Disabling Mitochondrial Peroxide Metabolism via Combinatorial Targeting of Peroxiredoxin 3 as an Effective Therapeutic Approach for Malignant Mesothelioma
Fig 3
TS adducts specific cysteines of rPRX3 in vitro.
(A) In vitro reaction between recombinant wild type (WT) PRX3 (100 μM) and 200 μM TS. Reactions contained either the Trx regeneration system or TCEP. The samples were treated with 6 successive additions of 100 μM H2O2 and 200 μM TS, as indicated. Samples were resolved by reducing SDS-PAGE and visualized by staining for total protein with GelCode Blue. (B) WT PRX3 or the indicated PRX3 mutants were incubated with or without TS as in panel A and formation of irreducible PRX3 dimers was visualized by staining for total protein after reducing SDS-PAGE. (C) WT PRX3 and the dimeric variant of PRX3 (EE Mut) were treated with 200 μM TS. (D) MS analysis of TS adducts. The EE Mut samples shown in lanes 3 and 4 of panel C were treated with 20 mM DTT to reduce disulfide bonds and to block further reactions of thiostrepton dehydroalanines with PRX3 thiols. The molecular weight of the resulting PRX3 adducts were determined by ESI-TOF MS. The signals at 21640.6 and 21672.5 atomic mass units (amu) correspond to the average molecular weight of the reduced monomer (-SH, 21640.6 amu) and sulfinic acid monomer (-SO2, 21672.6 amu), respectively. The peak at 43280.1 amu corresponds to a PRX3 dimer containing one disulfide (theoretical MW = 43,278.2) that presumably occurs as a result of trace amounts of oxidants present during buffer exchange prior to MS analysis. The signals at 44,944.9 and 46,612.3 amu correspond to 2 PRX3 monomers linked by 1 (44,945.0 amu) and 2 (46,609.8 amu) thiostrepton adducts, respectively. (E) Samples from (D) were digested with trypsin and peptides were analyzed by MALDI-TOF MS. The peptide at 7970.2 amu corresponds to a single thiostrepton linked to both the C108 and C229 peptides in PRX3 (7971.0 amu).