Oxidative Stress and Detoxication
Nitric oxide, NO°, is a gas and highly reactive molecule, long time considered as a noxious and harmful chemical until the mid-eighties when NO° was found to be a ubiquitous signalling molecule involved in many vital physiological processes. NO° became Science’s Breakthrough/Molecule of the Year 1992 and the 1998 Nobel Prize in Medicine was awarded for the discovery of NO° physiological role. Since then, NO° has been the topics of more than 170 000 scientific papers.
However as stated by Koshland in his editorial of the Science 1992 Molecule of the year issue, “it was a surprise to find such an unlikely agent, a labile and toxic gas, for such essential physiological roles”. Indeed, NO° is a redox and extremely reactive molecule. Redox (oxidoreduction) chemistry is a universal and extensive chemistry: upon biosynthesis, NO° will eventually get transformed in a large range of Reactive Nitrogen Species (RNSs) that will react with (almost) all biomolecules, from metabolites to lipids, DNA, proteins… Because of this large reactivity spectrum, NO° stands at the crossroad of many different (if not opposite) physiological activities, depending mostly on the physico-chemical properties of the milieu.
This is exemplified in mammals where NO° (and its derived RNSs) are involved in signalling processes (such as neural communication, vascular tone control, NANC signalling for the regulation of GI tract, cardiac or erectile processes) but also in the fine-tuning of the multifaceted endothelial homeostasis (antioxidant, platelet aggregation, neutrophil adhesion, vascular permeability, angiogenesis, proliferation…). However, NO° is also directly used as a cytotoxic agent in non-specific immune defence. In this context, NO° and RNS are among the most relevant molecules in oxidative stress related pathologies. When the oxidative activity of NO°/RNS become out of control, these ubiquitous redox molecules once engaged in signalling processes convert into self-harming compounds leading to major pathological conditions that account for inflammatory, neurodegenerative and cardiovascular diseases, but also directly related to cancer, asthma, diabetes…
This pleiotropic and unpredictable activity of a physiological molecule such as NO° was a kind of revolution in the field of chemical biology. This led the path to reassess the biological role of Redox active species (ROS, RSSs) such as H2O2, H2S… In this context, others and we have proposed a new theoretical framework to address the multiple activities and functions of reactive species. We proposed the existence of a Reactive Species Interactome (RSI), a redox system that connects all (bio)chemical pathways generating reactive species with cellular intermediary metabolism, bioenergetics, and the extracellular environment. By interconnecting all redox active molecules, the RSI is shaping a Redox Landscape that synchronizes all redox-based processes and allows living entities to sense and to adapt to changes in the chemical composition of intra- and extracellular milieu.
Using philosophy of science and biosemiotics backgrounds, we have been investigating how redox chemistry has been fashioning biological communication system. Redox chemistry constitutes a universal molecular language, a lingua franca that bridges the inanimate and living realms, connecting major bio-geochemical cycles. We proposed that redox processes have co-evolved with biological complexification, giving rise to a multi-layered system of increasingly complex redox architectures. All redox-based structures, processes and functions that are taking place at different levels of biological organisation are integrated within what we defined as the ‘Redox Interactome’. This Redox Interactome is the communication system that enables self-regulation of each redox-based process along with the synchronisation of multiple physiological processes taking place in different tissues. This complex Redox Architecture provides the organism with an integrative, multi-scale Redox Regulation System that allows accommodation of different stresses within and across all levels of biological organisation.
This approach led us to revisit the concept of redox homeostasis that is increasingly utilized in the scientific literature but remains a catchall concept that is poorly defined with neither clear meaning nor relevant usage. We proposed that the molecular bases of homeostatic behaviours must be precisely characterised and considered within the frame of redox biochemical systems. The Redox Interactome framework seems therefore required to understand the integration of isolated redox processes within the context of any physio/pathological condition.
