsoLG for 1 h, but not 24 h, prior to incubating with PON1 resulted in significant loss of PON1 hyperactivity. The initial reaction of IsoLG with lysines forms pyrrole adducts that can still react to crosslink other proteins, but over time these pyrrole adduct oxidize to form non-reactive lactam adducts. Therefore, modification of ApoAI by IsoLG only appears to result in reduced PON1 activity prior to oxidation of ApoAI adducts to non-reactive lactam adducts, suggesting that crosslinking of ApoAI to PON1 drives loss of PON1 activity. Future studies will examine whether small molecule IsoLG scavengers can block MPO mediated inhibition of PON1 activity.