Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels
Gulsah Sevimli,
Matthew J. Smith,
Tuba Akgul Caglar,
Şükriye Bilir,
Melike Secilmis,
Hamza Y. Altun,
Esra N. Yigit,
Fan Yang,
Thomas P. Keeley,
Roland Malli,
Gürkan Öztürk,
Giovanni E. Mann,
Emrah Eroglu
Affiliations
Gulsah Sevimli
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
Matthew J. Smith
King's British Heart Foundation Centre of Research Excellence, School of Cardiovascular and Metabolic Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK
Tuba Akgul Caglar
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey
Şükriye Bilir
Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey
Melike Secilmis
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
Hamza Y. Altun
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey
Esra N. Yigit
Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey; Department of Biotechnology, Gebze Technical University, Kocaeli, Turkey
Fan Yang
King's British Heart Foundation Centre of Research Excellence, School of Cardiovascular and Metabolic Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK
Thomas P. Keeley
Target Discovery Institute, University of Oxford, Oxford, OX3 7FZ, UK
Roland Malli
Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010, Graz, Austria; BioTechMed Graz, Mozartgasse 12/II, 8010, Graz, Austria
Gürkan Öztürk
Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey; Physiology Department, International School of Medicine, Istanbul Medipol University, Istanbul, Turkey
Giovanni E. Mann
King's British Heart Foundation Centre of Research Excellence, School of Cardiovascular and Metabolic Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UK; Corresponding author.
Emrah Eroglu
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey; Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul, Turkey; Corresponding author. Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul, Turkey.
Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.