We also applied our Redox System’s approach to analyse the new COVID-19 disease, as an example of a stress response that challenges the integrity of redox balance at the whole-body level. We considered COVID-19 as a redox disease and explored its impact on stress signalling and adaptation in relation to the body’s ability to maintain homeostasis. Using our RSI framework, we highlighted the way acute and prolonged oxidative stresses lead to a progressive failure of the redox regulation systems, disturbing all redox-based signalling processes, and leading to catastrophic cascades of “oxidative storm”. This complete and profound redox system failure might help understanding the molecular foundations of long-Covid pathologies and fostering precise therapeutic interventions.
 Cortese-Krott et al. “The Reactive Species Interactome: Evolutionary Emergence, Biological Significance, and Opportunities for Redox Metabolomics and Personalized Medicine” 2017. Antioxidant Redox Signalling. Cortese-Krott et al. 2020 “The Reactive Species Interactome” 2020. In Oxidative Stress, Eustress and Distress. Helmut Sies Editor. Academic Press.
 Santolini & Feelisch. « Redox Chemistry as Biology Lingua Franca ». 2019. First International Conference “Bridging the Philosophies of Biology and Chemistry”
 Santolini et al. “The Redox Architecture of physiological function” 2019. Current Opinion Physiology.
 Cumpstey et al. “COVID-19 – A Redox Disease” 2021. Antioxidants and Redox Signaling. Under revision.
The versatility of redox chemistry and the tight imbrication of redox processes within a unique redox system are the major parameters to be taken into account when addressing the bioactivity and physiological effect of NO°/RNS. This system’s biochemistry approach represents the conceptual framework of our scientific project : how can we explain the multiple and various activities of NO° in the living kingdom ?
The LSOD expertise and international recognition has been built on the 20 years old investigation of the structure and mechanism of the NO°-producing enzyme: NO-Synthase (NOS). Using a wide array of biophysical (Raman, EPR…) and biochemical (sited-directed mutagenesis, fast-kinetics enzymology) approaches, we have been characterizing for many years the structure and sophisticated molecular mechanism of the canonical mammalian NO-Synthase. In the recent years, we have started investigating new NO-Synthases as they became identified through genomic approaches. Indeed, NOS that were first identified in mammals have been found throughout the tree of life in archae, bacteria, protists, fungi, insects… Our series of results led us to conclude that bacterial NO-Synthases share specific structural features with mammalian NOSs but do not achieve the same catalytic mechanism and, on that ground, should not be considered as genuine NO synthases. Confronted to the high (structural, phylogenetic, ecological…) diversity of new NOSs we then achieved a general description of this new family of proteins laying the scope for investigating their evolution, and function in so many different phyla. This led us to highlight a series of new biological interrogations:
- what is the evolutionary story of NO° biology within each kingdom of life
- what is the specific biological activity of NO° in any of these organisms: antioxidant, signalling, redox homeostasis, oxidative stress…
- what is the catalytic functioning, biochemical activity and biological role of these various NO-Synthases
- how can we address the deep structural heterogeneity of the NO-Synthases protein family
- what is the influence of ecological milieu in NOSs evolution and functioning
We decided to focus on a particular family of NOS protein : NOSs of the green lineage. Indeed NOSs are found in all the major kingdoms of life but that of land plant. Whereas NO° is a major signalling molecule in land plants, NO-Synthases are indeed absent from these organisms – and the origin of NO° biosynthesis within land plants remains yet to be elucidated. However, NOSs are present in some algae. NOSs sequences can be found in the genomes of species belonging to all classes of algae (but heterogeneously distributed within this phylum) and inhabiting various ecological milieus (marine/freshwater, pelagic/benthic) (ref). Our team was able to characterize for the first time a plant NOS, that from the algae Ostreococcus tauri (otNOS). We analyse otNOS structure and functioning and showed that otNOS was a genuine NO° Synthase.
