Redox western blot technique7/13/2023 ![]() ![]() Expression of TRX in mammalian cells is enhanced by a variety of stresses such as H 2O 2, x-ray and UV exposure, or anticancer drug treatment ( 4, 9, 10, 11, 12). Subsequent studies have shown that TRX is released from several cell types through a leaderless pathway in response to various stimulations ( 6, 7), and that APCs release TRX to provide the reducing environment for the activation of T lymphocytes ( 8). Human TRX was originally cloned as a soluble factor produced by human T cell leukemia virus type I (HTLV-I)-transformed ATL2 cells ( 3, 4) or as an autocrine growth factor produced by EBV-transformed B cells ( 5). Thioredoxin (TRX) 3 is a small (12 kDa) ubiquitous protein containing a conserved active site, -Cys-Gly-Pro-Cys-, and plays a variety of redox-related roles in organisms ranging from Escherichia coli to humans ( 1, 2). These results collectively suggest that the oxidative stress-induced TRX release from T lymphocytes depends on a redox-sensitive event and may be regulated by negative feedback loops using reactive oxygen species-mediated signal transductions. Confocal microscopy and Western blot analysis show that extracellular rTRX-WT added to the culture does not obviously enter T lymphocytes until 24 h. Preincubation of the transfectant with rTRX-WT for 1 h at 37☌, but not 0☌, results in a significant suppression of the TRX release, reactive oxygen species, and caspase-3 activity induced by H 2O 2, respectively. H 2O 2-induced release of TRX from the transfectant is inhibited by the presence of rTRX-WT in a dose-dependent manner. In contrast, another type of transfectant expressing the tagged TRX mutant (C32S/C35S CS) fails to release the protein. In the culture supernatant of a Jurkat transfectant expressing the tagged TRX-wild type (WT), the tagged TRX protein is rapidly released at 1 h and kept at a constant level until 6 h after the addition of H 2O 2. The level of TRX release is augmented upon the addition of H 2O 2, but suppressed upon the addition of N-acetylcysteine. Human T cell leukemia virus type I-transformed T lymphocytes constitutively release a large amount of TRX. We show here that a redox-active site in TRX is essential for its release from T lymphocytes in response to H 2O 2 and extracellular TRX regulates its own H 2O 2-induced release. Thioredoxin (TRX) is released from various types of mammalian cells despite no typical secretory signal sequence. ![]()
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