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The reactive species interactome
CHAPTER
The reactive species interactome
4
Miriam M. Cortese-Krotta, Jerome Santolinib, Steve A. Woottonc, Alan A. Jacksonc, Martin Feelischd a
Department of Cardiology, Pulmonology and Angiology, Medical Faculty, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany b Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette Cedex, France c Human Nutrition, University of Southampton and University Hospital Southampton, Southampton, United Kingdom d Clinical & Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
Abstract Enzymatically generated reactive species are emerging as endogenous signaling molecules, targeting specific thiol/metal redox switches in proteins and organelles, thereby affecting cell metabolism and survival. To better characterize the complex network of interactions of reactive species with themselves and their biological targets and to understand their significance for metabolic control, we recently introduced the “reactive species interactome” (RSI) concept. The RSI is a primeval redox system that connects the (bio)chemical pathways that generate reactive species with cellular intermediary metabolism, bioenergetics, and the extracellular environment. The main characteristics of the RSI are (a) robustness and flexibility, (b) adaptability and rapid responsiveness, and (c) the ability to sense the chemical composition of intraand extracellular milieu. These properties enable cells/organs to communicate and sense and adapt to changes in environmental conditions and metabolic demand. As conceptual framework, the RSI may enable to better understand how complex life forms operate and respond to challenges at the whole organism level, potentially enabling us to harness this information for medical use in health and disease. Keywords: Nitric oxide, Hydrogen sulfide, Redox regulation, Systems biology
Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00004-3 © 2020 Elsevier Inc. All rights reserved.
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Introduction For a long time, “reactive species” were considered to be toxic by-products of the metabolism of oxygen (O2), nitric oxide (NO•), and hydrogen sulfide (H2S)/sulfane sulfura containing species. More recently, it has become evident that the physiological functions of reactive species can only be understood when viewed as part of an integrated whole since they are interconnected by chemical reactions, produced— in an apparently purposeful manner—by specific enzymes within cells and tissues and have the potential to modulate common intra- and extracellular targets. Wellknown examples of such chemical interactions include the production of peroxynitrite (ONOO−) from enzymatically generated NO• and superoxide (Beckman, Beckman, Chen, Marshall, & Freeman, 1990) and the synthesis of nitrosopersulfide (SSNO−) and S-N from NO• and H2S (Cortese-Krott et al., 2015). Importantly, the products of these chemical interactions tend to trigger biological effects that are distinct from and independent of the effects induced by the parent molecules from which they originate. The richness and diversity of the physicochemical properties and lifetimes of the intermediary products generated by these (bio)chemical interactions are harnessed by Nature to enable and/or support physiological functions of considerable complexity. In an effort to integrate the chemical biology and biochemistry of these reactive species and the (patho)physiological significance of their interactions, we introduced the concept of the “reactive species interactome (RSI)” (Cortese-Krott et al., 2017). Specifically, we defined the RSI as a redox system consisting of chemical interactions of reactive species among themselves and with downstream biological targets such as thiols and metal centers (Fig. 1). This chapter aims to provide a summary
FIG. 1 Constituting elements of the reactive species interactome. See text for details. Modified from Cortese-Krott, M. M., Koning, A., Kuhnle, G. G. C., Nagy, P., Bianco, C. L., et al. (2017). The reactive species interactome: Evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized medicine. Antioxidants & Redox Signaling 27, 684–712. a
Sulfane sulfur denotes sulfur that is bound covalently only to (an)other sulfur atom(s).
Chemical interactions among reactive species
of the main constituents of this system, explain why it is an essential contributor to cellular and integrative redox signaling, and outline its potential significance for physiology and redox diseases in complex organisms.
Chemical interactions among reactive species Comprehensive treatments of the fundamental chemistry of reactive species including their kinetics of formation, chemical reactivities, action radii and interactions with common biological targets are available elsewhere (Basudhar et al., 2016; Cortese-Krott, Butler, Woollins, & Feelisch, 2016; Cortese-Krott, Fernandez, Kelm, Butler, & Feelisch, 2015; Fukuto et al., 2012; Halliwell & Gutteridge, 2015; Jacob & Winyard, 2009). The main oxygen-, nitrogen-, and sulfur-related reactive entities are therefore listed here only in tabular form according to their predominant chemical attributes (Table 1), and their possible interactions are only discussed in very general terms. Table 1 Exemplary reactive oxygen, nitrogen, and sulfur species and related hybrid molecules. Species
Occurrence/ formation
Chemical properties
Reactive oxygen species, ROS Dioxygen, O2
Aerobic life
Superoxide, O2•−
1e− reduction of O2
Hydrogen peroxide, H2O2 Hydroxyl radical, HO•
1e− reduction of O2− 1e− reduction of H2O2
Has unpaired electrons, reacts readily with other radicals, poor 1e− oxidant, but otherwise easily reduced by 2, 3, and 4e− Has one unpaired electron. Good reductant. Under acidic conditions can be an oxidant. Reacts with other radicals Not a radical species. Two-electron oxidant. Electrophilic. Can modify RSH (below) Potent 1e− oxidant. Short-lived radical species capable of abstracting an e− or hydrogen atom from most biological molecules
Reactive nitrogen species, RNS Nitric oxide, NO
Enzymatic, NO2− reduction
Nitrogen dioxide, NO2
Oxidation of NO
Dinitrogen trioxide, N2O3
Oxidation of NO by O2
Has unpaired e−. Poor oxidant. Reacts with other radicals (O2 and O2– and other organic radicals [R•]). Can act as an antioxidant by quenching radical chemistry Radical species. Good 1e− oxidant. Reacts with other radicals (can make R-NO2 when reacted with R•) Not a radical. Electrophilic and can nitrosate nucleophiles (add equivalent of “NO+”). Synthesis only relevant at high concentrations of NO Continued
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Table 1 Exemplary reactive oxygen, nitrogen, and sulfur species and related hybrid molecules—cont'd Species
Occurrence/ formation
Peroxynitrite, ONOO−
Reaction of NO and O2−
Nitroxyl, HNO
S-nitrosothiol reduction
Nitrite, NO2−
Oxidation of NO, dietary
Chemical properties Not a radical but can generate both HO• and NO2. Rearranges to give nitrate (NO3−). Can oxidize by 2e− (via peroxide-like chemistry) Reacts readily with thiols. A good hydrogen atom donor. Can act as an antioxidant by quenching radical reactions Unreactive at neutral pH. Not a radical. Nitrosating agent under acidic conditions. Can be reduced to NO (under acidic conditions)
Reactive sulfur species, RSS Thiol, RSH
Endogenous (e.g., cysteine)
Thiyl radical, RS·
1e− oxidation of RSH Oxidation of RSH Geochemical, enzymatic
Disulfide, RSSR Hydrogen sulfide, H 2S Sulfenic acid, RSOH Hydropersulfide, RSSH
2e− oxidation of RSH Enzymatic
Hydropolysulfide, RSSnH Dialkyl polysulfide, RSSnR
Oxidation of RSH Oxidation of RSH
Not a radical. Good metal ligand. Can be oxidized to give other biologically relevant sulfur species Radical species. Good 1e− oxidant. Will react with other radical species such as NO Not a radical. Electrophilic. Can be reduced back to RSH under biological conditions Not a radical. Good metal ligand. Can react with other biological electrophilic sulfur species (e.g., RSSR, RSOH) Not a radical. Electrophilic (reacts with other thiols to give disulfides, RSSR) Not a radical. Carries a sulfane sulfur. Nucleophile; can be electrophilic (akin to RSSR). Good 1e− reductant. Readily reduced back to RSH Properties similar to RSSH (although enhanced in all aspects) Properties similar to RSSR (although enhanced in all aspects). Can also be nucleophilic
S-N hybrid molecules S-Nitrosothiols, RSNO
Nitrosation of RSH
SSNO−
Unknown, to be determined Unknown
SNO− (and isomers) Sulfi/NO
Unknown
Not a radical. Can be reduced to RSH and HNO. Can transfer “NO+” to another thiol (transnitrosation) Fairly stable. Appears to be a source of NO. Chemical properties pending Fleeting. Chemical properties pending Fairly stable. Appears to be a source of NO and HNO
Modified from Cortese-Krott, M. M., Koning, A., Kuhnle, G. G. C., Nagy, P., Bianco, C. L., et al. (2017). The reactive species interactome: Evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized medicine. Antioxidants & Redox Signaling, 27, 684–712.
