Intricate aspects of the thioredoxin system in redox signaling

Reversible modifications of redox sensitive protein thiols by reactive oxygen and nitrogen species have emerged as a major posttranslational mechanism that affects the function of the respective proteins and therewith all downstream events. These modifications can be reversed by redox catalysts of which the thioredoxin system forms one of the most prominent. It is ubiquitously expressed and consists of thioredoxin reductase (TrxR) that takes electrons from NADPH to reduce thioredoxin (Trx) as well as a myriad of other substrates. Within this thesis we have studied several aspects of cellular signaling pathways modulated by the Trx system.

Paper I. The Trx system is overexpressed in many types of cancers and considered to contribute to their survival by countering elevated ROS levels that are typical for these cells. Thus, inhibiting TrxR in order to attenuate the antioxidant capacity of cancer cells might tip the balance in favor of ROS induced cell death pathways as a principle of cancer therapy. TrxR1 is a particularly suitable target in this context due to its highly reactive and accessible selenocysteine (Sec) residue within its C-terminal active site. At physiological pH the Sec is mostly de-protonated and thus easily targeted by electrophilic compounds. In characterizing Au, Pt and Pd based salts we found that all inhibited the Sec-depended activity of the enzyme in a specific manner, with Au and Pd being more potent than Pt in vitro. In context of cellular TrxR1, however, inhibition and cytotoxicity were mainly dependent on the ligand substituents of the compounds and thus their cellular uptake and metabolism. We furthermore discovered cisplatin triggered covalent complex formation of TrxR1 with either Trx1 or TRP14 (thioredoxin like protein of 14 kDa), which potentially contributes to the mechanism of cisplatin mediated cytotoxicity.

Paper II. TrxR1 has in addition to its main isoform at least five minor splice variants that are distinguished by their N-terminal extensions. These may directly influence the activity of the TrxR1 core module or mediate subcellular localization via potential translocation signals. One of these variants, named “v3” (carrying a unique glutaredoxin domain), was previously shown to associate with the plasma membrane where it provoked dynamic filopodia. Within this study we found that v3 associates with specific membrane raft microdomains upon N-myristoylation and palmitoylation. These membrane structures were shown to serve as signaling platforms, including redox dependent processes, suggesting that v3 is potentially involved in redox signaling.

Paper III. Transcription factors are a specific group of proteins that regulate the rapid transcription of genes. Many are functionally intertwined, activated under redox perturbing conditions and highly controlled by regulatory networks like the thioredoxin and glutathione systems. Signaling pathways leading to their activation are complex and expected to be modulated by numerous factors. In order to simultaneously characterize several transcription factors on single cell level we developed a method that is based on a three-colored fluorescence-based reporter plasmid (pTRAF). We demonstrated the use by quantifying responses of the three medically important transcription factors Nrf2, HIF and NFB, utilizing HEK293 cells that were subjected to diverse stimulants.

In conclusion, we studied TrxR1 targeting by noble metal based compounds and characterized their ability to transform TrxR1 into its pro-oxidant SecTRAP form as a principle of anti-cancer therapy. We also identified the mechanism behind the intracellular localization of “v3” and show that it is targeted to lipid rafts where it is a potentially important regulator of signaling processes. Finally we developed a tool to study the activation of three redox sensitive, intertwined transcription factors.

List of scientific papers

I. Stefanie Prast-Nielsen, Marcus Cebula, Irina Pader, Elias S. J. Arnér. Noble metal targeting of thioredoxin reductase – covalent complexes with thioredoxin and thioredoxin-related protein of 14 kDa triggered by cisplatin. Free Radic Biol Med. 2010;49:1765-78.
https://doi.org/10.1016/j.freeradbiomed.2010.09.008

II. Marcus Cebula, Naazneen Moolla, Alexio Capovilla and Elias S. J. Arnér. TXNRD1-encoded v3 is targeted to membrane rafts by N-acylation and induces filopodia independently of its redox active site integrity. J Biol Chem. 2013;288:10002-11.
https://doi.org/10.1074/jbc.M112.445932

III. Katarina Johansson, Marcus Cebula, Olle Rengby, Kristian Dreij, Kristmundur Sigmundsson, Elias S.J. Arnér. Simultaneous Determination of Nrf2, HIF and NFκB Activation at Single‐Cell Resolution. [Manuscript]