Molecular characterisation of the assembly and functioning of a membrane complex: the NADPH-oxidase – PROTOTYPE NOX

The aim of the project entitled « Prototype NOX » is to better understand the functioning of the phagocyte enzyme complex. This enzymatic complex consists of at least 5 soluble and membrane proteins (the latter being the catalytic subunit) and associates (i) dynamics of protein assembly, (ii) charge transfers, (iii) and enzymatic coupled reactions, in order to produce anion superoxide, precursor of ROS. To better understand the importance, the synchronization of these events and the regulatory factors, we developed an acellular oxidase system. This system reproduces in vitro the molecular architecture of the enzymatic complex allowing fine physical-chemistry studies in order to investigate its structure-function relationships. In parallel, studies of engineered catalytic subunit were performed in the cellular context of phagocytes to make the link between molecular properties of the NOX and their regulations by the cellular signaling. For the structural studies, the quantities of required proteins are such that we needed to find equivalent enzymes in the bacterial world, easier to produce in abundant quantities.

In this work, in an unexpected way, the catalytic part of the enzyme was shown to be able on its own to generate ROS, this being also the case of the bacterial counterparts discovered in this study. The modification of the membrane or the modulation of the assembly of various proteins can control the activity of the enzyme. This could be of importance for the immune system at the cellular level. The identified bacterial homolog of NOX enzymes were produced and biochemically characterized. Their structural studies and the obtained crystals open promising perspectives towards the first structure of an enzyme of this family.

By identifying for the first time prokaryotic members of this enzymatic family, enormous perspectives are born in terms of development of the studies at the structural level, one of the initial objectives of the project, but also of the function of these systems in the bacterial world.
The “in vitro” reconstruction of the integer complex, completely stemming from the engineered proteins, is capable, under control, of generating radicals in a comparable way in the “in vivo” situation. This tool allows us to circumvent the cellular constraints to approach easily its physico-chemical studies. The flexibility of this system allowed us to modulate the the complex composition, the membrane structure and the effectors (inhibitors, fatty acid). These studies showed that the interactions between protein-protein and lipid-protein can regulate the enzymatic activity and that the NOX complex is particularly sensitive to the stereochemistry of certain molecules. The yeast heterologous expression system opens us the way to provide a valuable tool to study the other members of the family of the NOX more difficult to manipulate.

Beyond the fundamental aspect of this project, these systems, in particular bacterial which is easier to produce, open the way to potential economic developments.

These results have been published in peer-reviewed journals. 6 publications concerning the study of the enzyme regulation were published (one being in revision and 2 under submission processes). The possible use of the bacterial enzymes identified by Pr. F Fieschi's team in pre-screening for the pharmaceutical industry led to the deposit of a patent, then to the market study and now a start-up phase of maturation (piloted by J Dupuy). The results stemming from this work are now going to be able to be published (2 articles in the course of submission and at least two others can be envisaged in the future).

The NADPH-oxidase is essential for the innate immune defence and is present in professional phagocyte cells. The activation of this complex is tightly regulated and involves phosphorylation events correlated to specific protein-protein interactions and chemical modifications. The phagocyte NADPH-oxidase has become the prototype of a family of electron transport system (NADPH-oxidase family) recently discovered in various tissues. They all share the capacity to produce superoxide radical from the reduction of molecular oxygen. The active enzyme is the result of the translocation of four cytosolic proteins to the membrane component, the so-called Flavocytochrome b558 (Cytb558). The Cytb558 is the catalytic core of the NADPH-oxidase that generates superoxide anion from oxygen by using reducing equivalent provided by NADPH via FAD and two hemes. A dysfunction of NADPH-oxidase leads to severe immune disease and to other important human pathologies.
Our aim is to understand the functioning of this dynamic membrane-bound electron-transferring enzyme by studying its functional and structural properties in a cell-free system (in vitro). Several important steps in the NADPH-oxidase functioning have been identified. However, at the molecular level, many questions remain. The limiting factor for functional and structural studies is the lack of sufficient amounts of the membrane flavoprotein in stable, pure and homogeneous form. We recently overcame this bottleneck by producing the recombinant Cytb558 in a yeast expression system. Associated with the recombinant cytosolic proteins, the Cytb558 forms a totally recombinant cell-free system, free of cell signaling constraints and in which the environment can be easily controlled. Our aim is to take advantage of this new tool to elucidate, at the molecular level, the mechanisms underlying the reactive oxygen species production (involving protein-protein interaction, fatty acid activation, electron transfer reaction,…). Using a wide range of expertises (molecular biology, biochemistry, fluorescence and absorption spectroscopy, radiation biology and structural biology), we will gain new insight into the key structural and functional features of the protein components that control the activation and inhibition of the enzyme assembly processes and the coordination of the different redox partners.
The methods like the stopped flow technique and pulsed radiolysis that we intend to use, have, to our knowledge, never been applied to analyze the NADPH-oxidase functioning. These are methods of choice to determine the reaction intermediates leading to the superoxide generation within the Cytb558 and to decipher the mechanism underlying the activation processes of the catalytic subunit. They are likely to yield unprecedented insight into the NADPH-oxidase biological functions. A fundamental aspect in this project is that the large scale production of the catalytic Cytb558 will enable us to make progress towards the determination of the three-dimensional structure of this membrane protein. This challenging project will combine a comprehensive set of biophysical, biochemical and structural approaches for the study of the NADPH-oxidase. These approaches will provide a better understanding of the molecular mechanisms underlying in the cellular process and will facilitate attempts at rational drug design where membrane proteins are often key target molecules.