Chemical interactions among reactive species
Of all the small molecule entities of biological relevance, the chemical biology of O2 is the most studied and established. Serving as ultimate electron acceptor, reduced oxygen species including superoxide ( O2•− ), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•) are unavoidable, nonenzymatically generated companions of aerobic life. However, the same molecules, collectively termed “reactive oxygen species” (ROS) (Halliwell & Gutteridge, 2015), are also produced enzymatically to elicit highly specific biological effects and serve as cell signaling agents. Akin to ROS, equivalent terminology is in use for “reactive nitrogen species” (RNS) and “reactive sulfur species” (RSS), denoting NO-derived and H2S/RSH-derived species, respectively (Halliwell & Gutteridge, 2015) (Jacob & Winyard, 2009). This classification does not take into consideration the widely varying and distinct chemical properties and reactivities of ROS, RNS, and RSS. The compounds listed in Table 1 cover an enormous chemical space, ranging from highly reducing (RSSH, O2•− ) to highly oxidizing (HO• and NO2), from highly electrophilic (RSOH, H2O2, and HNO) to highly nucleophilic (RSSH), and from good hydrogen atom donors (HNO and RSSH) to potent hydrogen atom abstractors (HO· and NO2). Among the RSS, hydropersulfides (RSSH) and polysulfides (RSxH) have recently received considerable attention (Fukuto et al., 2018; Steudel & Chivers, 2019). The generation of hydropersulfides from the corresponding thiol represents an oxidation process (the sulfur in RSSH is of the same oxidation state as that in a disulfide, RSSR) and can be mediated by several of the oxidants listed in Table 1 (e.g., H2O2 in the presence of H2S). Interestingly, a hydropersulfide is a superior reductant compared with the corresponding thiol. Thus, an extremely potent reductant (RSSH) is generated primarily under oxidizing conditions, a fact that seems to have been taken advantage of by nature as it has been proposed that RSSH formation can be protective against oxidative stress (Ono et al., 2014). Sulfur’s versatile redox chemistry is further enriched by complex interaction with reactive nitrogen (e.g., NO• and HNO) and reactive oxygen species (e.g., O2•− and H2O2) (see Fig. 1). In fact, both RNS and RSS react with O2 and ROS, giving rise to a variety of nitrogen oxides (•NO2, N2O3, ONOO−, NO2−, and NO3−) and sulfur oxides (SO2, SO32−, S2O32−, Sx(SO3)22−, and SO42−). RNS can also interact with RSS leading to the formation of S-N hybrid species (Cortese-Krott et al., 2016). Examples of these reactive species include S-nitrosothiols (RSNOs), nitrosopersulfide, and the dinitrosylated sulfite adduct, SULFI/NO, each displaying very different bioactivities. S-nitrosothiols are found in cells and tissues and can form via several different pathways (Cortese-Krott et al., 2016; Fukuto et al., 2012). One possibility is the reaction of a free thiol with a nitrosating species, that is, an entity that donates a nitrosonium (NO+) equivalent such as N2O3. Another possibility is the reaction of a thiyl radical (RS•) with NO•. Importantly, N2O3 generation is kinetically restricted (Wink, Darbyshire, Nims, Saavedra, & Ford, 1993) (for similar reasons as NO2 generation); as one-electron oxidants, thiyl radicals are very reactive, and their formation only occurs under very specific conditions, as in the active site of enzymes such as ribonucleotide reductase (Stubbe & Van Der Donk, 1998).
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For sensing/signaling purposes, the biochemical syntheses of ROS, RNS, and RSS are tightly controlled kinetically, temporally, and spatially. For example, NO• biosynthesis can occur via three primary pathways involving nitric oxide synthase (NOS) enzymes that are distinct with regard to their regulation and location (Förstermann & Sessa, 2012). Moreover, significant generation of ONOO− requires that both NO• and O2•− are made at the same place, rate, and time (Jourd'heuil et al., 2001); this requirement makes ONOO− generation rather difficult, possibly protecting cells from inadvertent formation and narrowing its action radius. The generation of NO2 from NO• is kinetically second order in NO• and first order in O2, indicating that significant NO2 levels (at least made via NO•/O2 chemistry) can only be produced in compartments possessing high levels of both precursors such as lipid membranes (Liu, Miller, Joshi, Thomas, & Lancaster, 1998) and lead to the formation of nitro fatty acids, molecules with potent pharmacological and biological activity (Rudolph & Freeman, 2009). Taken together, the chemical diversity of the reactive species of the RSI would have allowed the evolution of life to take advantage of widely varying chemistries provided by a limited number of biochemical precursors (such as O2, NO•, H2S, and derived species; this does not exclude the involvement of additional reactive small molecules such as hydrogen, ammonia, carbon monoxide, and formaldehyde). The strict chemical requirements for the effective formation of specific reaction products among an array of possibilities may offer selective advantages by limiting the generation of undesired species and ensuring that they are only formed under specific conditions (which can then be harnessed to fulfill a particular function in biology). By contrast, inadvertent or aberrant generation of ROS, RNS, or RSS carries the risk of having pathophysiological consequences.
Characteristics of the reactive species interactome Living entities are essentially open systems with a boundary that separates the internal from the external environment (Von Bertalanffy, 1968). The internal milieu is kept relatively constant at the expense of energy, whereas the external environment is often subject to rapid change. The ability to adjust the cellular/organismal metabolic machinery to environmental changes or variations in metabolic demand is essential for survival. The RSI represents the dynamic biochemical interface between the internal and external environment that allows those changes to be sensed and translated into an appropriate response that ensures living systems stay “fit for purpose” as conditions change. The RSI is bidirectional, facing outward to sense the environment and inward to allow chemical alterations to adjust metabolism and protein function. The principal reaction partners of reactive species are metal centers and protein thiols (thereby becoming primary “biological targets” during the course of evolution).
