Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. While many key players involved in oxidative protein folding are known, our understanding of how redox homeostasis in the ER is maintained and how EROs, the Cys residues of nascent proteins, and the luminal glutathione redox buffer interact is limited. Here, we isolated viable ero1 ero2 double mutants largely deficient in ERO activity, which rendered the mutants highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER lumen in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress, as induced by acute exposure to dithiothreitol, was delayed in ero1 ero2 mutants. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in the ero1 ero2 mutant reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.

Lactose and Casein Cause Changes on Biomarkers of Oxidative Damage and Dysbiosis in an Experimental Model of Multiple Sclerosis

Background and Objectives: Experimental autoimmune encephalomyelitis (EAE) in rats closely reproduces multiple sclerosis (MS), a disease characterized by neuroinflammation and oxidative stress, that also appears to extend to other organ compartments. The origin of MS is a matter for discussion, but it would seem that altering certain bacterial populations present in the gut may lead to a proinflammatory condition due to the bacterial lipopolysaccharides (LPS) in the so-called brain-gut axis. The casein and lactose in milk confer anti-inflammatory properties and immunomodulatory effects. The objectives of this study were: to evaluate the effects of administration of casein and lactose on the oxidative damage and the clinical status caused by EAE, and to verify whether both, casein and lactose, had any effect on the LPS and its transport protein -LBP-. Methods: Twenty male dark Agouti rats were divided into: control rats (control), EAE rats and EAE rats to which casein and lactose, EAE+casein and EAE+lactose, respectively, were administered. Fifty-one days after casein and lactose administration, the rats were sacrificed and different organs were studied (brain, spinal cord, blood, heart, liver, kidney, small and large intestine). In the latter, products derived from oxidative stress were studied (lipid peroxides and carbonylated proteins) as well as the glutathione redox system, various inflammation factors (total nitrite, Nuclear Factor-kappa B p065, the Rat Tumour Necrosis Factor-α) and the LPS and LBP values. Results and Conclusion: Casein and lactose administration improved the clinical aspect of the disease at the same time as reducing inflammation and oxidative stress, exerting its action on the glutathione redox system or increasing GPx levels.

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

<p>The functioning of coral reef systems, as biodiversity hotspots, is largely dependent on the symbiotic association between dinoflagellate symbionts (Symbiodinium spp.) and scleractinian coral hosts. The breakdown of this symbiosis (coral bleaching), as a result of global warming and other stressors, therefore has profound implications for the tropical marine environment. Corals associate with a variety of Symbiodinium genotypes, and it is this mosaic nature that contributes to the variable stress thresholds of corals. Research over the past 25 years has established that the generation and scavenging of reactive oxygen species (ROS) in both partners, under light and thermal stress, is a fundamental element of the bleaching response. However, while the existence of more thermally susceptible and tolerant symbiont types has been recognized, the differences in the antioxidant systems that may accompany these properties have received less attention. The purpose of this thesis was to explore the role of the antioxidant network in explaining the different thermal susceptibilities of various symbiont types and how the activity of key antioxidants in both partners under thermal stress relates to bleaching patterns in different corals. Thus, the specific objectives were to: (1) assess the antioxidant network response in different Symbiodinium types; (2) investigate the activity and structural diversity of key enzymatic antioxidants in different Symbiodinium types; (3) examine the regulation of these antioxidants at the transcriptomic and proteomic levels; and (4) contrast the symbiont’s and host’s antioxidant responses under bleaching conditions. Symbiodinium types in culture were found to differ significantly with regards to the concentration and activity of specific antioxidants, exhibiting magnitude scale differences in some of them. However, the response of the main removal pathway, involving superoxide dismutase (SOD) and ascorbate peroxidase (APX), under lethal thermal stress was fairly similar. Instead, the typespecific differences were found to lie in more downstream systems, and particularly in those associated with the maintenance of the glutathione redox state. A declining glutathione redox state was the common feature of the three thermally susceptible Symbiodinium types: B1, C1, and E. Indeed, in comparison to the most sensitive type (B1), the tolerant type F1 exhibited stronger antioxidant up-regulation and the successful preservation of the highly reduced glutathione pool. Comparing antioxidant gene orthologues from members of different Symbiodinium clades (A-E) revealed a higher degree of sequence variation at the amino acid level for peroxidases, which reflected the genetic radiation of the genus. In contrast, primary defences in the form of SOD isoforms were highly conserved. Sequence variations between Symbiodinium types involved residues that constitute binding sites of substrates and co-factors, and therefore likely affect the catalytic properties of these enzymes. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between transcriptomic expression and proteomic activity due to high variability in expression between replicates, and little response in their enzymatic activity over three days. In contrast to previous findings, up-regulation of antioxidant defences was not evident in Symbiodinium cells inside the host (i.e. in hospite). In fact, oxidative stress in the thermally sensitive corals Acropora millepora and Pocillopora damicornis was only apparent from increased host catalase activity, which interestingly preceded photosynthetic dysfunction of their symbionts. Baseline antioxidant activities of thermally tolerant and susceptible host species showed no differences, though the scavenging activities of the hosts were considerably higher than those of the symbionts. Baseline activities for the symbionts were different, however, with Symbiodinium C15 from the thermally tolerant coral Montipora digitata exhibiting the lowest activities for SOD and catalase peroxidase. This thesis provides significant findings with respect to the variability in antioxidant activity, structure, and network response in different Symbiodinium types in culture, and how these relate to thermal tolerance. What effect these differences have on the response in the intact symbiosis remains unclear, however, as the findings contradict the classic bleaching model of photoinhibition and symbiont-derived ROS. I argue, using previously published data, that heating rates might profoundly affect the way we perceive the antioxidant response of both partners to thermal stress, and that host antioxidant defences might not be as easily overwhelmed by symbiont ROS as suggested previously. This thesis reports important findings on the antioxidant system in different Symbiodinium types, but also raises new questions about the antioxidant response of the intact coral.</p>

