Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)
Krisztina Veszelyi1, Csilla Emese Németh2, Viola Varga1, Balázs Besztercei1, Éva Margittai1
1 Institute of Translational Medicine, Semmelweis University, Budapest, Hungary
2 Department of Molecular Biology, Semmelweis University, Budapest, Hungary
Introduction: The endoplasmic reticulum (ER) is the major site of protein thiol oxidation during post-translational modification. The lumen of the ER is usually considered as an oxidative environment, although some processes which require reducing agents, such as NADPH, are also found here. The colocalized thiol/disulfide and reduced/oxidized pyridine nucleotide systems are enzymatically linked in most compartments to form complex redox systems. However, the parallel occurrence of oxidized thiol-disulfides and reduced pyridine nucleotides may indicate that the ER lumen lacks the components that connect the two systems.
Aims: Our aim was to investigate the luminal presence of one of these linking systems, the thioredoxin (Trx) / thioredoxin reductase (TrxR) proteins.
Methods: Earlier, TrxR activity in each organelle was measured using a colorimetric kit and we examined the protein expression of Trx/TrxR isoforms on subcellular fractions by Western blot analysis. The intracellular distribution of Trx/TrxR isoforms was also examined by immunofluorescent microscopy. An in silico analysis was performed to analyze the predicted localization of each protein’s each isoform. Plasmids were constructed for the transient transfection of Hela cells to overexpress Trx1 and TrxR1 in the ER lumen.
Results: Our previous results showed that the specific activity of TrxR in the ER is around 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). Analysis of rat liver subcellular fractions revealed that Trx and TrxR isoforms are not expressed in the ER. Immunofluorescent analysis confirmed that Trx and TrxR isoforms did not show colocalization with ER-specific marker Grp94. In silico prediction analysis also predicted a very low probability of luminal localization for each isoform (0–5%).
Conclusions: Our results suggest 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 redox systems in the lumen, allowing the parallel presence of a reduced pyridine nucleotide pool and oxidized proteins.
e-mail address: firstname.lastname@example.org
School: Semmelweis University, Doctoral School of Theoretical and Translational Medicine
Supervisor: Dr. Margittai Éva