Role of the reactive species interactome
The RSI is characterized by the following: (a) Robustness and flexibility. The almost infinite possibilities of interactions between the reactive species, the redundancy of its constituents and targets, and the richness of chemical products generated by the RSI afford the unique robustness and flexibility of the system. (b) Rapid responsiveness and adaptability. The cross-reactivity and ability to rapidly interact with exogenous substances and endogenous targets render the RSI capable of responding rapidly to changes in external and internal conditions. The controlled formation and high reactivity of its constituents produce a unique fingerprint of reaction products that evolved to serve as both “carrier of information” and “transducing modality.” (c) Ability to sense the environment and transduce signals into functional changes. The products of the RSI are continuously generated by enzymatic reactions the rates of which depend on the availability of the precursors (sulfur-containing amino acids, arginine, and inorganic substrates such as oxygen, nitrite, and sulfate); this serves as an alternative way to sense changes in environmental conditions. Together with the pattern of reaction products produced at the outer surface of the boundary, the sensing process used by the RSI is thus not much different from the olfactory response; instead of relying on receptors, however, it uses a regulated chemical sensing process, enabling cells to actively “sniff out” their environment. The ability of the RSI to induce changes in enzymatic activity (e.g., by posttranslational protein modifications or regulation of gene expression) allows signals to be transduced by inhibiting or activating downstream effector pathways. It thereby serves an integrative function to sense and transduce multiple stressors and adjust bioenergetic needs and metabolic activities. In complex multicellular organisms, a need arises to keep processes synchronized in multiple cells and organs, energy requirements minimized, and whole body metabolism coordinated across space and time. To this end, the extracellular fluid (lymph and plasma) serves as communication conduits and pathway for exchange of precursors and reaction products (Cortese-Krott et al., 2017).
ole of the reactive species interactome in the response R to stress and evolution of life The term “stress” implies challenges to the ability of a cell (or an organism) to cope with changes in environmental conditions or metabolic demand; such stressors typically induce an adaptive response to better withstand future challenges (i.e., increase resilience). In earlier perspectives, we have discussed how the RSI may act as integral feature of the stress response and may thereby have participated in the evolutionary process (Cortese-Krott et al., 2017; Santolini, Wootton, Jackson, & Feelisch, 2019).
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We argued that the RSI must have originated already during the early stages of development of life on earth, when small molecules (gases, metals, and S/N/C-based compounds) participated in redox interactions that formed the bridge between inanimate and living matter. Life emerged in a sulfur-rich environment, long before the accumulation of oxygen in the environment (Cortese-Krott et al., 2017; Olson & Straub, 2015). The ability to effectively handle sulfur and RSS must therefore have played a fundamental role in the development of cellular mechanisms to deal with stressors, including the emergence of superoxide dismutase and catalase (Olson et al., 2017; Olson et al., 2018) before they assumed their contemporary antioxidant roles in handling O2•− and H2O2 (Cortese-Krott et al., 2017, Olson & Straub, 2015). NO• may have been the first gaseous molecule that was harnessed for intercellular communication and antioxidant protection, enabling the formation of symbiotic relationships (Feelisch & Martin, 1995). Sulfur, along with nitrogen and oxygen, would appear to be at the core of the redox code (Jones & Sies, 2015), allowing to exploit the opportunities offered by their electron configuration to build resilience (in the form of antioxidant enzymes, free thiols, and transcription factors essential for the stress response), to ensure a more efficient energy provision (e.g., via the Fe/S clusters in mitochondria) and to orchestrate the coordinated development of specialized functions in multicellular organisms (e.g., via regulation of protein function by thiol redox switches). Sulfur enables cells to harness the opportunities offered by this particular element in the form of inorganic (H2S and sulfate) and organic (cysteine and glutathione) redox molecules for communication and metabolic and energetic regulation. The utilization of sulfur for sensing and adaptation via the RSI has enabled lateral and vertical integration of the different levels of biological organization (Santolini et al., 2019), including complex intra- and intercellular communication required for growth and development and the various systems involved in maintaining the internal milieu relatively constant in the face of internal/external stressors.
The redox interactome The RSI is embedded within a wider redox network that connects the endogenous redox system of individual living systems with their environment and thus the biosphere to the O, N, S, and C cycles of the atmosphere, hydrosphere, and lithosphere. This is a reciprocal relationship by which geochemical redox alterations have influenced, over evolutionary timescales, all life on earth, while metabolic end products generated by the various life forms have shaped the redox landscape in which they evolved (Lovelock & Margulis, 1974). These considerations have been captured in the form of the redox interactome (Santolini et al., 2019). Originating from a small number of starting molecules, a self-organizing (and self-perpetuating) chemistry led to increasingly complex structures and chemistries, enabling increasingly complicated biological functions. Over time, the evolution of the geochemical environment and the diversification of life forms offered the
The RSI in the context of stratified medicine
p ossibility to explore new chemical combinations within highly compartmentalized structures. The resulting complexification in form and function led to a multiplication of reactions and possible interactions that may have been an important evolutionary driver for biological diversification and the development of specialized physiological functions in complex organisms. With all these coupled redox processes proceeding simultaneously at different levels of biological organization, from individual protein and subcellular organelle to the ecosystem level, this architecture allowed all metabolic activities within the organism to stay fully synchronized (Santolini et al., 2019).
