Sabine Heumüller

Publications of Sabine Heumüller

• Identification of structural elements in Nox1 and Nox4 controlling localization and activity.

  Authors: Ina Helmcke, Sabine Heumüller, Ritva Tikkanen, Katrin Schröder, Ralf P Brandes

  Antioxidants & redox signaling. 01/2009;

  Nox NADPH oxidases differ in their mode of activation, subcellular localization and physiological function. Nox1 releases superoxide anions (O2-) and depends on cytosolic activator proteins, whereasNox NADPH oxidases differ in their mode of activation, subcellular localization and physiological function. Nox1 releases superoxide anions (O2-) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H2O2) and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 which were expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 co-localized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma membrane localization was lost and the protein was retained in vesicle-like-structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H2O2 to O2-. Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.

• Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant.

  Authors: Sabine Heumüller, Sven Wind, Eduardo Barbosa-Sicard, Harald H H W Schmidt, Rudi Busse, Katrin Schröder, Ralf P Brandes

  Hypertension. 03/2008; 51(2):211-7.

  A large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, reliedA large body of literature suggest that vascular reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species. Many studies, however, relied on data obtained with the inhibitor apocynin (4'-hydroxy-3'methoxyacetophenone). Because the mode of action of apocynin, however, is elusive, we determined its mechanism of inhibition on vascular NADPH oxidases. In HEK293 cells overexpressing NADPH oxidase isoforms (Nox1, Nox2, or Nox4), apocynin failed to inhibit superoxide anion generation detected by lucigenin chemiluminescence. In contrast, apocynin interfered with the detection of reactive oxygen species in assay systems selective for hydrogen peroxide or hydroxyl radicals. Importantly, apocynin interfered directly with the detection of peroxides but not superoxide, if generated by xanthine/xanthine oxidase or nonenzymatic systems. In leukocytes, apocynin is a prodrug that is activated by myeloperoxidase, a process that results in the formation of apocynin dimers. Endothelial cells and smooth muscle cells failed to form these dimers and, therefore, are not able to activate apocynin. Dimer formation was, however, observed in Nox-overexpressing HEK293 cells when myeloperoxidase was supplemented. As a consequence, apocynin should only inhibit NADPH oxidase in leukocytes, whereas in vascular cells, the compound could act as an antioxidant. Indeed, in vascular smooth muscle cells, the activation of the redox-sensitive kinases p38-mitogen-activate protein kinase, Akt, and extracellular signal-regulated kinase 1/2 by hydrogen peroxide and by the intracellular radical generator menadione was prevented in the presence of apocynin. These observations indicate that apocynin predominantly acts as an antioxidant in endothelial cells and vascular smooth muscle cells and should not be used as an NADPH oxidase inhibitor in vascular systems.