MO_V_L: Molecular Sciences V. Lectures
Krisztina Veszelyi1, Viola Varga1, Csilla E. Németh2, Éva Margittai1
1 Semmelweis University, Institute of Translational Medicine, Budapest
2 Semmelweis University, Institute of Biochemistry and Molecular Biology, Budapest
Introduction: The endoplasmic reticulum (ER) is the major site of protein thiol oxidation during post-translational modification. Due to the dominance of oxidized proteins, 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 parallel occurrence of oxidized thiol-disulfides and reduced pyridine nucleotides may indicate that the ER lumen lacks components which connect the two systems.
Aims: Our aim was to investigate the luminal presence of the thioredoxin (Trx) / thioredoxin reductase (TrxR) proteins, capable to link the protein thiol and pyridine nucleotide systems.
Methods: Protein expression of Trx/TrxR isoforms was examined on subcellular fractions by Western blot analysis. TrxR activity in each organelle was measured using a colorimetric kit. 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 isoform.
Results: We 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 the two isoforms of Trx, and the three isoforms of TrxR 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 show 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.
Funding: EFOP-3.6.3-VEKOP-16-2017-00009, Az orvos-, egészségtudományi- és gyógyszerészképzés tudományos műhelyeinek fejlesztése
Semmelweis University, Doctoral School of Theoretical and Translational Medicine