The RSI in the context of stratified medicine The ideas of “stratification” and “personalized medicine” have come to be used in a clinical context as shorthand for acknowledging biological variability. As a normal feature of clinical diagnosis and care, patients are classified by disease entity to offer a cure, which is proportional to the degree of disease and “personalized.” Diagnosis is mainly based on the measurement of vital signs, biomarkers of organ damage and inflammation, and treatment on tackling “signs and symptoms” rather than the true causes of diseases. “Personalized medicine” is often used interchangeably with “precision medicine,” which purports to provide a specific and individualized therapy on the basis of a global analysis of the genetic and metabolic status of patients by applying genomics, transcriptomics, proteomics, and metabolomics. Although the technical developments related to carrying out such “omic” approaches and the analysis of “big data” produced by applying them are advancing rapidly, considerable knowledge gaps remain, preventing their implementation into routine clinical practice. Methods and procedures allowing a deep and reproducible analysis of redox signaling and redox responses as well as the diagnosis of specific “redox diseases,” which could be securely applied in a clinical context, are considerably less advanced as compared with the other “omics.” The main reasons for this are the short half-life of reactive species (compared with mRNA, proteins, lipids, and many metabolites) and a lack of understanding what each of the possible readouts may actually be indicative of. The RSI concept may offer a structured framework suited to disentangle the redox architecture of an organism and possibly an avenue into identifying the causes of redox diseases. We suggested earlier (Cortese-Krott et al., 2017) to apply an “omic” approach targeted at critical elements of the RSI, including (1) the precursors/substrates (e.g., O2, amino acids, and cofactors involved in redox control), (2) the transducing elements (cysteine-based redox relays and the dynamic interplay of circulating reduced and oxidized and protein-bound thiols), and (3) the stable end products of the RSI (S-,N-,and O-based stable metabolites). Combined with appropriate mathematical and statistical modeling approaches, such measurements may also help identifying central nodes of interactions and integrative markers of redox metabolism. One such example is homocysteine, which represents a combined readout of methionine and vitamin B6 and B12 availabilities, one‑carbon metabolism (methionine recycling
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and tetrahydrofolate pathways), flux through the trans‑sulfuration pathway, and formation of reactive sulfur species; another one is glycine, for example, the availability of which is paramount to porphyrin synthesis and glutathione production. Most biomarker studies focus on a few readouts of oxidative stress in plasma, serum, or urine in the context of disease-related organ damage and inflammation rather than a broad panel of redox-active compounds. Since the RSI concept itself is relatively new, few examples of its application to understanding redox processes in health and disease have so far been provided, and published examples (Abdulle et al., 2019; Bos et al., 2019; Horscroft et al., 2017; Van Der Graaf et al., 2016; Wandrag et al., 2017) are essentially observational. A more recent feasibility study in a small number of healthy individuals exposed to combined environmental and metabolic stressors (a moderate bicycle exercise under normoxic and hypoxic conditions), revealed marked differences in the pattern of changes in arteriovenous plasma concentrations of redox metabolites between individuals (see Fig. 2) even though the challenges participants were subjected to were identical (Cumpstey et al., 2019). Another remarkable feature was the dynamic nature of the alterations upon onset and cessation of the acute stressor (physical activity) superimposed onto the chronic stressor (hypobaric hypoxia). These results suggest that differences in gender, genetic makeup, and exposome history translate into quantifiable differences in redox responses to stressors. Naturally, the RSI is a highly dynamic system. To understand the acute status and reserve capacity of its constituent parts in a whole organisms (and possibly get a handle on individual metabolic fluxes), one would need to collect longitudinal data rather than cross-sectional snapshots only. At a bare minimum, one ought to compare steady-state concentrations of relevant readouts at baseline to concentrations following systematic perturbation of the entire system/whole body (in human studies, comparing, e.g., sedentary/exercise, normoxia/hypoxia, and starved/fed). Examples where such approaches have been used in human infectious disease settings have demonstrated the existence of hysteresis loops that appear to “map the disease space,” offering potential for tracking resilience to infections or tracing differences in health outcomes between individuals (Schneider, 2011; Torres et al., 2016).
Summary and conclusions The RSI is a primordial redox system that connects the (bio)chemical pathways involved in the generation of reactive species with cellular intermediary metabolism, bioenergetics, and the extracellular environment. The RSI is characterized by robustness and flexibility, rapid responsiveness, and adaptability, the ability to sense the environment and to transduce signals into functional changes. These properties allow cells/organisms to sense and adapt to changes in environmental conditions, allow interorgan communication and become an integral part of the stress response. A systematic analysis of the precursors/substrates, the transducing elements, and the stable end-products of the RSI may help identifying central nodes of interactions
P3
P1
TFT
TFT
HNE
HNE
cGMP
cGMP 8-isoPGF 8-isoPGF
NO2– NO3–
NO2– NO3–
RXNO
RXNO Cys free
Cys free Cys total
Cys total
GSH free
GSH free GSH total Sulfide free
GSH total Sulfide free Sulfide total
Sulfide total Sulfate
Sulfate
Thiosulf
Thiosulf 1
2
3
4
5
1
2
Sea level
3
4
5
1
2
3
4
5
1
2
Sea level
Altitude
P2
4
3
5
Altitude
P4 TFT TFT
HNE
HNE cGMP
cGMP
8-isoPGF
8-isoPGF
NO2– NO2–
NO3– RXNO
NO3–
Cys free
RXNO Cys free Cys total
Cys total
GSH free
GSH free GSH total
GSH total Sulfide free
Sulfide free Sulfide total
Sulfide total Sulfate
Sulfate Thiosulf
Thiosulf 1
2
3
Sea level
4
5
1
2
3 Altitude
4
5
1
2
3
Sea level
4
5
1
2
3
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5
Altitude
FIG. 2 The reactive species interactome in action: response heterogeneity between human study participants subjected to identical stressors. Differences in steady-state concentrations of various redox readouts covering ROS, RNS, and RSS chemistry between arterial and venous blood plasma (units omitted for the sake of clarity) during rest (T1), graded exercise (T2–T4), and early recovery (T5) in four study participants (P1–P4) during normoxia (sea level) and chronic hypoxia (high altitude). From Cumpstey, A. F., Minnion, M., Fernandez, B. O., Mikus-Lelinska, M., Mitchell, K., et al. (2019). Pushing arterial-venous plasma biomarkers to new heights: A model for personalised redox metabolomics? Redox Biology 21, 101113; please consult original publication for details.
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and integrative markers of redox metabolism. Being able to reliably capture multiple facets of the RSI across different compartments in a time-resolved manner will be fundamental to understanding how redox biology and physiology are regulated and affected by disease.
