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Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

Anestål K, Prast-Nielsen S, Cenas N, Arnér ES - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

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.

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Related in: MedlinePlus

Model for the formation and function of SecTRAPs.We propose that during most conditions of normal cell growth the Sec residue in TrxR1 is intact and the role of this enzyme is thus to sustain the many cellular functions of the thioredoxin system. This activity is dependent upon the redox active C-terminal Sec-containing active site in TrxR1. SecTRAPs may however be formed from TrxR1 if its Sec residue becomes compromised, either by removal or by derivatization with electrophilic compounds, while the FAD and N-terminal redox active CVNVGC motif of the enzyme are kept intact. SecTRAPs lack the Trx reducing activity of native TrxR1 but become potent inducers of cell death by a gain of function. This cell death is, as shown in the present study, rapid, does not require induction of protein synthesis, involves production of reactive oxygen species and is prevented by caspase inhibitors. If, on the other hand, both the CVNVGC motif and the C-terminal selenolthiol motif become inactivated, e.g. by certain types of TrxR1 inhibitors or if the overall expression of TrxR1 is diminished in cells, impaired cell function or cell death may still occur. In such cases the cellular consequences would however not be due to a gain of function in derivatives of TrxR1, but rather to hampered functions of the complete cellular thioredoxin system.
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pone-0001846-g009: Model for the formation and function of SecTRAPs.We propose that during most conditions of normal cell growth the Sec residue in TrxR1 is intact and the role of this enzyme is thus to sustain the many cellular functions of the thioredoxin system. This activity is dependent upon the redox active C-terminal Sec-containing active site in TrxR1. SecTRAPs may however be formed from TrxR1 if its Sec residue becomes compromised, either by removal or by derivatization with electrophilic compounds, while the FAD and N-terminal redox active CVNVGC motif of the enzyme are kept intact. SecTRAPs lack the Trx reducing activity of native TrxR1 but become potent inducers of cell death by a gain of function. This cell death is, as shown in the present study, rapid, does not require induction of protein synthesis, involves production of reactive oxygen species and is prevented by caspase inhibitors. If, on the other hand, both the CVNVGC motif and the C-terminal selenolthiol motif become inactivated, e.g. by certain types of TrxR1 inhibitors or if the overall expression of TrxR1 is diminished in cells, impaired cell function or cell death may still occur. In such cases the cellular consequences would however not be due to a gain of function in derivatives of TrxR1, but rather to hampered functions of the complete cellular thioredoxin system.

Mentions: Here we showed that SecTRAPs, devoid of the natural Sec-containing active site of TrxR1, could still have their FAD cofactor efficiently reduced by NADPH. Upon reduction by NADPH, SecTRAPs could also form the charge-transfer complex between the FAD moiety and the CVNVGC redox active disulfide/dithiol-containing site. The fact that the cell-killing SecTRAP properties evidently depend upon an intact FAD/CVNVGC-containing redox active motif and correlate to a prooxidant capacity has additional implications. A potential drug or compound that would inhibit the redox activity of this N-terminal motif, and thereby block the reductive half reaction of TrxR1-derived protein, would according to his notion not give rise to the formation of SecTRAPs. However, such potential drugs may still have major effects on cellular function as a result of the lowered capacity of the thioredoxin system, reminiscent to the effects of knocking down TrxR1 expression by siRNA [60], [68], [74], [75]. This emphasizes the delicate balance between different cellular effects that can be governed by either an intact thioredoxin system, by a loss of thioredoxin reductase activity, or by the formation of SecTRAPs. This concept is summarized in the scheme shown in Figure 9.


Cell death by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.

Anestål K, Prast-Nielsen S, Cenas N, Arnér ES - PLoS ONE (2008)

Model for the formation and function of SecTRAPs.We propose that during most conditions of normal cell growth the Sec residue in TrxR1 is intact and the role of this enzyme is thus to sustain the many cellular functions of the thioredoxin system. This activity is dependent upon the redox active C-terminal Sec-containing active site in TrxR1. SecTRAPs may however be formed from TrxR1 if its Sec residue becomes compromised, either by removal or by derivatization with electrophilic compounds, while the FAD and N-terminal redox active CVNVGC motif of the enzyme are kept intact. SecTRAPs lack the Trx reducing activity of native TrxR1 but become potent inducers of cell death by a gain of function. This cell death is, as shown in the present study, rapid, does not require induction of protein synthesis, involves production of reactive oxygen species and is prevented by caspase inhibitors. If, on the other hand, both the CVNVGC motif and the C-terminal selenolthiol motif become inactivated, e.g. by certain types of TrxR1 inhibitors or if the overall expression of TrxR1 is diminished in cells, impaired cell function or cell death may still occur. In such cases the cellular consequences would however not be due to a gain of function in derivatives of TrxR1, but rather to hampered functions of the complete cellular thioredoxin system.
© Copyright Policy
Related In: Results  -  Collection

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pone-0001846-g009: Model for the formation and function of SecTRAPs.We propose that during most conditions of normal cell growth the Sec residue in TrxR1 is intact and the role of this enzyme is thus to sustain the many cellular functions of the thioredoxin system. This activity is dependent upon the redox active C-terminal Sec-containing active site in TrxR1. SecTRAPs may however be formed from TrxR1 if its Sec residue becomes compromised, either by removal or by derivatization with electrophilic compounds, while the FAD and N-terminal redox active CVNVGC motif of the enzyme are kept intact. SecTRAPs lack the Trx reducing activity of native TrxR1 but become potent inducers of cell death by a gain of function. This cell death is, as shown in the present study, rapid, does not require induction of protein synthesis, involves production of reactive oxygen species and is prevented by caspase inhibitors. If, on the other hand, both the CVNVGC motif and the C-terminal selenolthiol motif become inactivated, e.g. by certain types of TrxR1 inhibitors or if the overall expression of TrxR1 is diminished in cells, impaired cell function or cell death may still occur. In such cases the cellular consequences would however not be due to a gain of function in derivatives of TrxR1, but rather to hampered functions of the complete cellular thioredoxin system.
Mentions: Here we showed that SecTRAPs, devoid of the natural Sec-containing active site of TrxR1, could still have their FAD cofactor efficiently reduced by NADPH. Upon reduction by NADPH, SecTRAPs could also form the charge-transfer complex between the FAD moiety and the CVNVGC redox active disulfide/dithiol-containing site. The fact that the cell-killing SecTRAP properties evidently depend upon an intact FAD/CVNVGC-containing redox active motif and correlate to a prooxidant capacity has additional implications. A potential drug or compound that would inhibit the redox activity of this N-terminal motif, and thereby block the reductive half reaction of TrxR1-derived protein, would according to his notion not give rise to the formation of SecTRAPs. However, such potential drugs may still have major effects on cellular function as a result of the lowered capacity of the thioredoxin system, reminiscent to the effects of knocking down TrxR1 expression by siRNA [60], [68], [74], [75]. This emphasizes the delicate balance between different cellular effects that can be governed by either an intact thioredoxin system, by a loss of thioredoxin reductase activity, or by the formation of SecTRAPs. This concept is summarized in the scheme shown in Figure 9.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus