- Email: [email protected]
Investigating the Redox Toxicology of Ambient Air Pollutants
resin-assisted quantitative redox proteomics. The stoichiometry information of SSG and total oxidation on a total of ~4,100 Cys sites was quantified. The basal average oxidation level of protein thiols was ~12%, which is consistent with the overall reducing status in vivo. The average percentage of SSG in total oxidation was 32.0% at basal, supporting that SSG is one major type of thiol modifications. Interesting, we observed that the redox potential of a cellular organelle was well correlated with the level of oxidation in the organelle. For example, endoplasmic reticulum and lysosome have much higher oxidation levels than those from reducing organelles, such as nucleus and mitochondrion. In contrast, the reducing organelles have an average higher percentage of SSG in total oxidation, suggesting SSG is the dominant modification and plays an important regulatory role in these organelles. Moreover, the integration of stoichiometric and dynamic data reveals potential evidence of inhibitory and activation sites via redox modifications
doi: 10.1016/j.freeradbiomed.2016.10.117 77 Janus-Faced Sestrin2 Controls ROS and MTOR Signalling Through Two Separate Functional Domains Seung-Hyun Ro1,2, Hanseong Kim2, Sojin An2, Uhn-Soo Cho2, and Jun Hee Lee2 1 University of Nebraska-Lincoln, USA, 2University of Michigan, Ann Arbor, USA Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is a twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.
doi: 10.1016/j.freeradbiomed.2016.10.118 78 LPS Infection Increasing ROS Level Regulates Stress Signaling Cascades, Inflammation and Apoptosis in Keratinocyte after 2-Chloroethyl Ethyl Sulphide Challenge Satish Sagar1, Soumya Ranjan Parida1, and Arttatrana Pal1 1 KIIT School of Biotechnology, Bhubaneswar, India 2-chloroethyl ethyl sulphide (CEES), a monofunctional analog of sulfur mustard (SM), is a well-known chemical warfare agent that induces vesicles or blisters on cutaneous toxicity in exposed individuals. CEES form highly reactive electrophilic sulphonium
ions, which attack nucleophilic centers of biomolecules especially nucleic acid, caused damage in epithelial layers of skin, lungs and eye. Infection with the bacterial endotoxin lipopolysaccharides (LPS) are important immune system infection cues in epithelial layers of skin, but it remains unknown the molecular mechanisms to activate signaling cascades regulates inflammation and loss of cell-cell communication after CEES exposure. In the present study, we investigate the molecular mechanisms of inflammation and loss of cell-cell communication after LPS infection with CEES exposure on keratinocytes. Our results shows that LPS infection with CEES exposure enhanced accumulation of reactive oxygen species (ROS), resulting in the activation of nuclear factor κB (NF-κB) via ERK1/2MAPK/Akt/tuberin-mTOR pathways, which subsequently subdue the pro-inflammatory mediators, apoptosis and lose of cellcell communication, leading to biomolecules damage. Protection against LPS infection with CEES toxicity could also be performed by blocking of ROS activation with antioxidant N-acetyl-L-cysteine (NAC), and ERK1/2MAPK (PD98059, U0126) and Akt inhibitors (LY294002, Wortmannin), which inhibit the intracellular redoxsensitive signaling pathways regulates inflammation and cell-cell communication. These results illustrate that accumulated ROS in mouse keratinocytes functions as a key NF-B signaling cascade via ERK1/2MAPK/Akt/tuberin-mTOR regulatory pathways induced by CEES after LPS infection, leading to inflammation, apoptosis and loss of cell-cell communication, which is attenuated by antioxidant NAC, ERK1/2MAPK and Akt inhibitors. This work was supported by Indian Council of Medical Research (ICMR), Grant: 53/21/2010-CMB/BMS/ICMR, Government of India to Dr. Arttatrana Pal.