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Halliwell, B., & Gutteridge, J. M. (2015). Free radicals in biology and medicine. USA: Oxford University Press. Horscroft, J. A., Kotwica, A. O., Laner, V., West, J. A., Hennis, P. J., et al. (2017). Metabolic basis to Sherpa altitude adaptation. Proceedings of the National Academy of Sciences of the United States of America, 114, 6382–6387. Jacob, C., & Winyard, P. G. (2009). Redox signaling and regulation in biology and medicine. John Wiley & Sons. Jones, D. P., & Sies, H. (2015). The redox code. Antioxidants & Redox Signaling, 23, 734–746. Jourd’heuil, D., Jourd’heuil, F. L., Kutchukian, P. S., Musah, R. A., Wink, D. A., et al. (2001). Reaction of superoxide and nitric oxide with peroxynitrite implications for peroxynitrite-mediated oxidation reactions in vivo. The Journal of Biological Chemistry, 276, 28799–28805. Liu, X., Miller, M. J., Joshi, M. S., Thomas, D. D., & Lancaster, J. R. (1998). Accelerated reaction of nitric oxide with O2 within the hydrophobic interior of biological membranes. Proceedings of the National Academy of Sciences of the United States of America, 95, 2175–2179. Lovelock, J. E., & Margulis, L. (1974). Atmospheric homeostasis by and for the biosphere: The gaia hypothesis. Tellus, 26, 1–10. Olson, K. R., Gao, Y., Arif, F., Arora, K., Patel, S., et al. (2018). Metabolism of hydrogen sulfide (H2S) and production of reactive sulfur species (RSS) by superoxide dismutase. Redox Biology, 15, 74–85. Olson, K. R., Gao, Y., Deleon, E. R., Arif, M., Arif, F., et al. (2017). Catalase as a sulfide-sulfur oxido-reductase: An ancient (and modern?) regulator of reactive sulfur species (RSS). Redox Biology, 12, 325–339. Olson, K. R., & Straub, K. D. (2015). The role of hydrogen sulfide in evolution and the evolution of hydrogen sulfide in metabolism and signaling. Physiology, 31, 60–72. Ono, K., Akaike, T., Sawa, T., Kumagai, Y., Wink, D. A., et al. (2014). Redox chemistry and chemical biology of H2S, hydropersulfides, and derived species: Implications of their possible biological activity and utility. Free Radical Biology & Medicine, 77, 82–94. Rudolph, V., & Freeman, B. A. (2009). Cardiovascular consequences when nitric oxide and lipid signaling converge. Circulation Research, 105, 511–522. Santolini, J., Wootton, S. A., Jackson, A. A., & Feelisch, M. (2019). The redox architecture of physiological function. Current Opinion in Physiology, 9, 34–47. Schneider, D. S. (2011). Tracing personalized health curves during infections. PLoS Biology, 9, e1001158. Steudel, R., & Chivers, T. (2019). The role of polysulfide dianions and radical anions in the chemical, physical and biological sciences, including sulfur-based batteries. Chemical Society Reviews. in press. https://doi.org/10.1039/c8cs00826d. Stubbe, J., & Van Der Donk, W. A. (1998). Protein radicals in enzyme catalysis. Chemical Reviews, 98, 705–762. Torres, B. Y., Oliveira, J. H. M., Thomas Tate, A., Rath, P., Cumnock, K., et al. (2016). Tracking resilience to infections by mapping disease space. PLoS Biology, 14, e1002436. Van Der Graaf, A. M., Paauw, N. D., Toering, T. J., Feelisch, M., Faas, M. M., et al. (2016). Impaired sodium-dependent adaptation of arterial stiffness in formerly preeclamptic women: The RETAP-vascular study. American Journal of Physiology. Heart and Circulatory Physiology, 310, H1827–H1833. Von Bertalanffy, L. (1968). General System Theory—Foundations, Development, Applications. New York: George Braziller, Inc., 289 pp.
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Wandrag, L., Siervo, M., Riley, H. L., Khosravi, M., Fernandez, B. O., et al. (2017). Does hypoxia play a role in the development of sarcopenia in humans? Mechanistic insights from the Caudwell Xtreme Everest expedition. Redox Biology, 13, 60–68. Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., & Ford, P. C. (1993). Reactions of the bioregulatory agent nitric oxide in oxygenated aqueous media: Determination of the kinetics for oxidation and nitrosation by intermediates generated in the nitric oxide/oxygen reaction. Chemical Research in Toxicology, 6, 23–27.
The reactive species interactome
4
Miriam M. Cortese-Krotta, Jerome Santolinib, Steve A. Woottonc, Alan A. Jacksonc, Martin Feelischd a
Department of Cardiology, Pulmonology and Angiology, Medical Faculty, Heinrich Heine University of Düsseldorf, Düsseldorf, Germany b Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette Cedex, France c Human Nutrition, University of Southampton and University Hospital Southampton, Southampton, United Kingdom d Clinical & Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton, United Kingdom
Abstract Enzymatically generated reactive species are emerging as endogenous signaling molecules, targeting specific thiol/metal redox switches in proteins and organelles, thereby affecting cell metabolism and survival. To better characterize the complex network of interactions of reactive species with themselves and their biological targets and to understand their significance for metabolic control, we recently introduced the “reactive species interactome” (RSI) concept. The RSI is a primeval redox system that connects the (bio)chemical pathways that generate reactive species with cellular intermediary metabolism, bioenergetics, and the extracellular environment. The main characteristics of the RSI are (a) robustness and flexibility, (b) adaptability and rapid responsiveness, and (c) the ability to sense the chemical composition of intraand extracellular milieu. These properties enable cells/organs to communicate and sense and adapt to changes in environmental conditions and metabolic demand. As conceptual framework, the RSI may enable to better understand how complex life forms operate and respond to challenges at the whole organism level, potentially enabling us to harness this information for medical use in health and disease. Keywords: Nitric oxide, Hydrogen sulfide, Redox regulation, Systems biology
Oxidative Stress. https://doi.org/10.1016/B978-0-12-818606-0.00004-3 © 2020 Elsevier Inc. All rights reserved.
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Introduction For a long time, “reactive species” were considered to be toxic by-products of the metabolism of oxygen (O2), nitric oxide (NO•), and hydrogen sulfide (H2S)/sulfane sulfura containing species. More recently, it has become evident that the physiological functions of reactive species can only be understood when viewed as part of an integrated whole since they are interconnected by chemical reactions, produced— in an apparently purposeful manner—by specific enzymes within cells and tissues and have the potential to modulate common intra- and extracellular targets. Wellknown examples of such chemical interactions include the production of peroxynitrite (ONOO−) from enzymatically generated NO• and superoxide (Beckman, Beckman, Chen, Marshall, & Freeman, 1990) and the synthesis of nitrosopersulfide (SSNO−) and S-N from NO• and H2S (Cortese-Krott et al., 2015). Importantly, the products of these chemical interactions tend to trigger biological effects that are distinct from and independent of the effects induced by the parent molecules from which they originate. The richness and diversity of the physicochemical properties and lifetimes of the intermediary products generated by these (bio)chemical interactions are harnessed by Nature to enable and/or support physiological functions of considerable complexity. In an effort to integrate the chemical biology and biochemistry of these reactive species and the (patho)physiological significance of their interactions, we introduced the concept of the “reactive species interactome (RSI)” (Cortese-Krott et al., 2017). Specifically, we defined the RSI as a redox system consisting of chemical interactions of reactive species among themselves and with downstream biological targets such as thiols and metal centers (Fig. 1). This chapter aims to provide a summary
FIG. 1 Constituting elements of the reactive species interactome. See text for details. Modified from Cortese-Krott, M. M., Koning, A., Kuhnle, G. G. C., Nagy, P., Bianco, C. L., et al. (2017). The reactive species interactome: Evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized medicine. Antioxidants & Redox Signaling 27, 684–712. a
Sulfane sulfur denotes sulfur that is bound covalently only to (an)other sulfur atom(s).
Chemical interactions among reactive species
of the main constituents of this system, explain why it is an essential contributor to cellular and integrative redox signaling, and outline its potential significance for physiology and redox diseases in complex organisms.