Symbiont diversity and coral bleaching: An antioxidant view on thermal stress

<p>The functioning of coral reef systems, as biodiversity hotspots, is largely dependent on the symbiotic association between dinoflagellate symbionts (Symbiodinium spp.) and scleractinian coral hosts. The breakdown of this symbiosis (coral bleaching), as a result of global warming and other stressors, therefore has profound implications for the tropical marine environment. Corals associate with a variety of Symbiodinium genotypes, and it is this mosaic nature that contributes to the variable stress thresholds of corals. Research over the past 25 years has established that the generation and scavenging of reactive oxygen species (ROS) in both partners, under light and thermal stress, is a fundamental element of the bleaching response. However, while the existence of more thermally susceptible and tolerant symbiont types has been recognized, the differences in the antioxidant systems that may accompany these properties have received less attention. The purpose of this thesis was to explore the role of the antioxidant network in explaining the different thermal susceptibilities of various symbiont types and how the activity of key antioxidants in both partners under thermal stress relates to bleaching patterns in different corals. Thus, the specific objectives were to: (1) assess the antioxidant network response in different Symbiodinium types; (2) investigate the activity and structural diversity of key enzymatic antioxidants in different Symbiodinium types; (3) examine the regulation of these antioxidants at the transcriptomic and proteomic levels; and (4) contrast the symbiont’s and host’s antioxidant responses under bleaching conditions. Symbiodinium types in culture were found to differ significantly with regards to the concentration and activity of specific antioxidants, exhibiting magnitude scale differences in some of them. However, the response of the main removal pathway, involving superoxide dismutase (SOD) and ascorbate peroxidase (APX), under lethal thermal stress was fairly similar. Instead, the typespecific differences were found to lie in more downstream systems, and particularly in those associated with the maintenance of the glutathione redox state. A declining glutathione redox state was the common feature of the three thermally susceptible Symbiodinium types: B1, C1, and E. Indeed, in comparison to the most sensitive type (B1), the tolerant type F1 exhibited stronger antioxidant up-regulation and the successful preservation of the highly reduced glutathione pool. Comparing antioxidant gene orthologues from members of different Symbiodinium clades (A-E) revealed a higher degree of sequence variation at the amino acid level for peroxidases, which reflected the genetic radiation of the genus. In contrast, primary defences in the form of SOD isoforms were highly conserved. Sequence variations between Symbiodinium types involved residues that constitute binding sites of substrates and co-factors, and therefore likely affect the catalytic properties of these enzymes. While expression of antioxidant genes was successfully measured in Symbiodinium B1, it was not possible to assess the link between transcriptomic expression and proteomic activity due to high variability in expression between replicates, and little response in their enzymatic activity over three days. In contrast to previous findings, up-regulation of antioxidant defences was not evident in Symbiodinium cells inside the host (i.e. in hospite). In fact, oxidative stress in the thermally sensitive corals Acropora millepora and Pocillopora damicornis was only apparent from increased host catalase activity, which interestingly preceded photosynthetic dysfunction of their symbionts. Baseline antioxidant activities of thermally tolerant and susceptible host species showed no differences, though the scavenging activities of the hosts were considerably higher than those of the symbionts. Baseline activities for the symbionts were different, however, with Symbiodinium C15 from the thermally tolerant coral Montipora digitata exhibiting the lowest activities for SOD and catalase peroxidase. This thesis provides significant findings with respect to the variability in antioxidant activity, structure, and network response in different Symbiodinium types in culture, and how these relate to thermal tolerance. What effect these differences have on the response in the intact symbiosis remains unclear, however, as the findings contradict the classic bleaching model of photoinhibition and symbiont-derived ROS. I argue, using previously published data, that heating rates might profoundly affect the way we perceive the antioxidant response of both partners to thermal stress, and that host antioxidant defences might not be as easily overwhelmed by symbiont ROS as suggested previously. This thesis reports important findings on the antioxidant system in different Symbiodinium types, but also raises new questions about the antioxidant response of the intact coral.</p>

Genetically Encoded Biosensors to Monitor Intracellular Reactive Oxygen and Nitrogen Species and Glutathione Redox Potential in Skeletal Muscle Cells

Reactive oxygen and nitrogen species (RONS) play an important role in the pathophysiology of skeletal muscle and are involved in the regulation of intracellular signaling pathways, which drive metabolism, regeneration, and adaptation in skeletal muscle. However, the molecular mechanisms underlying these processes are unknown or partially uncovered. We implemented a combination of methodological approaches that are funded for the use of genetically encoded biosensors associated with quantitative fluorescence microscopy imaging to study redox biology in skeletal muscle. Therefore, it was possible to detect and monitor RONS and glutathione redox potential with high specificity and spatio-temporal resolution in two models, isolated skeletal muscle fibers and C2C12 myoblasts/myotubes. Biosensors HyPer3 and roGFP2-Orp1 were examined for the detection of cytosolic hydrogen peroxide; HyPer-mito and HyPer-nuc for the detection of mitochondrial and nuclear hydrogen peroxide; Mito-Grx1-roGFP2 and cyto-Grx1-roGFP2 were used for registration of the glutathione redox potential in mitochondria and cytosol. G-geNOp was proven to detect cytosolic nitric oxide. The fluorescence emitted by the biosensors is affected by pH, and this might have masked the results; therefore, environmental CO2 must be controlled to avoid pH fluctuations. In conclusion, genetically encoded biosensors and quantitative fluorescence microscopy provide a robust methodology to investigate the pathophysiological processes associated with the redox biology of skeletal muscle.

Effects of microgravity exposure and fructo-oligosaccharide ingestion on the proteome of soleus and extensor digitorum longus muscles in developing mice

Short-chain fatty acids produced by the gut bacterial fermentation of non-digestible carbohydrates, e.g., fructo-oligosaccharide (FOS), contribute to the maintenance of skeletal muscle mass and oxidative metabolic capacity. We evaluated the effect of FOS ingestion on protein expression of soleus (Sol) and extensor digitorum longus muscles in mice exposed to microgravity (μ-g). Twelve 9-week-old male C57BL/6J mice were raised individually on the International Space Station under μ-g or artificial 1-g and fed a diet with or without FOS (n = 3/group). Regardless of FOS ingestion, the absolute wet weights of both muscles tended to decrease, and the fiber phenotype in Sol muscles shifted toward fast-twitch type following μ-g exposure. However, FOS ingestion tended to mitigate the μ-g-exposure-related decrease in oxidative metabolism and enhance glutathione redox detoxification in Sol muscles. These results indicate that FOS ingestion mildly suppresses metabolic changes and oxidative stress in antigravity Sol muscles during spaceflight.

Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.