However the NOSs that are present in algae exhibit some structural diversity that questions their ability to produce NO°. We therefore launched a research program focusing on this new family of protein – the plant NOSs. The program aimed at determining the structure of a selection of algae NOSs – through structural biology, biophysics and molecular modelling, at elucidating the catalytic functioning and biochemical activity of these NOSs, and at characterizing their function and physiological role in algae. This project is achieved in collaboration with a Team from the Dijon-based Agroecologt UMR. David Wendehenne’s team is a world-renown lab of plant physiology that has a strong expertise in NO° signalling in plants. The collaborative project, supported by an ANR funding, will allow shedding light on the evolution of NO° biology in the green lineage and the particular relationship between redox homeostasis and Signalling in plants..
 Tejero et al. “Mechanism and regulation of ferrous heme-nitric oxide (NO) oxidation in NO synthases”. 2019 J. Biol. Chem. Lang et al. “ Reaction Intermediates and Molecular Mechanism of Peroxynitrite Activation by NO Synthases” 2016. Biophys. J. Brunel et al. “ Oxygen activation in NO synthases: evidence for a direct role of the substrate” 2016. FEBS openBio. Santolini “The molecular mechanism of mammalian NO-synthases: a story of electrons and protons.” 2011. J. Inorg. Biochem.
 Weisslocker-schatzel et al. “Revisiting the Val/Ile Mutation in Mammalian and Bacterial Nitric Oxide Synthases: A Spectroscopic and Kinetic Study” 2017 Biochemistry. Lang et al. “The conserved Trp-Cys hydrogen bond dampens the “push effect” of the heme cysteinate proximal ligand during the first catalytic cycle of nitric oxide synthase”. 2011. Biochemistry. Brunet et al. “The proximal hydrogen bond network modulates Bacillus subtilis nitric-oxide synthase electronic and structural properties”. 2011 J. Biol. Chem.
 Santolini “What does “NO-Synthase” stand for ?” 2019 Frontiers in Bioscience. Santolini et al. “Nitric oxide synthase in plants: Where do we stand?” 2017 Nitric Oxide
 Astier et al. “ The evolution of nitric oxide signalling diverges between animal and green lineages.” 2019 J Exp Bot. Weisslocker-schatzel et al. “The NOS-like protein from the microalgae Ostreococcus tauri is a genuine and ultrafast NO-producing enzyme” 2017 Plant Cell.
Nitric Oxide Synthases (NOSs) and Cytochromes P450 (CYPs), both involved in oxidative stress in a large variety of organisms, are highly related proteins as monooxygenase enzymes sharing a similar active site (a thiolate-bound heme). They were mostly identified first in mammals, but they are found omnipresent in the tree of life in the post-genomic era.
The increasing number of sequenced genomes in the recent years revealed a fascinating diversity of sequences of NOS and CYPs, likely associated to various functions, maybe divergent. The diversity of NOS is mainly found in the global organization of the protein domains, with noticeable divergence in structural domains (absence/presence of reductase domain, nature and order of domains). This contrasts with the strong conservation of the NOS oxygenase domain (more than 40% of sequence identity in average).
At the opposite, the CYPs are structurally highly conserved despite the low average sequence identity (as low as 20%), showing a structural uniformity, but they show a very large variety of ligands, either endogenous or xenobiotics, that lead to a large variety of biological functions or biotransformations. There is a new interest in the study of unknown NOS and CYPs found in genomes, that are not yet characterized or even not isolated.
