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Characterization of unique products in reactions of GSNO with H2S
Y. Zhou et al. / Free Radical Biology and Medicine 128 (2018) S47–S60
S51
85
87
Heme biosynthesis and trafficking
Characterization of unique products in reactions of GSNO with H2S
Jonathan Dietz 1,*, Oleh Khalimonchuk 2, Iryna Bohovych 2, Amit Reddi 3, Amy Medlock 4, Osiris Martinez-Guzman 3 1
University of Nebraska, USA University of Nebraska-Lincoln, USA 3 Georgia Institute of Technology, USA 4 University of Georgia, USA
Patrick Farmer*, Murugaeson Kumar Baylor University, USA
2
A new peroxisome-targeted two-photon fluorescent probe for imaging superoxide anion in mice with depression phenotypes
The interplay between NO and H2S signaling has attracted much interest in redox proteomics of nitrosative and oxidative stress. Both small molecules appear to target similar pathways in organisms, by reacting with protein free thiols or metalloenzymes to alter or enhance function. We have previously investigated the N-based product speciation in the reaction of GSNO with H2S, specifically noting that the N-based gas product distributions are analogous to those of reactions of GSNO with reductants (e.g. ascorbic acid). We have also identified a plethora of unique glutathione-based products by LC-HRMS analysis. Single Ion Chromatograms (SICs) confirm that these species are present in the reaction mixture and are separated on the LC column prior to ionization. A number of small reactive thiols can be trapped with iodoacetamide (IA), including polysulfides (SnH2), glutathione polysulfides (GSSnH) and trithionitrates, GSN(SnH)2. Unusual trithionitrates are also observed as acetamide adducts, GSN(SnA)2, but only at low ratios of H2S to GSNO. Competition experiments suggest one initial product, the sulfenic acid GSOH, acts as precursor to persulfide and oxidized glutathione-based products. Addition of a non-electrophilic radical trap, vinyl cyclopropane, has little effect on the product distributions, suggesting radical intermediates are shortlived. Other unique dimedone adducts were observed that are attributable to the sulfinyl (GS(O)H) tautomer. We also observe small S-oxoacids, HSOH and S(OH)2, as the derivatized ISnD and DSnD adducts. As with GSOH, unique thioacetals are observed which derive from the sulfenyl tautomers of both HS(O)H and GS(O)H. These unique mixed-valence sulfides have little precedent in the literature but remain observable in reaction mixtures after many hours on the bench.
Ping Li, Qi Ding*, Xin Wang, Zhang Wen, Bo Tang
https://doi.org/10.1016/j.freeradbiomed.2018.10.090
Heme is an essential cofactor involved in a plethora of vital functions. Heme is required to be in most subcellular compartments, but the way, in which it is transported from the mitochondria - its site of synthesis - to those compartments is poorly understood. Utilizing ratiometric, fluorescent heme sensors, we found that the GTPases in control of mitochondrial fusion, Mgm1 and fission, Dnm1, are positive and negative regulators of heme trafficking to the nucleus, respectively. Furthermore, we discovered that mitochondrial network morphology is under the control of heme biosynthesis. Our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.
https://doi.org/10.1016/j.freeradbiomed.2018.10.088
86
Shandong Normal University, China
Depression is a devastating mental disease with high morbidity and mortality rates, intimately linked with oxidative stress. Peroxisomes can produce and scavenge reactive oxygen species (ROS), playing a pivotal role in – maintaining redox balance in cells. Superoxide anion (O2 ), as the first produced reactive oxygen species in living systems, of whose level directly reflects the degree of oxidative stress. Thus, it is critical to identify the – function of O2 in peroxisomes for understanding the etiology of depression. Herein, we fabricate a new peroxisome-targetable fluorescent probe termed – TCP for imaging O2 in the living cells and brains of mice with depressionlike behaviors. Upon excitation with 800 nm, TCP exhibits obvious fluores– cence increased stepwise depend on the changes of O2 . The imaging results – demonstrate that the O2 in peroxisomes of glutamate-stimulated PC12 cells significantly increased compared to that of normal cells. Importantly, – utilizing TCP, we also explore the fluctuation of peroxisomes’ O2 in brains of mice with depression phenotypes. Altogether, this work provides new ways to understand the the occurrence and development of depression.
https://doi.org/10.1016/j.freeradbiomed.2018.10.089
88
Protection against cell oxidative damage by a naturally-occurring quercetin oxidation metabolite Jocelyn Fuentes*, Fernanda Arias, Elias Atala,, Hernan Speisky Universidad de Chile, Chile Introduction: Quercetin oxidation is believed to ultimately result in the loss of its antioxidant properties. Recently, however, we demonstrated that upon its oxidation, a mixture of metabolites which largely surpasses its original antioxidant activity is formed, and that a single metabolite, identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (BZF) accounts for such incremented activity. Material & Methods: HPLC-DAD and UHPLC-ESI-MS/MS was use to investigate the occurrence of such metabolite in flesh and peel of 20 quercetin-containing plant foods, and Hs68/Caco2 cells exposed to ROS were used to assess their antioxidant properties. Results: The BZF was found only in the dry outer-scale of certain type of Allium cepa. An aqueous extract of onion protected (EC80) against oxidative damage within the 165 mg/L to 16.5mL/L range. The concentrations of BZF corresponding to the latter extract varied between 1.3 nM and 130 nM. The specific removal of BZF from such extract led to the total loss of its antioxidant properties. However, an 80% protection was seen again, when a pure BZF preparation was added to the cells at the above-referred nM concentrations.
