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

Abstract

BackgroundSecTRAPs (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 FindingsBoth 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.ConclusionsWe 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.

Crystal structure of the human thioredoxin reductase–thioredoxin complex

“…Furthermore, we determined increased NADPH-oxidation with high concentrations of hTrxR1 W114A,U498C in the presence and absence of hTrx1 C73S in the assay (Table 2). Our results are in accordance with the previously described findings that TrxR1 shows an enhanced NADPH oxidation if there is no electron transport to the substrate 20,21 . Our notion was further supported by the fact that with DTNB, a small artificial disulphide substrate 22 , hTrxR1…”

Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

“…The Cys-59 thiolateand charge-transfer-containing active site is thereby instead available for other types of reactions, including one-(or two-) electron reduction of quinones such as juglone. This line of reasoning is supported by the known high capacity of native, Sec-derivatized as well as Sec-lacking truncated forms of TrxR in reduction of juglone directly through the N-terminal redox active motif (38,39,68). Hence, without resonance effects of Tyr-116 and therefore a lack of an efficient reductive half-reaction, the result would easily be the observed increased propensity for juglone reduction but diminished activity with Trx as substrate.…”

“…However, considering that juglone can be efficiently reduced by either the C-terminal selenolthiol motif or directly by the N-terminal FAD/dithiol motif of TrxR (38,39,68) and considering the plausible roles of Tyr-116 in the reductive and oxidative half-reactions, as described above, the effects of Tyr-116 mutations become explainable. Hence, we propose that the Tyr-116 substitutions diminish the possibility for the Cys-59 thiolate to attack Sec-498 of the selenenylsulfide, thereby mainly leaving the selenenylsulfide in the inactive oxidized state.…”

The Selenium-independent Inherent Pro-oxidant NADPH Oxidase Activity of Mammalian Thioredoxin Reductase and Its Selenium-dependent Direct Peroxidase Activities

“…The first class of inhibitors described for this enzyme (dinitrohalobenzenes) was also found to facilitate and increase its NADPH oxidase activity, presumably by redox cycling of the covalently attached dinitrophenyl groups (11,20). Subsequently, a similar induction of NADPH oxidase activity of TrxR was shown for other low molecular weight compounds, including juglone (19,21), curcumin (22), and motexafen gadolinium (23). However, the catalytic mechanisms of the non-induced inherent NADPH oxidase activity of TrxR have not yet been elucidated.…”

“…The inhibition of the active site of TrxR may promote pro-oxidant effects in cells, which may directly promote cell death as selenium-compromised thioredoxin reductase-derived apoptotic proteins (SecTRAPs) (1,19). These cellular effects are not yet well understood, and it is not clear if all inhibitors of TrxR promote these effects.…”