Lipid Raft–Mediated Uptake of Cysteine-Modified Thioredoxin-1: Apoptosis Enhancement by Inhibiting the Endogenous Thioredoxin-1

Abstract

Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus-transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-beta-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.

Protective effects of oral administration of yeast thioredoxin against gastric mucosal injury

“…We infer that yeast TRX affects the gastric cells mainly from the extracellular side in this study. In the previous report, recombinant human TRX was observed to enter into ATL2 cells gradually over 24 h. [30][31][32] We show that yeast TRX is digested within only several hours (Fig. 1), and thus it is unlikely that large fraction of active yeast TRX without denaturation enters the gastric cells.…”

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

“…In particular, a TRX mutant, TRX-C35S (with replacement of cysteine 35 by serine), was found to bind rapidly to the cell surface and be internalized into the cells through LRs in the plasma membrane. This indicates that the cysteine at the active site of TRX is important for the internalization and signal transduction of extracellular TRX through LRs (156,210).…”

Direct Association of Thioredoxin-1 (TRX) with Macrophage Migration Inhibitory Factor (MIF): Regulatory Role of TRX on MIF Internalization and Signaling

“…These results strongly suggested that formation of disulfide bond between MIF and cell surface TRX is involved in the internalization of MIF. Previously, we reported that TRX-C35S bound to ATL-2 cells much effectively than TRX-WT (20). There is evidence that a disulfide bond is involved in the binding of TRX-C35S to cell surface molecules.…”

“…Since endogenous TRX is expressed on the surface of ATL-2 cells and endothelial cells (10,20,24), we assumed that cell surface TRX is involved in the internalization process of MIF into the cells. We assessed the expression of TRX on the surface of ATL-2 cells or Jurkat T cells by flow cytometry.…”