Delivery of the selenoprotein thioredoxin reductase 1 to mammalian cells

David E Wright; Tarana Siddika; Ilka U Heinemann; Patrick O'Donoghue

doi:10.3389/fmolb.2022.1031756

Delivery of the selenoprotein thioredoxin reductase 1 to mammalian cells

Front Mol Biosci. 2022 Oct 11:9:1031756. doi: 10.3389/fmolb.2022.1031756. eCollection 2022.

Authors

David E Wright¹, Tarana Siddika¹, Ilka U Heinemann¹, Patrick O'Donoghue¹

Affiliation

¹ Departments of Biochemistry and Chemistry, The University of Western Ontario, London, ON, Canada.

Abstract

Over-expression of genetically encoded thioredoxin reductase 1 (TrxR1) TrxR1 can be toxic to cells due to the formation of a truncated version of the enzyme. We developed a new mammalian cell-based model to investigate TrxR1 activity. Fusion of the HIV-derived cell penetrating peptide (TAT) enabled efficient cellular uptake of purified TrxR1 containing 21 genetically encoded amino acids, including selenocysteine. The TAT peptide did not significantly alter the catalytic activity of TrxR1 in vitro. We monitored TrxR1-dependent redox activity in human cells using a TrxR1-specific red fluorescent live-cell reporter. Using programmed selenocysteine incorporation in Escherichia coli, our approach allowed efficient production of active recombinant human selenoprotein TrxR1 for delivery to the homologous context of the mammalian cell. The delivered TAT-TrxR1 showed robust activity in live cells and provided a novel platform to study TrxR1 biology in human cells.

Keywords: cell biology; cell penetrating peptide; enzymology; genetic code expansion; redox biology; selenocysteine; thioredoxin reductase; trans-activator of transcription peptide.