Chemical interactions among reactive species Comprehensive treatments of the fundamental chemistry of reactive species including their kinetics of formation, chemical reactivities, action radii and interactions with common biological targets are available elsewhere (Basudhar et al., 2016; Cortese-Krott, Butler, Woollins, & Feelisch, 2016; Cortese-Krott, Fernandez, Kelm, Butler, & Feelisch, 2015; Fukuto et al., 2012; Halliwell & Gutteridge, 2015; Jacob & Winyard, 2009). The main oxygen-, nitrogen-, and sulfur-related reactive entities are therefore listed here only in tabular form according to their predominant chemical attributes (Table 1), and their possible interactions are only discussed in very general terms. Table 1 Exemplary reactive oxygen, nitrogen, and sulfur species and related hybrid molecules. Species
Occurrence/ formation
Chemical properties
Reactive oxygen species, ROS Dioxygen, O2
Aerobic life
Superoxide, O2•−
1e− reduction of O2
Hydrogen peroxide, H2O2 Hydroxyl radical, HO•
1e− reduction of O2− 1e− reduction of H2O2
Has unpaired electrons, reacts readily with other radicals, poor 1e− oxidant, but otherwise easily reduced by 2, 3, and 4e− Has one unpaired electron. Good reductant. Under acidic conditions can be an oxidant. Reacts with other radicals Not a radical species. Two-electron oxidant. Electrophilic. Can modify RSH (below) Potent 1e− oxidant. Short-lived radical species capable of abstracting an e− or hydrogen atom from most biological molecules
Reactive nitrogen species, RNS Nitric oxide, NO
Enzymatic, NO2− reduction
Nitrogen dioxide, NO2
Oxidation of NO
Dinitrogen trioxide, N2O3
Oxidation of NO by O2
Has unpaired e−. Poor oxidant. Reacts with other radicals (O2 and O2– and other organic radicals [R•]). Can act as an antioxidant by quenching radical chemistry Radical species. Good 1e− oxidant. Reacts with other radicals (can make R-NO2 when reacted with R•) Not a radical. Electrophilic and can nitrosate nucleophiles (add equivalent of “NO+”). Synthesis only relevant at high concentrations of NO Continued
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Table 1 Exemplary reactive oxygen, nitrogen, and sulfur species and related hybrid molecules—cont'd Species
Occurrence/ formation
Peroxynitrite, ONOO−
Reaction of NO and O2−
Nitroxyl, HNO
S-nitrosothiol reduction
Nitrite, NO2−
Oxidation of NO, dietary
Chemical properties Not a radical but can generate both HO• and NO2. Rearranges to give nitrate (NO3−). Can oxidize by 2e− (via peroxide-like chemistry) Reacts readily with thiols. A good hydrogen atom donor. Can act as an antioxidant by quenching radical reactions Unreactive at neutral pH. Not a radical. Nitrosating agent under acidic conditions. Can be reduced to NO (under acidic conditions)
Reactive sulfur species, RSS Thiol, RSH
Endogenous (e.g., cysteine)
Thiyl radical, RS·
1e− oxidation of RSH Oxidation of RSH Geochemical, enzymatic
Disulfide, RSSR Hydrogen sulfide, H 2S Sulfenic acid, RSOH Hydropersulfide, RSSH
2e− oxidation of RSH Enzymatic
Hydropolysulfide, RSSnH Dialkyl polysulfide, RSSnR
Oxidation of RSH Oxidation of RSH
Not a radical. Good metal ligand. Can be oxidized to give other biologically relevant sulfur species Radical species. Good 1e− oxidant. Will react with other radical species such as NO Not a radical. Electrophilic. Can be reduced back to RSH under biological conditions Not a radical. Good metal ligand. Can react with other biological electrophilic sulfur species (e.g., RSSR, RSOH) Not a radical. Electrophilic (reacts with other thiols to give disulfides, RSSR) Not a radical. Carries a sulfane sulfur. Nucleophile; can be electrophilic (akin to RSSR). Good 1e− reductant. Readily reduced back to RSH Properties similar to RSSH (although enhanced in all aspects) Properties similar to RSSR (although enhanced in all aspects). Can also be nucleophilic
S-N hybrid molecules S-Nitrosothiols, RSNO
Nitrosation of RSH
SSNO−
Unknown, to be determined Unknown
SNO− (and isomers) Sulfi/NO
Unknown
Not a radical. Can be reduced to RSH and HNO. Can transfer “NO+” to another thiol (transnitrosation) Fairly stable. Appears to be a source of NO. Chemical properties pending Fleeting. Chemical properties pending Fairly stable. Appears to be a source of NO and HNO
Modified from Cortese-Krott, M. M., Koning, A., Kuhnle, G. G. C., Nagy, P., Bianco, C. L., et al. (2017). The reactive species interactome: Evolutionary emergence, biological significance, and opportunities for redox metabolomics and personalized medicine. Antioxidants & Redox Signaling, 27, 684–712.
Chemical interactions among reactive species
Of all the small molecule entities of biological relevance, the chemical biology of O2 is the most studied and established. Serving as ultimate electron acceptor, reduced oxygen species including superoxide ( O2•− ), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•) are unavoidable, nonenzymatically generated companions of aerobic life. However, the same molecules, collectively termed “reactive oxygen species” (ROS) (Halliwell & Gutteridge, 2015), are also produced enzymatically to elicit highly specific biological effects and serve as cell signaling agents. Akin to ROS, equivalent terminology is in use for “reactive nitrogen species” (RNS) and “reactive sulfur species” (RSS), denoting NO-derived and H2S/RSH-derived species, respectively (Halliwell & Gutteridge, 2015) (Jacob & Winyard, 2009). This classification does not take into consideration the widely varying and distinct chemical properties and reactivities of ROS, RNS, and RSS. The compounds listed in Table 1 cover an enormous chemical space, ranging from highly reducing (RSSH, O2•− ) to highly oxidizing (HO• and NO2), from highly electrophilic (RSOH, H2O2, and HNO) to highly nucleophilic (RSSH), and from good hydrogen atom donors (HNO and RSSH) to potent hydrogen atom abstractors (HO· and NO2). Among the RSS, hydropersulfides (RSSH) and polysulfides (RSxH) have recently received considerable attention (Fukuto et al., 2018; Steudel & Chivers, 2019). The generation of hydropersulfides from the corresponding thiol represents an oxidation process (the sulfur in RSSH is of the same oxidation state as that in a disulfide, RSSR) and can be mediated by several of the oxidants listed in Table 1 (e.g., H2O2 in the presence of H2S). Interestingly, a hydropersulfide is a superior reductant compared with the corresponding thiol. Thus, an extremely potent reductant (RSSH) is generated primarily under oxidizing conditions, a fact that seems to have been taken advantage of by nature as it has been proposed that RSSH formation can be protective against oxidative stress (Ono et al., 2014). Sulfur’s versatile redox chemistry is further enriched by complex interaction with reactive nitrogen (e.g., NO• and HNO) and reactive oxygen species (e.g., O2•− and H2O2) (see Fig. 1). In fact, both RNS and RSS react with O2 and ROS, giving rise to a variety of nitrogen oxides (•NO2, N2O3, ONOO−, NO2−, and NO3−) and sulfur oxides (SO2, SO32−, S2O32−, Sx(SO3)22−, and SO42−). RNS can also interact with RSS leading to the formation of S-N hybrid species (Cortese-Krott et al., 2016). Examples of these reactive species include S-nitrosothiols (RSNOs), nitrosopersulfide, and the dinitrosylated sulfite adduct, SULFI/NO, each displaying very different bioactivities. S-nitrosothiols are found in cells and tissues and can form via several different pathways (Cortese-Krott et al., 2016; Fukuto et al., 2012). One possibility is the reaction of a free thiol with a nitrosating species, that is, an entity that donates a nitrosonium (NO+) equivalent such as N2O3. Another possibility is the reaction of a thiyl radical (RS•) with NO•. Importantly, N2O3 generation is kinetically restricted (Wink, Darbyshire, Nims, Saavedra, & Ford, 1993) (for similar reasons as NO2 generation); as one-electron oxidants, thiyl radicals are very reactive, and their formation only occurs under very specific conditions, as in the active site of enzymes such as ribonucleotide reductase (Stubbe & Van Der Donk, 1998).