doi: 10.1016/j.freeradbiomed.2016.10.119 79 Investigating the Redox Toxicology of Ambient Air Pollutants
James M Samet1 1 US EPA, Chapel Hill, USA Ambient air pollution is a leading global cause of morbidity and mortality. Millions of Americans live in areas in which levels of tropospheric ozone exceed air quality standards, while exposure to particulate matter (PM2.5) alone results in 3.2 million excess deaths annually worldwide. Evidence from epidemiological and clinical studies points to a link between inhalation of air pollutants and adverse cardiovascular and pulmonary outcomes. Laboratory studies support an inflammatory basis for these effects, as exposure to a wide variety of environmental agents leads to inflammatory signaling and gene expression in human lung cells. Investigations aimed at elucidating the mechanisms of signaling activation in cells exposed to broadly disparate ambient air contaminants converge on protein sulfenylation and a loss of protein tyrosine phosphatase activity as key initiating events that are dependent on an accompanying elevation in intracellular concentrations of H2O2. Interventions with ectopic expression of catalase and metabolic inhibitors implicate both mitochondrial respiration and redox cycling as potential sources of the H2O2 response. Studies using various model particles show that in addition to bearing adsorbed electrophiles, surface moieties on carbonaceous particles can directly present radical and non-radical oxidative stress to cells. Investigating the redox toxicology of environmental electrophiles (e.g., ozone, metal ions, quinones) presents unique pitfalls and potential artifacts that can confound methodological approaches, including fluorogenic live-cell imaging and extracellular flux analyses. Nonetheless, an increasing body of evidence points to oxidative dysregulation of intracellular signaling in the initiation of the inflammatory effects of air pollution inhalation. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT
SfRBM / SFRRI 2016
S45
NECESSARILY REFLECT EPA POLICY.
doi: 10.1016/j.freeradbiomed.2016.10.120 80 Redox Biology of Thiosulfate as a Sulfide Donor in Endothelial Cells
Anna Leskova1, Sibile Pardue1, John D. Glawe1, Christopher G. Kevil 1, and Xinggui Shen1 1 LSUHSC-Shreveport, USA Recent reports have revealed that hydrogen sulfide (H2S) has emerged as the most recent endogenous gasotransmitter involved many physiological and pathological processes. The pathways involved in the production, consumption, and mechanism of action of H2S appear to be sensitive to alterations in the redox stress. Thiosulfate (S2O32–) is one of oxidized products of the sulfide pathway, and has been used extensively and safely to treat calcific uremic arteriopathy (a disease, in part due to calcification of small arteries) in dialysis patients. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In this study, we examined the effect of thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). We found that thiosulfate is a low-dose, slow-releasing sulfide donor that utilizes a glutathione-dependent pathway in a time-dependent manner. Also, it was found that thiosulfate reduces HUVECS proliferation treated with vascular endothelial growth factor (VEGF) in normoxic and hypoxic conditions. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions through non-enzymatic and enzymatic pathways and that this is a beneficial sulfide donor. Keywords: thiosulfate, sulfide, anti-angiogenic, glutathione, donor
doi: 10.1016/j.freeradbiomed.2016.10.121 81 Regulation of Vascular Tone and Blood Pressure by a Tryptophan-Derived Tricyclic Hydroperoxide Ghassan J Maghzal1, Christopher Stanley1, Sudhir Shengule1, Andrew M Giltrap2, Preet C Chadha1, Sasha Prysyazhna 3, Fernanda M Prado4, Yorihiro Yamamoto5, Paolo di Mascio4, Richard J Payne2, Philip Eaton3, and Roland Stocker1,6 1 Victor Chang Cardiac Research Institute, Australia, 2University of Sydney, Australia, 3King's College London, United Kingdom, 4 Universidade de São Paulo, Brazil, 5Tokyo University of Technology, Japan, 6University of New South Wales, Australia Vascular inflammation is associated with increased arterial O2●– /H2O2 that affect vascular tone and disease. We reported previously that in inflammation, vascular endothelial cells express indoleamine 2,3-dioxygenase 1 (IDO1, a heme-containing enzyme that oxidizes Trp to N-formyl-kynurenine) and that IDO1-mediated metabolism of Trp regulates vascular tone and blood pressure (Nat Med 16:279). Here we show that in the presence of H 2O2 purified IDO1 forms singlet oxygen (1O2), as assessed by the conversion of specific probes to their endoperoxides, and light emission at 1270 nm. 1O2 formed by isolated IDO1/H2O2 stereospecifically converts Trp to a tricyclic hydroperoxide (cis-WOOH) that decays to Nformyl-kynurenine. Formation of cis-WOOH is via an oxidative dioxygenase reaction, distinct from the known reductive dioxygenase and peroxidase activities of IDO1. In vitro, cis-WOOH
S46
but not trans-WOOH oxidizes protein kinase G1 (PKG1) to a dimer in a reaction dependent on Cys42. Similarly, exposure of endothelium-denuded arteries to cis-WOOH dimerizes PKG1. Such cis-WOOH-induced PKG1dimerization is associated with arterial relaxation, whereas arteries from “redox dead” PKG1C42S knock-in mice are refractory to relaxation by cis-WOOH. Moreover, Trp-induced relaxation of IDO1-expressing and endothelium-intact arteries depends on PKG1-Cys42 as corresponding segments from PKG1-C42S knock-in mice do not relax, and it is attenuated by pretreatment with PEG-catalase. Together, our data indicate that in ‘inflamed arteries’, endothelial IDO1/H2O2 generate light (i.e., 1O2) in a dark reaction. This results in formation of a tricyclic Trp-derived hydroperoxide that signals relaxation via oxidative activation of PKG1 in vascular smooth muscle cells.
