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Identification of novel cytosolic thioredoxin-1 target proteins in mammalian cells by mechanism-based kinetic trapping
Reactive oxygen species (ROS) have been reported to be locally produced after stimulation of various transmembrane receptors, including those for cytokines, growth factors and hormones. Transient ROS generation leads to the reversible oxidation of redox-sensitive proteins, which may either result in their transient activation or inactivation. Even though the role of ROS as a signal transducer is widely accepted, little is known about the identity of redox-sensitive proteins and the reduction mechanism by which they are re-generated. Thioredoxin-1 (Trx1) is an oxidoreductase known to influence a variety of cellular processes including proliferation, apoptosis and gene expression. In higher plants and cyanobacteria different proteomic approaches allowed for the identification of various thioredoxin target proteins. However, no comprehensive study of Trx1 interactions in the cytosol of mammalian cells has been performed so far. In this thesis, mechanism-based kinetic trapping was applied to identify proteins forming mixed disulfide intermediates with Trx1 in the cytosol of a human T cell leukemia line. Most previously established target proteins of Trx1 could be identified, including ribonucleotide reductase, peroxiredoxins and annexin-2, thus confirming the validity of the approach. Interestingly, a substantial number of newly identified proteins currently lacks functional annotation, suggesting that several thioredoxin-regulated pathways still await initial characterization. Of those newly identified proteins with known function, a significant portion is associated with cell cycle control and regulation of apoptosis, while others participate in various signal transduction pathways, cytoskeleton and membrane dynamics, metabolism and transcriptional control. These results strongly support the long-held belief that Trx1 not only plays a role in ROS scavenging and reductive metabolism but is also extensively involved in interactions with key regulatory proteins associated with cellular behavior and fate. While Trx1-interactions are clearly pleiotropic in that they affect a variety of processes simultaneously, we have found strong evidence that they are nevertheless highly target specific. The newly identified Trx1 target proteins cdk6 and caspase-2 were selected for further study. For cdk6 the thioredoxin-interacting cysteine residue was identified and for caspase-2 redox regulation of activity by Trx1 could be demonstrated in vitro, lending further support to the biological relevance of Trx-based regulation.
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HTML Summary of #8923 Identification of novel cytosolic thioredoxin-1 target proteins in mammalian cells by mechanism-based kinetic trapping
570 Biowissenschaften, Biologie
570 Life sciences