NOS and BH4 as a redox ‘hub’ in cardiovascular homeostasis and disease.
NO-Redox balance in cardiovascular disease
NO and ROS signal through chemical reactions with specific atoms of target proteins that lead to modification of metal centres and to covalent protein modifications. This notion of NO–Redox balance may be defined by the idea that reactive nitrogen species (RNS) and ROS work together in biological systems to achieve optimal signalling. NO-Redox imbalance arises when cellular signalling is disrupted by either increased ROS or decreased RNS. Moreover, cross-talk exists between the enzymes that produce ROS and RNS, so NO deficiency can in some cases result in increased ROS production. Thus, the interactions between ROS and RNS are multifaceted and strike a balance that can be disrupted at both the cell and organ levels in cardiovascular disease states, as within the dysfunctional endothelium.
A key concept in understanding redox balance in the cardiovascular system is that the effects of ROS and RNS depend on the location, amount, and timing of their production. A major objective of our group is to use novel approaches to investigate whether redox effects are ‘transduced’ throughout the cell having wider signalling implications in other loci, or whether redox signals remain localized around where the NO/ROS is produced.
Tetrahydrobiopterin (BH4) as a redox sensor and effector
The redox cofactor BH4 is a critical regulator of NOS function and NO-ROS signalling in cardiovascular disease, and provides an exemplar for studying the effects of altered NO and ROS action. In NOS catalysis, BH4 controls ‘coupling’ of the haem-oxygen intermediate to L-arginine oxidation, thus controlling the generation of either NO or superoxide and hence is a critical determinant of NO-ROS balance. Superoxide can be rapidly converted to other ROS such as peroxynitrite or hydrogen peroxide (H2O2), and the balance of NO vs. ROS production also influences NO redox state.
We are using models of altered BH4 availability to address the impact of perturbed NO-Redox balance on downstream redox-sensitive protein signalling to answer questions on the functional consequences that these effects may have on cardiovascular homeostasis and disease. These effects include, but are not limited to, protein oxidation, sulfenic acid modification of cysteine residues, and protein S-nitrosylation. Each regulatory mechanism may have unique downstream targets and result in BH4- and redox-dependent signalling via a variety of pathways. Recent findings from our laboratory have revealed that BH4 also has important non-canonical roles on cellular metabolism and mitochondrial function, independent of NOS and NO availability. Thus, BH4 regulation expands the repertoire of redox signalling to include not only NO, but also multiple ROS and NO-ROS effects that are potentially loci specific and important in cardiovascular disease pathogenesis.
We are using Mass Spectrometry based proteomics approaches, combined with the use of fluorescent biosensors to investigate how NO/ROS balance affects cell signalling, with the aim of identifying specific mechanisms and proteins that will provide new targets for future cardiovascular drug treatments. We are particularly interested in the wider impact of subcellular-specific changes on NO-redox balance and the discovery of new redox-related proteins and pathways that govern these effects.
How does subcellular localization of NO-ROS production affect downstream redox effects?
What is the role of the mitochondria in NO-Redox balance?
What novel proteins and pathways are specifically regulated by changes in NO-Redox signalling?
What are the non-canonical roles of BH4 in regulating cellular homeostasis?
Benedikt Kessler and Roman Fischer (Mass Spec core, TDI, Oxford)
James McCullagh (Chemistry, Oxford)
Harry Ischiropoulos (University of Pennsylvania, USA)
Phil Eaton (King’s College, London)
Mike Murphy (Cambridge)
In vitro and in vivo models of BH4 deficiency
Proteomics by Mass Spectrometry
Metabolomics by Mass Spectrometry