Rapid Induction of Cell Death by Selenium-compromised Thioredoxin Reductase 1 but Not by the Fully Active Enzyme Containing Selenocysteine

Elias S.J Arnér,Karin Anestål

doi:10.1074/jbc.m210733200

Elias S.J Arnér, Karin Anestål

Open Access

https://doi.org/10.1074/jbc.m210733200

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Abstract

Mammalian thioredoxin reductases are selenoproteins. For native catalytic activity, these enzymes utilize a C-terminal -Gly-Cys-Sec-Gly-COOH sequence (where Sec is selenocysteine) forming a redox active selenenylsulfide/selenolthiol motif. A range of cellular systems depend upon or are regulated by thioredoxin reductase and its major protein substrate thioredoxin, including apoptosis signal-regulating kinase 1, peroxiredoxins, methionine sulfoxide reductase, and several transcription factors. Cytosolic thioredoxin reductase 1 (TrxR1) is moreover inhibited by various electrophilic anticancer compounds. TrxR1 is hence generally considered to promote cell viability. However, several recent studies have suggested that TrxR1 may promote apoptosis, and the enzyme was identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12). Transient transfection with GRIM-12/TrxR1 was also shown to directly induce cell death. To further analyze such effects, we have here employed lipid-mediated delivery of recombinant TrxR1 preparations into human A549 cells, thereby bypassing selenoprotein translation to facilitate assessment of the protein-related effects on cell viability. We found that selenium-deficient TrxR1, having a two-amino acid-truncated C-terminal -Gly-Cys-COOH motif, rapidly induced cell death (38 +/- 29% apoptotic cells after 4 h; p < 0.005 compared with controls). Cell death induction was also promoted by selenium-compromised TrxR1 derivatized with either cis-diamminedichloroplatinum (II) (cisplatin) or dinitrophenyl moieties but not by the structurally related non-selenoprotein glutathione reductase. In contrast, TrxR1 with intact selenocysteine could not promote cell death. The direct cellular effects of selenium-compromised forms of TrxR1 may be important for the pathophysiology of selenium deficiency as well as for the efficacy of antiproliferative drugs targeting the selenocysteine moiety of this enzyme.

Highlights

Chondrial TrxR2, and a third isoform mainly located in testis [1,2,3,4,5]
To have well defined preparations of thioredoxin reductase 1 (TrxR1), we made use of purified recombinant enzyme produced in E. coli either using a system tailored for heterologous production of the full-length selenoprotein or a construct without a selenocysteine insertion sequence element, thereby producing the two-amino acid truncated selenium-deficient form of the enzyme [34]
We analyzed the effects of GR, which is a non-selenoprotein enzyme closely related to mammalian TrxR1 in both structure [8] and primary sequence [2] but naturally lacking the C-terminal Sec-containing redox active center [2, 7]

Summary

Introduction

Chondrial TrxR2, and a third isoform mainly located in testis [1,2,3,4,5]. All of these enzymes are selenoproteins and carry a selenocysteine (Sec) residue in a C-terminal redox active motif having the amino acid sequence -Gly-Cys-Sec-Gly-COOH. We were intrigued by the fact that transient transfection with TrxR1 constructs was shown to directly induce cell death in MCF-7 cells, with 25% cell death 72 h post-transfection compared with 8% in vector controls [27] or, in a subsequent study, up to 40% cell death 40 h post-transfection with similar results in T47D, COS-7, and HeLa cells [29] How could this selenoprotein, hitherto considered essential for cell viability, display such potent cell death-promoting effects? In an attempt to scrutinize whether the direct deleterious effects by TrxR1 on cell viability could be attributed to selenium-compromised forms of TrxR1, we have here made use of a recently developed methodology for lipid-mediated delivery of intact proteins into mammalian cells [33] This approach was chosen to avoid any production of a mixture of full-length and truncated TrxR1 that may result from inefficient selenoprotein synthesis using transfection experiments, even using constructs including the selenocysteine-encoding UGA codon and a correct mammalian selenocysteine insertion sequence element. We found that selenium-compromised TrxR1, either a selenium-deficient truncated form or an enzyme derivatized at the selenocysteine residue, rapidly induced cell death, whereas such direct effect on viability could not be seen using the full-length selenocysteine-containing enzymatically active form of the enzyme

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