The cytochrome P450s constitute one of the largest ubiquitous superfamily present in plants, animals, fungi, and prokaryotes, and, as such, represents a fascinating field of research for in silico investigations. The latest estimation of the “CYPome” comes to more than 300000 genes across all these kingdoms of life, and this number increases each year rapidly, mainly due to plant genomes sequencing projects, whereas a limited number of isoforms has been structurally determined (about 100 different isoforms available in PDB). A major unresolved issue thus remains the identification of physiological function of many of the newly discovered P450 enzymes, both in biosynthesis pathways and detoxification processes. The number of potentially recognized low molecular weight compounds (“ligandome”) is also challenging. Connecting the ligandome to the CYPome remains unexplored despite numerous publications devoted to CYP catalytic activities
In the huge CYPome, we adapted bioinformatics and modeling approaches for CYP “deorphanization” studies, i.e. modeling structurally unknown CYPs and predicting the palette of ligands potentially recognized and metabolized by these monoxygenases. In particular, specific bioinformatics tools have been developed for rebuilding reliable structures from low sequence identity templates (for example various plant CYPs and human orphan CYP2U1), and, in collaboration with University Paris-Diderot, cheminformatics tools for studying the channeling of substrates to the heme pocket which is particularly buried (Benkaidali, 2014, 2018).
With the new plant genomes programs (1kP, 10kP), we have today access to numerous plant CYP sequences most of them being of unknown function. Home-built models proved to be successful for predicting activity and ligand specificity in plants CYPs, which none structure are available in PDB: CYP76 isoforms (Hofer, 2013), CYP73 paralogs and orthologs (Renault, 2017; Boachon 2019, Abdollahi 2021), and CYP98 isoforms (Liu, 2016). In the latter case, the models rebuilt re-oriented the in vitro study, since the directed mutation of a CYP98 key residue identified by pure 3D-modeling changed the metabolic pathways in the seeds (Liu, 2016).
Human liver CYPs metabolism:
The study of ligand access through flexible tunnels gave also new insights in human liver metabolism by CYP3A4 (Benkaidali, 2017), and in CYP2B6-mediated cancer therapy studies (Touati, 2014). Lastly, we demonstrated the ability of our tools to “deorphanize” CYP2U1, a human isoform expressed in brain and functionally unknown but found mutated in patients affected by rare hereditary paraplegia (HSP). The rebuilt models (Ducassou, 2015) were used to identify potential ligands, either xenobiotics or endogenous (Dhers, 2017), and the whole structure rebuilt in model membrane could account for fatty acids recognition and specificity (Ducassou, 2017).
Phase III efflux of Xenobiotics by ABCB1 (P-gp)
P-glycoprotein (P-gp, ABCB1) is also another model of multispecific enzyme involved in mammalian cell detoxification. This primary active membrane transporter effluxes out of the cell a large number of various molecules (exo- or endogenous). Due to its physiological expression in various healthy tissues, it is also responsible for organism protection against toxic xenobiotics and, conversely, for the pharmacokinetic properties of many molecules of pharmacological interest, which often limits their efficacy. In particular, P-gp overexpression in tumor cells is responsible for cytotoxic drugs efflux, conferring multidrug resistance (MDR) phenotype to these cells, which leads to anticancer chemotherapy failures.
We study the question of molecular recognition of xeniobiotics by this membrane enzyme in different species, according to the availability of resolved structures in PDB. We used docking calculations (by Autodock) in C elegans P-gp structure released in 2012 and various mouse P-gp structures (2014-2018) to finely describe the 3D arrangement of all contact residues lining the large internal chamber of P-gp in its open inward-facing conformation that forms its multispecific transport site. This list of “hotspot” residues was established by combining enzymological studies to results of docking studies of various anthelmintinc drugs (David, 2016. Collaboration with S. Orlowski, I2BC, and A. Lespine, INRAe Toulouse). This study was extended to P-gp homologs in pathogenic nematodes, such as Hco-Pgp13 (from H contortus), in the frame of the study of resistance mechanisms to avermectins rapidly emerging (David, 2018).The results led to the indication that this previously uncharacterized ABC protein possibly contributes to anthelmintic drug resistance in H. contortus.
With the recent release of human P-gp high-resolution structures resolved by CryoEM (but still incomplete), it became possible to study the binding modes of various known drugs in different configurations of the protein. We are currently addressing ligand recognition and translocation mechanism by Molecular Dynamics simulations in complex membranes with models completed by homology modeling.