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For sensing/signaling purposes, the biochemical syntheses of ROS, RNS, and RSS are tightly controlled kinetically, temporally, and spatially. For example, NO• biosynthesis can occur via three primary pathways involving nitric oxide synthase (NOS) enzymes that are distinct with regard to their regulation and location (Förstermann & Sessa, 2012). Moreover, significant generation of ONOO− requires that both NO• and O2•− are made at the same place, rate, and time (Jourd'heuil et al., 2001); this requirement makes ONOO− generation rather difficult, possibly protecting cells from inadvertent formation and narrowing its action radius. The generation of NO2 from NO• is kinetically second order in NO• and first order in O2, indicating that significant NO2 levels (at least made via NO•/O2 chemistry) can only be produced in compartments possessing high levels of both precursors such as lipid membranes (Liu, Miller, Joshi, Thomas, & Lancaster, 1998) and lead to the formation of nitro fatty acids, molecules with potent pharmacological and biological activity (Rudolph & Freeman, 2009). Taken together, the chemical diversity of the reactive species of the RSI would have allowed the evolution of life to take advantage of widely varying chemistries provided by a limited number of biochemical precursors (such as O2, NO•, H2S, and derived species; this does not exclude the involvement of additional reactive small molecules such as hydrogen, ammonia, carbon monoxide, and formaldehyde). The strict chemical requirements for the effective formation of specific reaction products among an array of possibilities may offer selective advantages by limiting the generation of undesired species and ensuring that they are only formed under specific conditions (which can then be harnessed to fulfill a particular function in biology). By contrast, inadvertent or aberrant generation of ROS, RNS, or RSS carries the risk of having pathophysiological consequences.
Characteristics of the reactive species interactome Living entities are essentially open systems with a boundary that separates the internal from the external environment (Von Bertalanffy, 1968). The internal milieu is kept relatively constant at the expense of energy, whereas the external environment is often subject to rapid change. The ability to adjust the cellular/organismal metabolic machinery to environmental changes or variations in metabolic demand is essential for survival. The RSI represents the dynamic biochemical interface between the internal and external environment that allows those changes to be sensed and translated into an appropriate response that ensures living systems stay “fit for purpose” as conditions change. The RSI is bidirectional, facing outward to sense the environment and inward to allow chemical alterations to adjust metabolism and protein function. The principal reaction partners of reactive species are metal centers and protein thiols (thereby becoming primary “biological targets” during the course of evolution).
Role of the reactive species interactome
The RSI is characterized by the following: (a) Robustness and flexibility. The almost infinite possibilities of interactions between the reactive species, the redundancy of its constituents and targets, and the richness of chemical products generated by the RSI afford the unique robustness and flexibility of the system. (b) Rapid responsiveness and adaptability. The cross-reactivity and ability to rapidly interact with exogenous substances and endogenous targets render the RSI capable of responding rapidly to changes in external and internal conditions. The controlled formation and high reactivity of its constituents produce a unique fingerprint of reaction products that evolved to serve as both “carrier of information” and “transducing modality.” (c) Ability to sense the environment and transduce signals into functional changes. The products of the RSI are continuously generated by enzymatic reactions the rates of which depend on the availability of the precursors (sulfur-containing amino acids, arginine, and inorganic substrates such as oxygen, nitrite, and sulfate); this serves as an alternative way to sense changes in environmental conditions. Together with the pattern of reaction products produced at the outer surface of the boundary, the sensing process used by the RSI is thus not much different from the olfactory response; instead of relying on receptors, however, it uses a regulated chemical sensing process, enabling cells to actively “sniff out” their environment. The ability of the RSI to induce changes in enzymatic activity (e.g., by posttranslational protein modifications or regulation of gene expression) allows signals to be transduced by inhibiting or activating downstream effector pathways. It thereby serves an integrative function to sense and transduce multiple stressors and adjust bioenergetic needs and metabolic activities. In complex multicellular organisms, a need arises to keep processes synchronized in multiple cells and organs, energy requirements minimized, and whole body metabolism coordinated across space and time. To this end, the extracellular fluid (lymph and plasma) serves as communication conduits and pathway for exchange of precursors and reaction products (Cortese-Krott et al., 2017).
ole of the reactive species interactome in the response R to stress and evolution of life The term “stress” implies challenges to the ability of a cell (or an organism) to cope with changes in environmental conditions or metabolic demand; such stressors typically induce an adaptive response to better withstand future challenges (i.e., increase resilience). In earlier perspectives, we have discussed how the RSI may act as integral feature of the stress response and may thereby have participated in the evolutionary process (Cortese-Krott et al., 2017; Santolini, Wootton, Jackson, & Feelisch, 2019).
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We argued that the RSI must have originated already during the early stages of development of life on earth, when small molecules (gases, metals, and S/N/C-based compounds) participated in redox interactions that formed the bridge between inanimate and living matter. Life emerged in a sulfur-rich environment, long before the accumulation of oxygen in the environment (Cortese-Krott et al., 2017; Olson & Straub, 2015). The ability to effectively handle sulfur and RSS must therefore have played a fundamental role in the development of cellular mechanisms to deal with stressors, including the emergence of superoxide dismutase and catalase (Olson et al., 2017; Olson et al., 2018) before they assumed their contemporary antioxidant roles in handling O2•− and H2O2 (Cortese-Krott et al., 2017, Olson & Straub, 2015). NO• may have been the first gaseous molecule that was harnessed for intercellular communication and antioxidant protection, enabling the formation of symbiotic relationships (Feelisch & Martin, 1995). Sulfur, along with nitrogen and oxygen, would appear to be at the core of the redox code (Jones & Sies, 2015), allowing to exploit the opportunities offered by their electron configuration to build resilience (in the form of antioxidant enzymes, free thiols, and transcription factors essential for the stress response), to ensure a more efficient energy provision (e.g., via the Fe/S clusters in mitochondria) and to orchestrate the coordinated development of specialized functions in multicellular organisms (e.g., via regulation of protein function by thiol redox switches). Sulfur enables cells to harness the opportunities offered by this particular element in the form of inorganic (H2S and sulfate) and organic (cysteine and glutathione) redox molecules for communication and metabolic and energetic regulation. The utilization of sulfur for sensing and adaptation via the RSI has enabled lateral and vertical integration of the different levels of biological organization (Santolini et al., 2019), including complex intra- and intercellular communication required for growth and development and the various systems involved in maintaining the internal milieu relatively constant in the face of internal/external stressors.