doi: 10.1016/j.freeradbiomed.2016.10.122 82 Molecular Mechanism of Oxysterol Induced Survival Response: The Role of Autophagy and ROS Beyza Vurusaner1, Paola Gamba 2, Gabriella Testa2, Simona Gargiulo 2, Gabriella Leonarduzzi 2, Giuseppe Poli2, and Huveyda Basaga 1 1 Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Turkey, 2 Department of Clinical and Biological Sciences, University of Torino, Italy Oxysterols are a family of 27-carbon molecules originated from cholesterol oxidation and the atherogenic potential of oxysterols is linked to their ability to induce oxidative stress, inflammation and apoptotic cell death. Apparently, these compounds are able to modulate not only pro-apoptotic but also anti-apoptotic signals in targeted cells; however, their anti-apoptotic effect has not been investigated in depth. Hence, we aimed to elucidate the molecular mechanisms underlying the survival signaling elicited by 27hydroxycholesterol (27-OH) which is the most represented oxysterol in human blood. Using human promonocytic cells (U937) challenged with a relatively low concentration of 27-OH, a strong survival signaling through an early and transient increase of cellular reactive oxygen species (ROS) level, that enhanced MEKERK/PI3K-Akt phosphorylation, in turn responsible of a sustained quenching of ROS production was observed. Notably, involvement of antioxidant Nrf2 and its target genes, HO-1 and NQO-1 in this early survival response were shown. It thus appears that Nrf2 is responsible for the quenching of the oxidative imbalance exerted in 27-OH challenged cells. On the other hand, relatively high micromolar amount of 27- OH did not stimulate at all the investigated survival signaling with a constant prooxidant effect. The results of ongoing experiments on the actual role of autophagy in modulating the survival response will be also presented and discussed.The data obtained highlight oxysterols’ ability to promote cell survival that might contribute to the pathogenesis of inflammation-driven chronic diseases such as atherosclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.123
SfRBM / SFRRI 2016
doi: 10.1016/j.freeradbiomed.2016.10.117 77 Janus-Faced Sestrin2 Controls ROS and MTOR Signalling Through Two Separate Functional Domains Seung-Hyun Ro1,2, Hanseong Kim2, Sojin An2, Uhn-Soo Cho2, and Jun Hee Lee2 1 University of Nebraska-Lincoln, USA, 2University of Michigan, Ann Arbor, USA Sestrins are stress-inducible metabolic regulators with two seemingly unrelated but physiologically important functions: reduction of reactive oxygen species (ROS) and inhibition of the mechanistic target of rapamycin complex 1 (mTORC1). How Sestrins fulfil this dual role has remained elusive so far. Here we report the crystal structure of human Sestrin2 (hSesn2), and show that hSesn2 is a twofold pseudo-symmetric with two globular subdomains, which are structurally similar but functionally distinct from each other. While the N-terminal domain (Sesn-A) reduces alkylhydroperoxide radicals through its helix-turn-helix oxidoreductase motif, the C-terminal domain (Sesn-C) modified this motif to accommodate physical interaction with GATOR2 and subsequent inhibition of mTORC1. These findings clarify the molecular mechanism of how Sestrins can attenuate degenerative processes such as aging and diabetes by acting as a simultaneous inhibitor of ROS accumulation and mTORC1 activation.
