The Dual Oxidase Duox2 stabilized with DuoxA2 in an enzymatic complex at the surface of the cell produces extracellular H2O2 able to induce DNA damage in an inducible cellular model

Abstract

Thyroid hormone synthesis requires H 2 O 2 , produced by two NADPH oxidases, Duox1 and Duox2. To be fully active at the apical pole of the thyrocytes, these enzymes need additional maturation factors DuoxA1 and DuoxA2. The proteins have been shown to be localized at the cell surface, suggesting that they could form a complex with Duox counterparts. We have generated multiple HEK293 Tet-On3G cell lines that express various combinations of DuoxA upon doxycycline induction, in association with a constitutive expression of the Duox enzyme. We compared Duox specific activity, Duox/DuoxA cell surface interactions and the cellular consequences of sustained H 2 O 2 generation. By normalizing H 2 O 2 extracellular production by Duox or DuoxA membrane expression, we have demonstrated that the most active enzymatic complex is Duox2/DuoxA2, compared to Duox1/DuoxA1. A direct cell surface interaction was shown between Duox1/2 and both DuoxA1 and DuoxA2 using the Duolink® technology, Duox1/DuoxA1 and Duox2/DuoxA2 membrane complexes being more stable than the unpaired ones. A significant increase in DNA damage was observed in the nuclei of Duox2/DuoxA2 expressing cells after doxycycline induction and stimulation of Duox catalytic activity. The maturation and activity of Duox2 were drastically impaired when expressed with the glycosylation-defective maturation factor DuoxA2, while the impact of the unglycosylated DuoxA1 mutant on Duox1 membrane expression and activity was rather limited. The present data demonstrate for the first time that H 2 O 2 produced by the Duox2/DuoxA2 cell surface enzymatic complex could provoke potential mutagenic DNA damage in an inducible cellular model, and highlight the importance of the co-expressed partner in the activity and stability of Duox/DuoxA complexes.

Genome-wide meta-analysis of iron status biomarkers and the effect of iron on all-cause mortality in HUNT

“…We used Bayesian colocalization analysis to identify 94 unique pairs of GWAS loci and cisexpression quantitative trait locus (cis-eQTL) signals that showed sufficient overlap in at least one tissue to be consistent with a shared causal variant for the gene expression and the iron status biomarker (Supplemental Table 6). We found associations in a range of tissues 17,35 , and identified novel genes interacting with previously reported genes, for example DUOXA2 38 . Several iron status loci were also colocalized with cis-eQTL signals for genes in the major histocompatibility complex (MHC) other than HFE 39 , as well as with transcription regulators [40][41][42] , additional transporter proteins 43,44 and transferases 45,46 .…”

Intrathyroidal feedforward and feedback network regulating thyroid hormone synthesis and secretion

“…The maturation and function of DUOX1 and DUOX2 require DUOX maturation factor 1 (DUOXA1) and DUOXA2, respectively, which are chaperone proteins. By forming enzymatic complexes DUOX1/DUOXA1 and DUOX2/DUOXA2, the chaperones promote the correct maturation of DUOX1 and DUOX2 for their endoplasmic reticulum (ER) exit to the cell surface ( 57 ). Although similar in structure, the DUOX1 and DUOX2 proteins are encoded by two separate genes with fairly different regulatory mechanisms ( 58 ).…”

“…Although similar in structure, the DUOX1 and DUOX2 proteins are encoded by two separate genes with fairly different regulatory mechanisms ( 58 ). Compared to DUOX1/DUOXA1, DUOX2/DUOXA2 has higher enzymatic activities and is the predominant form in the thyroid ( 57 , 59 ). TPO utilizes H 2 O 2 generated by DUOX/DUOXA to catalyze the oxidation of I - and subsequent iodination of the tyrosine residues of soluble TG molecules nearby ( 60 ).…”