Figure 7
From: Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress
Dissection of proteasome inhibitor and oxidative activities of heme. (a) Hmox1 (−/−) MEFs were incubated with different metal porphyrins at 10 μM for 18 h. Expression of Sqstm1 and ubiquitin protein was probed by western blot. Accumulation of Sqstm1 and high-molecular-weight ubiquitin protein aggregates in cells treated with FePP and CoPP (Fe: iron; Sn: tin; Mn: manganese; Co: cobalt; Zn: zinc). (b) Accumulation of constitutively expressed SQSTM-myc in RAW264 cells after treatment with CoPP and bortezomib (Bor, 100 pM) for 12 h (western blot against myc-tag; β-actin as a loading control). (c) Inhibition of the proteolytic activity of purified 26S proteasome by CoPP and bortezomib was quantified by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc. All data for CoPP were corrected for fluorescence quenching by the porphyrin. Data represent mean±S.D. of six biologic replicates. (d) Linoleic acid oxidation by FePP but not by CoPP. Equal amounts of each porphyrin (final concentration 10 μM) were injected into stirred, airtight reaction vessels at 37°C containing linoleic acid as the reactant. Oxygen concentration was measured continuously during the reaction with a Clark type electrode. Data represent mean±S.D. (dashed lines) of three independent experiments. (e) Formation of cellular protein-lipid adducts in FePP-treated (20 μM, 5 h) or CoPP-treated (20 μM, 5 h) Hmox1 (−/−) MEFs detected by click chemistry facilitated biotinylation of cell lysates and subsequent western blot detection by HRP streptavidin. (f) Glutathione (GSH) in Hmox1 (−/−) MEF cells after incubation with different concentrations of FePP and CoPP for 12 h. Data indicate mean±S.D. of luminescence from six biologic replicates. (g, left) Heme (30 μM)-triggered lipid peroxidation (TBARS) in soybean lecithin micelles and inhibition by Trolox (10 mM). Data represent mean±S.D. of three biologic replicates. (g, right) Enzymatic activity of purified 26S proteasome as assessed by monitoring the release of fluorescent AMP from the proteasome substrate peptide Suc-LLVT-Amc in the presence or absence of heme (30 μM), bortezomib (100 pM)±the antioxidant Trolox (10 mM). Data were corrected for fluorescence quenching by heme, and represent mean±S.D. of six independent replicates