doi: 10.1016/j.freeradbiomed.2016.10.118 78 LPS Infection Increasing ROS Level Regulates Stress Signaling Cascades, Inflammation and Apoptosis in Keratinocyte after 2-Chloroethyl Ethyl Sulphide Challenge Satish Sagar1, Soumya Ranjan Parida1, and Arttatrana Pal1 1 KIIT School of Biotechnology, Bhubaneswar, India 2-chloroethyl ethyl sulphide (CEES), a monofunctional analog of sulfur mustard (SM), is a well-known chemical warfare agent that induces vesicles or blisters on cutaneous toxicity in exposed individuals. CEES form highly reactive electrophilic sulphonium
ions, which attack nucleophilic centers of biomolecules especially nucleic acid, caused damage in epithelial layers of skin, lungs and eye. Infection with the bacterial endotoxin lipopolysaccharides (LPS) are important immune system infection cues in epithelial layers of skin, but it remains unknown the molecular mechanisms to activate signaling cascades regulates inflammation and loss of cell-cell communication after CEES exposure. In the present study, we investigate the molecular mechanisms of inflammation and loss of cell-cell communication after LPS infection with CEES exposure on keratinocytes. Our results shows that LPS infection with CEES exposure enhanced accumulation of reactive oxygen species (ROS), resulting in the activation of nuclear factor κB (NF-κB) via ERK1/2MAPK/Akt/tuberin-mTOR pathways, which subsequently subdue the pro-inflammatory mediators, apoptosis and lose of cellcell communication, leading to biomolecules damage. Protection against LPS infection with CEES toxicity could also be performed by blocking of ROS activation with antioxidant N-acetyl-L-cysteine (NAC), and ERK1/2MAPK (PD98059, U0126) and Akt inhibitors (LY294002, Wortmannin), which inhibit the intracellular redoxsensitive signaling pathways regulates inflammation and cell-cell communication. These results illustrate that accumulated ROS in mouse keratinocytes functions as a key NF-B signaling cascade via ERK1/2MAPK/Akt/tuberin-mTOR regulatory pathways induced by CEES after LPS infection, leading to inflammation, apoptosis and loss of cell-cell communication, which is attenuated by antioxidant NAC, ERK1/2MAPK and Akt inhibitors. This work was supported by Indian Council of Medical Research (ICMR), Grant: 53/21/2010-CMB/BMS/ICMR, Government of India to Dr. Arttatrana Pal.
doi: 10.1016/j.freeradbiomed.2016.10.119 79 Investigating the Redox Toxicology of Ambient Air Pollutants
James M Samet1 1 US EPA, Chapel Hill, USA Ambient air pollution is a leading global cause of morbidity and mortality. Millions of Americans live in areas in which levels of tropospheric ozone exceed air quality standards, while exposure to particulate matter (PM2.5) alone results in 3.2 million excess deaths annually worldwide. Evidence from epidemiological and clinical studies points to a link between inhalation of air pollutants and adverse cardiovascular and pulmonary outcomes. Laboratory studies support an inflammatory basis for these effects, as exposure to a wide variety of environmental agents leads to inflammatory signaling and gene expression in human lung cells. Investigations aimed at elucidating the mechanisms of signaling activation in cells exposed to broadly disparate ambient air contaminants converge on protein sulfenylation and a loss of protein tyrosine phosphatase activity as key initiating events that are dependent on an accompanying elevation in intracellular concentrations of H2O2. Interventions with ectopic expression of catalase and metabolic inhibitors implicate both mitochondrial respiration and redox cycling as potential sources of the H2O2 response. Studies using various model particles show that in addition to bearing adsorbed electrophiles, surface moieties on carbonaceous particles can directly present radical and non-radical oxidative stress to cells. Investigating the redox toxicology of environmental electrophiles (e.g., ozone, metal ions, quinones) presents unique pitfalls and potential artifacts that can confound methodological approaches, including fluorogenic live-cell imaging and extracellular flux analyses. Nonetheless, an increasing body of evidence points to oxidative dysregulation of intracellular signaling in the initiation of the inflammatory effects of air pollution inhalation. THIS ABSTRACT OF A PROPOSED PRESENTATION DOES NOT
SfRBM / SFRRI 2016
S45
NECESSARILY REFLECT EPA POLICY.
