PhD Scientific Days 2024

Budapest, 9-10 July 2024

Poster Session F - Molecular Medicine 3.

Endoplasmic Reticulum Redox Homeostasis - the Potential Absence of the Thioredoxin / Thioredoxin Reductase System in the Lumen


Krisztina Nóra Veszelyi1, Csilla Emese Németh2, Viola Varga3, Balázs Besztercei3, Éva Margittai3
1: Semmelweis Egyetem ÁOK Transzlációs Medicina Intézet
2: Department of Molecular Biology, Semmelweis University, Budapest, Hungary
3: Institute of Translational Medicine, Semmelweis University, Budapest, Hungary

Text of the abstract

Introduction: The endoplasmic reticulum (ER) is the major site for protein thiol oxidation during post-translational modification. The lumen of the ER is typically regarded as an oxidative environment, despite the presence of some processes requiring reducing agents like NADPH. In most cellular compartments, the thiol/disulfide and reduced/oxidized pyridine nucleotide systems are enzymatically linked to form complex redox networks. However, the parallel presence of oxidized thiol-disulfides and reduced pyridine nucleotides suggests that the ER lumen may lack elements that link these two systems together.
Aims: Our aim was to confirm the luminal deficiency of one of these coupling systems, the thioredoxin (Trx) / thioredoxin reductase (TrxR) proteins, and to investigate the effects of their artificial expression.
Methods: We performed an in silico analysis to predict the localization of each Trx/TrxR isoform. We examined the protein expression of each isoform in subcellular fractions by Western blot analysis. Their intracellular distribution was further analyzed by immunofluorescence microscopy. TrxR activity was measured with a colorimetric assay in various organelles. We measured cell viability of transiently transfected cells expressing Trx1 and TrxR1 in the ER and examined the induction of apoptosis by Western blot.
Results: In silico prediction analysis showed a very low probability of luminal localization for each isoform (0–5%). Analysis of rat liver subcellular fractions revealed that none of the Trx/TrxR isoforms are expressed in the ER. Immunofluorescence analysis confirmed that Trx and TrxR isoforms did not colocalize with the ER-marker Grp94. The specific activity of TrxR in the ER was nearly zero (0,02 U/mg ± 0,01), while we measured higher activities in the cytoplasm (1,26 U/mg ± 0,11) and mitochondria (1,57 U/mg ± 0,19). Transient transfection of cells with ER-targeted Trx1 and TrxR1 resulted in a rapid decrease in cell viability and induction of apoptosis.
Conclusions: Our results indicate that none of the components of the Trx/TrxR system is expressed in the ER lumen. The absence of this electron transfer chain may explain the uncoupling of the luminal redox systems, allowing the parallel occurrence of a reduced pyridine nucleotide pool and oxidized proteins.
Funding: Semmelweis 250+ Kiválósági PhD Ösztöndíj