Structure-Function Relationship, Membranes, and Complex Assembly
Laura Baciou (Senior Researcher), Chantal Houée (Associate Professor), Tania Bizouarn (Researcher-HDR), Marie Erard (Professor), Oliver Nüsse (Professor), Xavier Serfaty (Postdoctoral Researcher)
Alumni: Sophie Dupré-Crochet (Professor at UVSQ since 2023), Aicha Bouraoui (PhD Candidate 2016–2019), Dina Al Abyad (PhD Candidate 2019–2022), Sana Aimeur (PhD Candidate 2020–2023), Elodie Hudik (Research Assistant)
Our goal is to understand how the stability and activity of the NADPH oxidase complex are influenced by the dynamics of its assembly and lipid-protein interactions. These phenomena drive the transition from the inactive to the active state of the NADPH oxidase, evolving over time or under various physiological conditions. Since any dysregulation of its activity can lead to or contribute to serious pathologies (immunological or neurodegenerative), exploring these mechanisms at the molecular level is essential for identifying effective strategies to control its function. To address the complexity of these assembly and activation processes of NADPH oxidase, we employ a range of complementary approaches from biology, biochemistry, and biophysics.
Functional Studies
Using molecular biology and biochemical techniques, the various components of the NADPH oxidase complex are produced in bacteria, yeast, or isolated from phagocytic cells obtained from blood donors (French Blood Establishment). These components are used to reconstitute functional in vitro systems with controlled compositions and environments, avoiding constraints or biases typical of cellular studies. We study different levels of complexity of the NADPH oxidase complex, ranging from isolated systems (in detergents or polymers) to artificial membrane vesicles (small unilamellar vesicles (SUV) or giant unilamellar vesicles (GUV)), or biological membranes (neutrophils, cell lines). We continuously optimize our study objects to minimize experimental detection and measurement biases [Serfaty2020].
By employing techniques such as oxymetry and spectroscopies (steady-state and time-resolved absorption/fluorescence, stopped-flow, bilayer interferometry, ITC, (Far) Western Blot, confocal microscopy), we characterize the enzymatic properties (Km, KI, reaction rates) and assembly dynamics of the NADPH oxidase complex.
NADPH oxidase proteins are purified and assembled in vitro into membrane vesicles. Their activity is monitored using several complementary spectroscopic methods [Xavier Serfaty’s thesis, 2019].
This allows us to explore the role of the membrane environment on enzyme assembly and activity (cholesterol, phosphoinositides, etc.) [Baciou2018, AlAbyad2023], as well as identify new protein components that may regulate its activity [Bouraoui2023].
Spinning disk confocal fluorescence microscopy images of prototype GFP trimera binding to giant liposome membranes in the presence of arachidonic acid (AA) [AlAbyad2023].
Effect of membrane composition on NADPH oxidase activity [Baciou 2018].
Structural Studies
To decipher the conformations and their changes during the activation of the enzymatic complex, we combine functional studies of the complex with structural studies using circular dichroism techniques and Small-angle X-ray Scattering (SAXS) at synchrotrons, along with approaches utilizing artificial intelligence strategies (AlphaFold) [Aimeur2024, Versini2023].
AlphaFold model of the functional NADPH oxidase complex [Aimeur2024]
The resting system has been studied in cells. In this state, the cytosolic proteins p47phox, p67phox, and p40phox are retained in the cytosol as a heterotrimer to prevent their interaction with membrane components. Using FRET-FLIM microscopy coupled with SAXS data, we have demonstrated their assembly mode in cells and proposed the first 3D model of this heterotrimer [Ziegler2019].