α, ß–unsaturated carbonyls covalently modify cellular PTEN.

Abstract

<p>(<b>A</b>) Diagram of the procedure to identify PTEN with an oxidized or alkylated thiol in cells exposed to ROS or α, ß–unsaturated carbonyls. The anti-PTEN immunoblot shows oxidized or carbonylated PTEN (NA Pulldown) relative to total PTEN (Cell Lysate) isolated from MCF-7 cells treated 30 min with vehicle (DMSO), 10 µM Δ12-PGJ₂ or 4-HNE versus 10 min with 100 µM H₂O₂; an immunoblot from a separate experiment shows oxidized, carbonylated and total PTEN in cells treated for 30 min with vehicle or 20 µM acrolein versus 10 min with 100 µM H₂O₂. (<b>B</b>) Anti-PTEN immunoblot of MCF-7 cell lysates fractionated by SDS-PAGE under non-reducing conditions. PTEN oxidized to a Cys¹²⁴-Cys⁷¹ disulfide appears as a faster migrating species (PTEN oxidized disulfide) only in cells treated with H₂O₂. This species was undetectable in MCF-7cells treated 30 min with DMSO vehicle or 20 µM each Δ12-PGJ₂, 4-HNE, acrolein or 15-HpETE. (<b>C</b>) Chemical structures of typical α, ß–unsaturated carbonyls. Acrolein and 4-HNE are α, β enals; Δ12PGJ₂ is an α,β enone. Electrophilic β carbons are denoted with δ⁺. Blots are representative of results obtained in at least three independent experiments.</p

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