טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTavori Hagai
SubjectA Mutual Association between Paraoxonase 1 (PON1) and the
Atherosclerotic Lesion: In Search after a Specific
Lactone as a Substrate for PON1
DepartmentDepartment of Medicine
Supervisors Professor Emeritus Michael Aviram
Professor Jacob Vaya
Full Thesis textFull thesis text - English Version


Abstract


Paraoxonase 1 (PON1) is a member of the three Paraoxonases gene family (PON1, PON2, and PON3). PON1 plays a significant role in protection against atherosclerotic plaque initiation and progression. In parallel, PON1 activity is depressed under excessive oxidative stress conditions. Both PON1 native substrate and the mechanisms of the enzyme anti-atherogenicity are not clear yet.

The aim of the present study was:  to better understand the mutual interactions between PON1 and the human carotid atherosclerotic lesion in search for potential PON1 substrates.

The oxidizing potency of the human carotid lesion lipid extract (LLE) and the anti-atherogenic role of PON1 on LLE oxidation competence were next examined. Our findings show that the LLE oxidized both LDL and macrophages. In addition, LLE decreases HDL-mediated cholesterol efflux from macrophages. Incubation of LLE with PON1 beneficially affected LLE composition and reduced LLE atherogenic properties. We next isolated and identified LLE oxidizing agent, as well as the PON1 inhibitor in LLE. The LLE was shown to contain five main lipid fractions, out of them only fraction #2 (F2) was able to promote macrophages oxidation which was shown to proceed via a mitochondrial pathway Incubation of F2 with PON1 resulted in a significant reduction in F2 peroxide levels. Furthermore, PON1 reduced F2 ability to oxidize macrophages. F2 was found to be a triglyceride composed from palmitic, oleic, and linoleic acids and 0.3% of its linoleic acid oxidized.

PON1 inhibitor in the lesion was observed in fraction #5, subfraction C (F5c) which differs from the lesion oxidizing agent in F2. The lesion PON1 inhibitor molecular structure was identified as linoleic acid hydroperoxide (LA-OOH). During its inactivation by the lesion, PON1 behave as a peroxidase enzyme, reducing LA-OOH to LA- OH (hydroxide) via its free cysteine group at position 284 of the protein. A similar reaction indeed occurred when LA-OOH was incubated with pure cysteine. The presence of cysteine as well as other thiol compounds, also prevented PON1 inactivation and restored the enzyme activity (after PON1 inhibition).


We thus conclude that the anti- atherosclerotic properties of HDL could be attributed, at list in part, to two major mechanism of action of HDL-associated PON1; (i) Protecting macrophages from TG - induced oxidative modification in a process that involves a  mitochondrial pathway. (ii) Reduction of LLE peroxide levels via specific reaction between the PON1 free thiol group and lesion LA-OOHs.