S51
85
87
Heme biosynthesis and trafficking
Characterization of unique products in reactions of GSNO with H2S
Jonathan Dietz 1,*, Oleh Khalimonchuk 2, Iryna Bohovych 2, Amit Reddi 3, Amy Medlock 4, Osiris Martinez-Guzman 3 1
University of Nebraska, USA University of Nebraska-Lincoln, USA 3 Georgia Institute of Technology, USA 4 University of Georgia, USA
Patrick Farmer*, Murugaeson Kumar Baylor University, USA
2
A new peroxisome-targeted two-photon fluorescent probe for imaging superoxide anion in mice with depression phenotypes
The interplay between NO and H2S signaling has attracted much interest in redox proteomics of nitrosative and oxidative stress. Both small molecules appear to target similar pathways in organisms, by reacting with protein free thiols or metalloenzymes to alter or enhance function. We have previously investigated the N-based product speciation in the reaction of GSNO with H2S, specifically noting that the N-based gas product distributions are analogous to those of reactions of GSNO with reductants (e.g. ascorbic acid). We have also identified a plethora of unique glutathione-based products by LC-HRMS analysis. Single Ion Chromatograms (SICs) confirm that these species are present in the reaction mixture and are separated on the LC column prior to ionization. A number of small reactive thiols can be trapped with iodoacetamide (IA), including polysulfides (SnH2), glutathione polysulfides (GSSnH) and trithionitrates, GSN(SnH)2. Unusual trithionitrates are also observed as acetamide adducts, GSN(SnA)2, but only at low ratios of H2S to GSNO. Competition experiments suggest one initial product, the sulfenic acid GSOH, acts as precursor to persulfide and oxidized glutathione-based products. Addition of a non-electrophilic radical trap, vinyl cyclopropane, has little effect on the product distributions, suggesting radical intermediates are shortlived. Other unique dimedone adducts were observed that are attributable to the sulfinyl (GS(O)H) tautomer. We also observe small S-oxoacids, HSOH and S(OH)2, as the derivatized ISnD and DSnD adducts. As with GSOH, unique thioacetals are observed which derive from the sulfenyl tautomers of both HS(O)H and GS(O)H. These unique mixed-valence sulfides have little precedent in the literature but remain observable in reaction mixtures after many hours on the bench.
Ping Li, Qi Ding*, Xin Wang, Zhang Wen, Bo Tang
https://doi.org/10.1016/j.freeradbiomed.2018.10.090
Heme is an essential cofactor involved in a plethora of vital functions. Heme is required to be in most subcellular compartments, but the way, in which it is transported from the mitochondria - its site of synthesis - to those compartments is poorly understood. Utilizing ratiometric, fluorescent heme sensors, we found that the GTPases in control of mitochondrial fusion, Mgm1 and fission, Dnm1, are positive and negative regulators of heme trafficking to the nucleus, respectively. Furthermore, we discovered that mitochondrial network morphology is under the control of heme biosynthesis. Our results indicate that mitochondrial dynamics and heme trafficking are integrally coupled.
https://doi.org/10.1016/j.freeradbiomed.2018.10.088
86
Shandong Normal University, China
Depression is a devastating mental disease with high morbidity and mortality rates, intimately linked with oxidative stress. Peroxisomes can produce and scavenge reactive oxygen species (ROS), playing a pivotal role in – maintaining redox balance in cells. Superoxide anion (O2 ), as the first produced reactive oxygen species in living systems, of whose level directly reflects the degree of oxidative stress. Thus, it is critical to identify the – function of O2 in peroxisomes for understanding the etiology of depression. Herein, we fabricate a new peroxisome-targetable fluorescent probe termed – TCP for imaging O2 in the living cells and brains of mice with depressionlike behaviors. Upon excitation with 800 nm, TCP exhibits obvious fluores– cence increased stepwise depend on the changes of O2 . The imaging results – demonstrate that the O2 in peroxisomes of glutamate-stimulated PC12 cells significantly increased compared to that of normal cells. Importantly, – utilizing TCP, we also explore the fluctuation of peroxisomes’ O2 in brains of mice with depression phenotypes. Altogether, this work provides new ways to understand the the occurrence and development of depression.
https://doi.org/10.1016/j.freeradbiomed.2018.10.089
88
Protection against cell oxidative damage by a naturally-occurring quercetin oxidation metabolite Jocelyn Fuentes*, Fernanda Arias, Elias Atala,, Hernan Speisky Universidad de Chile, Chile Introduction: Quercetin oxidation is believed to ultimately result in the loss of its antioxidant properties. Recently, however, we demonstrated that upon its oxidation, a mixture of metabolites which largely surpasses its original antioxidant activity is formed, and that a single metabolite, identified as 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxy-3(2H)-benzofuranone (BZF) accounts for such incremented activity. Material & Methods: HPLC-DAD and UHPLC-ESI-MS/MS was use to investigate the occurrence of such metabolite in flesh and peel of 20 quercetin-containing plant foods, and Hs68/Caco2 cells exposed to ROS were used to assess their antioxidant properties. Results: The BZF was found only in the dry outer-scale of certain type of Allium cepa. An aqueous extract of onion protected (EC80) against oxidative damage within the 165 mg/L to 16.5mL/L range. The concentrations of BZF corresponding to the latter extract varied between 1.3 nM and 130 nM. The specific removal of BZF from such extract led to the total loss of its antioxidant properties. However, an 80% protection was seen again, when a pure BZF preparation was added to the cells at the above-referred nM concentrations.