doi: 10.1016/j.freeradbiomed.2016.10.120 80 Redox Biology of Thiosulfate as a Sulfide Donor in Endothelial Cells
Anna Leskova1, Sibile Pardue1, John D. Glawe1, Christopher G. Kevil 1, and Xinggui Shen1 1 LSUHSC-Shreveport, USA Recent reports have revealed that hydrogen sulfide (H2S) has emerged as the most recent endogenous gasotransmitter involved many physiological and pathological processes. The pathways involved in the production, consumption, and mechanism of action of H2S appear to be sensitive to alterations in the redox stress. Thiosulfate (S2O32–) is one of oxidized products of the sulfide pathway, and has been used extensively and safely to treat calcific uremic arteriopathy (a disease, in part due to calcification of small arteries) in dialysis patients. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In this study, we examined the effect of thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). We found that thiosulfate is a low-dose, slow-releasing sulfide donor that utilizes a glutathione-dependent pathway in a time-dependent manner. Also, it was found that thiosulfate reduces HUVECS proliferation treated with vascular endothelial growth factor (VEGF) in normoxic and hypoxic conditions. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions through non-enzymatic and enzymatic pathways and that this is a beneficial sulfide donor. Keywords: thiosulfate, sulfide, anti-angiogenic, glutathione, donor
doi: 10.1016/j.freeradbiomed.2016.10.121 81 Regulation of Vascular Tone and Blood Pressure by a Tryptophan-Derived Tricyclic Hydroperoxide Ghassan J Maghzal1, Christopher Stanley1, Sudhir Shengule1, Andrew M Giltrap2, Preet C Chadha1, Sasha Prysyazhna 3, Fernanda M Prado4, Yorihiro Yamamoto5, Paolo di Mascio4, Richard J Payne2, Philip Eaton3, and Roland Stocker1,6 1 Victor Chang Cardiac Research Institute, Australia, 2University of Sydney, Australia, 3King's College London, United Kingdom, 4 Universidade de São Paulo, Brazil, 5Tokyo University of Technology, Japan, 6University of New South Wales, Australia Vascular inflammation is associated with increased arterial O2●– /H2O2 that affect vascular tone and disease. We reported previously that in inflammation, vascular endothelial cells express indoleamine 2,3-dioxygenase 1 (IDO1, a heme-containing enzyme that oxidizes Trp to N-formyl-kynurenine) and that IDO1-mediated metabolism of Trp regulates vascular tone and blood pressure (Nat Med 16:279). Here we show that in the presence of H 2O2 purified IDO1 forms singlet oxygen (1O2), as assessed by the conversion of specific probes to their endoperoxides, and light emission at 1270 nm. 1O2 formed by isolated IDO1/H2O2 stereospecifically converts Trp to a tricyclic hydroperoxide (cis-WOOH) that decays to Nformyl-kynurenine. Formation of cis-WOOH is via an oxidative dioxygenase reaction, distinct from the known reductive dioxygenase and peroxidase activities of IDO1. In vitro, cis-WOOH
S46
but not trans-WOOH oxidizes protein kinase G1 (PKG1) to a dimer in a reaction dependent on Cys42. Similarly, exposure of endothelium-denuded arteries to cis-WOOH dimerizes PKG1. Such cis-WOOH-induced PKG1dimerization is associated with arterial relaxation, whereas arteries from “redox dead” PKG1C42S knock-in mice are refractory to relaxation by cis-WOOH. Moreover, Trp-induced relaxation of IDO1-expressing and endothelium-intact arteries depends on PKG1-Cys42 as corresponding segments from PKG1-C42S knock-in mice do not relax, and it is attenuated by pretreatment with PEG-catalase. Together, our data indicate that in ‘inflamed arteries’, endothelial IDO1/H2O2 generate light (i.e., 1O2) in a dark reaction. This results in formation of a tricyclic Trp-derived hydroperoxide that signals relaxation via oxidative activation of PKG1 in vascular smooth muscle cells.
doi: 10.1016/j.freeradbiomed.2016.10.122 82 Molecular Mechanism of Oxysterol Induced Survival Response: The Role of Autophagy and ROS Beyza Vurusaner1, Paola Gamba 2, Gabriella Testa2, Simona Gargiulo 2, Gabriella Leonarduzzi 2, Giuseppe Poli2, and Huveyda Basaga 1 1 Biological Sciences and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Turkey, 2 Department of Clinical and Biological Sciences, University of Torino, Italy Oxysterols are a family of 27-carbon molecules originated from cholesterol oxidation and the atherogenic potential of oxysterols is linked to their ability to induce oxidative stress, inflammation and apoptotic cell death. Apparently, these compounds are able to modulate not only pro-apoptotic but also anti-apoptotic signals in targeted cells; however, their anti-apoptotic effect has not been investigated in depth. Hence, we aimed to elucidate the molecular mechanisms underlying the survival signaling elicited by 27hydroxycholesterol (27-OH) which is the most represented oxysterol in human blood. Using human promonocytic cells (U937) challenged with a relatively low concentration of 27-OH, a strong survival signaling through an early and transient increase of cellular reactive oxygen species (ROS) level, that enhanced MEKERK/PI3K-Akt phosphorylation, in turn responsible of a sustained quenching of ROS production was observed. Notably, involvement of antioxidant Nrf2 and its target genes, HO-1 and NQO-1 in this early survival response were shown. It thus appears that Nrf2 is responsible for the quenching of the oxidative imbalance exerted in 27-OH challenged cells. On the other hand, relatively high micromolar amount of 27- OH did not stimulate at all the investigated survival signaling with a constant prooxidant effect. The results of ongoing experiments on the actual role of autophagy in modulating the survival response will be also presented and discussed.The data obtained highlight oxysterols’ ability to promote cell survival that might contribute to the pathogenesis of inflammation-driven chronic diseases such as atherosclerosis.
doi: 10.1016/j.freeradbiomed.2016.10.123
SfRBM / SFRRI 2016