Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

Reductive and oxidative half-reactions of glutathione reductase from escherichia coli

Glutathione reductase catalyzes the reduction of glutathione disulfide by NADPH and has a redox active disulfide and an FAD cofactor in each monomer. In the reductive half-reaction, FAD is reduced by NADPH and electrons pass from the reduced flavin to the redox active disulfide. The oxidative half-reaction is dithiol-disulfide interchange between the enzyme dithiol and glutathione disulfide. We have investigated the reductive and oxidative half-reactions using wild-type glutathione reductase from Escherichia coli and in an altered form of the enzyme in which the active site acid-base catalyst, His439, has been changed to an alanine residue (H439A). H439A has 0.3% activity in the NADPH/GSSG assay. The replacement affects both the oxidative half-reaction, as expected, and the reductive half-reaction-specifically, the passage of electrons from reduced flavin to the disulfide. Reduction of H439A by NADPH allows direct observation of flavin reduction. The NADPH-FAD charge transfer complex is formed in the dead time. Reduction of FAD, at a limiting rate of 250 s-1, is observed as a decrease at 460 nm and an increase at 670 nm (FADH--NADP+ charge transfer). Subsequent passage of electrons from FADH- to the disulfide (increase at 460 nm and a decrease at 670 nm) is very slow (6-7 s-1) and concentration independent in H439A. The monophasic oxidative half-reaction is very slow, as expected for reduced H439A. The limiting rate of the reductive half-reaction in wild-type enzyme is independent of the NADPH concentration and determined to be 110s-1, while the limiting rate of the oxidative half-reaction has been estimated as 490 s-1, and is dependent on the glutathione disulfide concentration. Thus, the acid-base catalyst participates in the disulfide reduction step by stabilizing the nascent thiolate and in the oxidative half-reaction by protonating the dissociating glutathione thiolate anion. Both roles are consistent with proposed mechanisms. © 1994 American Chemical Society

Desmoplakin Cardiomyopathy, a Fibrotic and Inflammatory Form of Cardiomyopathy Distinct from Typical Dilated or Arrhythmogenic Right Ventricular Cardiomyopathy

Background: Mutations in desmoplakin (DSP), the primary force transducer between cardiac desmosomes and intermediate filaments, cause an arrhythmogenic form of cardiomyopathy that has been variably associated with arrhythmogenic right ventricular cardiomyopathy. Clinical correlates of DSP cardiomyopathy have been limited to small case series. Methods: Clinical and genetic data were collected on 107 patients with pathogenic DSP mutations and 81 patients with pathogenic plakophilin 2 (PKP2) mutations as a comparison cohort. A composite outcome of severe ventricular arrhythmia was assessed. Results: DSP and PKP2 cohorts included similar proportions of probands (41% versus 42%) and patients with truncating mutations (98% versus 100%). Left ventricular (LV) predominant cardiomyopathy was exclusively present among patients with DSP (55% versus 0% for PKP2, P<0.001), whereas right ventricular cardiomyopathy was present in only 14% of patients with DSP versus 40% for PKP2 (P<0.001). Arrhythmogenic right ventricular cardiomyopathy diagnostic criteria had poor sensitivity for DSP cardiomyopathy. LV late gadolinium enhancement was present in a primarily subepicardial distribution in 40% of patients with DSP (23/57 with magnetic resonance images). LV late gadolinium enhancement occurred with normal LV systolic function in 35% (8/23) of patients with DSP. Episodes of acute myocardial injury (chest pain with troponin elevation and normal coronary angiography) occurred in 15% of patients with DSP and were strongly associated with LV late gadolinium enhancement (90%), even in cases of acute myocardial injury with normal ventricular function (4/5, 80% with late gadolinium enhancement). In 4 DSP cases with 18F-fluorodeoxyglucose positron emission tomography scans, acute LV myocardial injury was associated with myocardial inflammation misdiagnosed initially as cardiac sarcoidosis or myocarditis. Left ventricle ejection fraction <55% was strongly associated with severe ventricular arrhythmias for DSP cases (P<0.001, sensitivity 85%, specificity 53%). Right ventricular ejection fraction <45% was associated with severe arrhythmias for PKP2 cases (P<0.001) but was poorly associated for DSP cases (P=0.8). Frequent premature ventricular contractions were common among patients with severe arrhythmias for both DSP (80%) and PKP2 (91%) groups (P=non-significant). Conclusions: DSP cardiomyopathy is a distinct form of arrhythmogenic cardiomyopathy characterized by episodic myocardial injury, left ventricular fibrosis that precedes systolic dysfunction, and a high incidence of ventricular arrhythmias. A genotype-specific approach for diagnosis and risk stratification should be used.http://deepblue.lib.umich.edu/bitstream/2027.42/175344/2/CIRCULATIONAHA.119.044934 (2).pdfPublished versionDescription of CIRCULATIONAHA.119.044934 (2).pdf : Published versio

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Selenoproteins and the Thioredoxin System

The thioredoxin system, comprising NADPH, flavoprotein thioredoxin reductase (TrxR), and 12 kDa thioredoxin (Trx) with a catalytic dithiol/disulfide, is the ubiquitous biological cellular disulfide reduction system with major functions in DNA synthesis, defense against oxidative stress, and thiol redox control. In mammalian cells Trx system activity is controlled by the three TrxR isoenzymes which are large homodimeric selenoproteins. The availability of selenium affects not only the Trx system activity but also the existing form of TrxR. Selenium-deficient conditions cause the increase of a low activity form of TrxR, in which a cysteine residue substitutes for selenocysteine (Sec). On the other hand, the Trx system can reduce selenite into selenide, which is required for Sec residue synthesis in proteins. Trx system activity in turn may regulate the redox state and subcellular translocation of Sec insertion sequence element-binding protein 2 (SBP2) and Sec incorporation efficiency in all selenoproteins. The overall structure of TrxR is similar to that of glutathione reductase, but with a C-terminal elongation of 16 residues containing the conserved C-terminal active-site sequence –Gly–Cys–Sec–Gly. In oxidized TrxR, the active site is a selenenylsulfide, which is reduced to a catalytic selenolthiol by electrons from the redox-active disulfide/dithiol of the other subunit, as revealed by three-dimensional structures of the rat TrxR1 enzymes. The critical role of Sec in TrxR and its accessible location and reactivity in the C-terminal active site provide promising pharmaceutical drug targets for various human diseases such as malignant cancer and rheumatoid arthritis