The redox interactome The RSI is embedded within a wider redox network that connects the endogenous redox system of individual living systems with their environment and thus the biosphere to the O, N, S, and C cycles of the atmosphere, hydrosphere, and lithosphere. This is a reciprocal relationship by which geochemical redox alterations have influenced, over evolutionary timescales, all life on earth, while metabolic end products generated by the various life forms have shaped the redox landscape in which they evolved (Lovelock & Margulis, 1974). These considerations have been captured in the form of the redox interactome (Santolini et al., 2019). Originating from a small number of starting molecules, a self-organizing (and self-perpetuating) chemistry led to increasingly complex structures and chemistries, enabling increasingly complicated biological functions. Over time, the evolution of the geochemical environment and the diversification of life forms offered the
The RSI in the context of stratified medicine
p ossibility to explore new chemical combinations within highly compartmentalized structures. The resulting complexification in form and function led to a multiplication of reactions and possible interactions that may have been an important evolutionary driver for biological diversification and the development of specialized physiological functions in complex organisms. With all these coupled redox processes proceeding simultaneously at different levels of biological organization, from individual protein and subcellular organelle to the ecosystem level, this architecture allowed all metabolic activities within the organism to stay fully synchronized (Santolini et al., 2019).
The RSI in the context of stratified medicine The ideas of “stratification” and “personalized medicine” have come to be used in a clinical context as shorthand for acknowledging biological variability. As a normal feature of clinical diagnosis and care, patients are classified by disease entity to offer a cure, which is proportional to the degree of disease and “personalized.” Diagnosis is mainly based on the measurement of vital signs, biomarkers of organ damage and inflammation, and treatment on tackling “signs and symptoms” rather than the true causes of diseases. “Personalized medicine” is often used interchangeably with “precision medicine,” which purports to provide a specific and individualized therapy on the basis of a global analysis of the genetic and metabolic status of patients by applying genomics, transcriptomics, proteomics, and metabolomics. Although the technical developments related to carrying out such “omic” approaches and the analysis of “big data” produced by applying them are advancing rapidly, considerable knowledge gaps remain, preventing their implementation into routine clinical practice. Methods and procedures allowing a deep and reproducible analysis of redox signaling and redox responses as well as the diagnosis of specific “redox diseases,” which could be securely applied in a clinical context, are considerably less advanced as compared with the other “omics.” The main reasons for this are the short half-life of reactive species (compared with mRNA, proteins, lipids, and many metabolites) and a lack of understanding what each of the possible readouts may actually be indicative of. The RSI concept may offer a structured framework suited to disentangle the redox architecture of an organism and possibly an avenue into identifying the causes of redox diseases. We suggested earlier (Cortese-Krott et al., 2017) to apply an “omic” approach targeted at critical elements of the RSI, including (1) the precursors/substrates (e.g., O2, amino acids, and cofactors involved in redox control), (2) the transducing elements (cysteine-based redox relays and the dynamic interplay of circulating reduced and oxidized and protein-bound thiols), and (3) the stable end products of the RSI (S-,N-,and O-based stable metabolites). Combined with appropriate mathematical and statistical modeling approaches, such measurements may also help identifying central nodes of interactions and integrative markers of redox metabolism. One such example is homocysteine, which represents a combined readout of methionine and vitamin B6 and B12 availabilities, one‑carbon metabolism (methionine recycling
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and tetrahydrofolate pathways), flux through the trans‑sulfuration pathway, and formation of reactive sulfur species; another one is glycine, for example, the availability of which is paramount to porphyrin synthesis and glutathione production. Most biomarker studies focus on a few readouts of oxidative stress in plasma, serum, or urine in the context of disease-related organ damage and inflammation rather than a broad panel of redox-active compounds. Since the RSI concept itself is relatively new, few examples of its application to understanding redox processes in health and disease have so far been provided, and published examples (Abdulle et al., 2019; Bos et al., 2019; Horscroft et al., 2017; Van Der Graaf et al., 2016; Wandrag et al., 2017) are essentially observational. A more recent feasibility study in a small number of healthy individuals exposed to combined environmental and metabolic stressors (a moderate bicycle exercise under normoxic and hypoxic conditions), revealed marked differences in the pattern of changes in arteriovenous plasma concentrations of redox metabolites between individuals (see Fig. 2) even though the challenges participants were subjected to were identical (Cumpstey et al., 2019). Another remarkable feature was the dynamic nature of the alterations upon onset and cessation of the acute stressor (physical activity) superimposed onto the chronic stressor (hypobaric hypoxia). These results suggest that differences in gender, genetic makeup, and exposome history translate into quantifiable differences in redox responses to stressors. Naturally, the RSI is a highly dynamic system. To understand the acute status and reserve capacity of its constituent parts in a whole organisms (and possibly get a handle on individual metabolic fluxes), one would need to collect longitudinal data rather than cross-sectional snapshots only. At a bare minimum, one ought to compare steady-state concentrations of relevant readouts at baseline to concentrations following systematic perturbation of the entire system/whole body (in human studies, comparing, e.g., sedentary/exercise, normoxia/hypoxia, and starved/fed). Examples where such approaches have been used in human infectious disease settings have demonstrated the existence of hysteresis loops that appear to “map the disease space,” offering potential for tracking resilience to infections or tracing differences in health outcomes between individuals (Schneider, 2011; Torres et al., 2016).
Summary and conclusions The RSI is a primordial redox system that connects the (bio)chemical pathways involved in the generation of reactive species with cellular intermediary metabolism, bioenergetics, and the extracellular environment. The RSI is characterized by robustness and flexibility, rapid responsiveness, and adaptability, the ability to sense the environment and to transduce signals into functional changes. These properties allow cells/organisms to sense and adapt to changes in environmental conditions, allow interorgan communication and become an integral part of the stress response. A systematic analysis of the precursors/substrates, the transducing elements, and the stable end-products of the RSI may help identifying central nodes of interactions
P3
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FIG. 2 The reactive species interactome in action: response heterogeneity between human study participants subjected to identical stressors. Differences in steady-state concentrations of various redox readouts covering ROS, RNS, and RSS chemistry between arterial and venous blood plasma (units omitted for the sake of clarity) during rest (T1), graded exercise (T2–T4), and early recovery (T5) in four study participants (P1–P4) during normoxia (sea level) and chronic hypoxia (high altitude). From Cumpstey, A. F., Minnion, M., Fernandez, B. O., Mikus-Lelinska, M., Mitchell, K., et al. (2019). Pushing arterial-venous plasma biomarkers to new heights: A model for personalised redox metabolomics? Redox Biology 21, 101113; please consult original publication for details.
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and integrative markers of redox metabolism. Being able to reliably capture multiple facets of the RSI across different compartments in a time-resolved manner will be fundamental to understanding how redox biology and physiology are regulated and affected by disease.
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