Identification of Novel Nuclear Targets of Human Thioredoxin 1

Abstract

The dysregulation of protein oxidative post-translational modifications has been implicated in stress-related diseases. Trx1 is a key reductase that reduces specific disulfide bonds and other cysteine post-translational modifications. Although commonly in the cytoplasm, Trx1 can also modulate transcription in the nucleus. However, few Trx1 nuclear targets have been identified because of the low Trx1 abundance in the nucleus. Here, we report the large-scale proteomics identification of nuclear Trx1 targets in human neuroblastoma cells using an affinity capture strategy wherein a Trx1C35S mutant is expressed. The wild-type Trx1 contains a conserved C32XXC35 motif, and the C32 thiol initiates the reduction of a target disulfide bond by forming an intermolecular disulfide with one of the oxidized target cysteines, resulting in a transient Trx1-target protein complex. The reduction is rapidly consummated by the donation of a C35 proton to the target molecule, forming a Trx1 C32-C35 disulfide, and results in the concurrent release of the target protein containing reduced thiols. By introducing a point mutation (C35 to S35) in Trx1, we ablated the rapid dissociation of Trx1 from its reduction targets, thereby allowing the identification of 45 putative nuclear Trx1 targets. Unexpectedly, we found that PSIP1, also known as LEDGF, was sensitive to both oxidation and Trx1 reduction at Cys 204. LEDGF is a transcription activator that is vital for regulating cell survival during HIV-1 infection. Overall, this study suggests that Trx1 may play a broader role than previously believed that might include regulating transcription, RNA processing, and nuclear pore function in human cells. Molecular

Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes

“…This was accompanied by increased ROS levels and an imbalance of members of the Bcl-2 family. Although increased ROS may contribute to this strong knockout phenotype, it should be recognized that thioredoxins can directly interact with protein complexes and transcription factors (29) and mediate alternative redox reactions such as trans-nitrosylation (30), demonstrating that their role extends beyond H 2 O 2 scavenging alone. This pleiotropic role of mitochondrial TXNRD2 may explain why early homozygous knockout results in embryonic lethality in mice (31).…”

Modulation of signaling mechanisms in the heart by thioredoxin 1

“…Using this technique, they have identified AMPK as an oxidoreductase substrate of Trx1. Wu et al utilized this strategy to identify additional targets of Trx1 in the nucleus [18]. Recently, Booze et al have modified this trapping mutant transgenic mouse model by including a LoxP–STOP–LoxP cassette between the promoter and Flag-Trx1 C35S to allow temporal and cell-specific transgene expression [19].…”

A novel mouse model for the identification of thioredoxin-1 protein interactions

“…Studies from our laboratory have successfully used the Trx1 substrate trap in lung adenocarcinoma cells [17]. Novel nuclear targets, such as the transcription factor PC4 and SFRS1 interaction protein 1 (PSIP1), have been identified by fusing a canonical nuclear localization signal to the substrate trap transgene [18]. However, there are caveats with using this substrate trap in vitro or in cellular models since they do not recapitulate complex physiologies of multicellular organisms.…”