Zahia Touat-Hamici1, Anne-Laure Bulteau2, Juliusz Bianga3, Hélène Jean-Jacques4, Joanna Szpunar3, Ryszard Lobinski3, Laurent Chavatte5. 1. From the Centre de Génétique Moléculaire, CGM, CNRS, UPR3404, Gif-sur-Yvette 91198, France. 2. Institut de Génomique Fonctionnelle de Lyon, IGFL, CNRS/ENS UMR5242, 69007 Lyon, France. 3. CNRS/Univ Pau & Pays Adour, Institut des Sciences Analytiques et de Physico-chimie pour l'Environnement et les Matériaux, IPREM-UMR5254, 64000 Pau, France. 4. Institut de Biologie Intégrative de la Cellule, I2BC, 91198 Gif-sur-Yvette, France. 5. Centre International de Recherche en Infectiologie, CIRI, 69007 Lyon, France; INSERM U1111, 69007 Lyon, France; CNRS/ENS/UCBL1 UMR5308, 69007 Lyon, France. Electronic address: laurent.chavatte@ens-lyon.fr.
Abstract
BACKGROUND: Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. METHODS: We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. RESULTS: We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. CONCLUSIONS: We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. GENERAL SIGNIFICANCE: We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture.
BACKGROUND: Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy. METHODS: We investigated the hierarchy of selenoprotein expression in response to selenium concentration variation in four cell lines originating from kidney (HEK293, immortalized), prostate (LNCaP, cancer), skin (HaCaT, immortalized) and liver (HepG2, cancer), using complementary analytical methods. We performed (i) enzymatic activity, (ii) RT-qPCR, (iii) immuno-detection, (iv) selenium-specific mass spectrometric detection after non-radioactive 76Se labeling of selenoproteins, and (v) luciferase-based reporter constructs in various cell extracts. RESULTS: We characterized cell-line specific alterations of the selenoproteome in response to selenium variation that, in most of the cases, resulted from a translational control of gene expression. We established that UGA-selenocysteine recoding efficiency, which depends on the nature of the SECIS element, dictates the response to selenium variation. CONCLUSIONS: We characterized that selenoprotein hierarchy is cell-line specific with conserved features. This analysis should be done prior to any experiments in a novel cell line. GENERAL SIGNIFICANCE: We reported a strategy based on complementary methods to evaluate selenoproteome regulation in human cells in culture.
Authors: Sarah Tauber; Maria Katharina Sieckmann; Katrin Erler; Wilhelm Stahl; Lars-Oliver Klotz; Holger Steinbrenner Journal: Antioxidants (Basel) Date: 2021-01-23