- Email: [email protected]
An EPR Study of the Interaction of NTA with Transferrin and Albumin
run the same experiments with NTA. We present EPR data which indicates that NTA does not remove iron from transferrin and thus the detected EPR intensity in a plasma should be from NTBI. EPR studies are in progress to determine the degree to which NTBI is removed from human albumin by NTA.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.284 239 ,QKLELWLRQRI3HUR[LGDVLQ0HGLDWHG&ROODJHQ,9 &URVVOLQNLQJ Bee Bathish1, Anthony Kettle1, and Christine Winterbourn1 1 University of Otago, Christchurch, New Zealand
Cross-linking of collagen IV is necessary for formation of extracellular matrix. Strong evidence suggest that the crosslinking reaction is catalysed by peroxidasin, a recently discovered human peroxidase. The crosslink forms between juxtaposed methionine and lysine residues on two collagen IV protomers. This linkage was shown to be a sulfilimine bond that forms when hypohalous acid (HOX) reacts with sulfur atom on methionine. HOX is generated by peroxidase-catalysed reaction between hydrogen peroxide and halide. Peroxidasin catalyses the same reaction with higher affinity for Br- than Cl- as a substrate. We investigated the effect of several endogenous and exogenous substrates and inhibitors of other peroxidases on the activity of peroxidasin and the cross-linking of collagen IV in cell culture. Our results show that peroxidasin is secreted from the cells and able to form the crosslink when supplied with H2O2 and Br- as substrates. Endogenous and exogenous peroxidase substrates and inhibitors interfered with the crosslinking. Catalase and methionine, which scavenge H2O2 and HOX, respectively, interfered with the activity of peroxidasin. Moreover, cross-linking of collagen IV is susceptible to inhibition when treated with thiocyanate and acetaminophen. Thiocyanate inhibited the activity of peroxidasin at physiological concentrations. Acetaminophen, a widely used pharmacological analgesic, also inhibited peroxidasin at therapeutic concentrations. Given the essential role of collagen IV for structural integrity of extracellular matrix, this data suggests that pathological consequences of physiological levels of thiocyanate or therapeutic uses of acetaminophen should be considered.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.285 240 373%2[LGDWLRQ3URPRWHVD1RYHO,QWHUDFWLRQZLWK ȗ
Alexandre Bergeron1,2, Gérald Coulis1,2, Seung Jun Kim3, Nicholas K. Tonks4, and Benoit Boivin1,2 1 Montreal Heart Institute, Canada, 2Université de Montréal, Canada, 3Korea Research Institute of Bioscience and Biotechnology, Republic of Korea, 4Cold Spring Harbor Laboratory, USA Despite the contribution of cardiac hypertrophy to heart failure, molecular mechanisms involved in this transition remain unclear. However, work from several groups point towards a critical role for reactive oxygen species (ROS) in this process. Importantly, the inactivation of the catalytic cysteine of Protein Tyrosine Phosphatases (PTPs) by ROS inhibits their ability for phosphotyrosyl hydrolysis, perturbs the delicate balance between the actions of PTPs and protein kinases, and potentially
integrates ROS signalling to control signal transduction in cardiac hypertrophy and heart failure. We explored whether PTPs were inactivated in hypertrophying hearts and we identified PTP1B as a target of ROS in these conditions. We modeled the tridimensional structure of the reversibly oxidized form of PTP1B and synthetized a peptide that is exposed to the cytosol in these conditions. We used this peptide to pull-down, purify and identify new interacting partners. Using this approach, we identified 14-33] by mass spectrometry as a major interacting partner. We then induced PTP1B oxidation in cells using EGF, and confirmed that 14-3-3] interacted with PTP1B in a transient manner. As expected, this interaction was prevented by treating cells with Nacetylcysteine. Moreover, a stable interaction was detected between 14-3-3] and PTP1B (C215A, S216A), a mutant known to adopt the oxidized conformation of the enzyme. Finally, GSTμ was also identified as a potential partner of the protein complex. We are currently exploring whether a protein complex formed of 14-3-3] and GSTμ participate in the regulated reactivation of PTP1B and how this affects cardiac hypertrophy.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.286 241 3RVVLEOH5HODWLRQVKLSEHWZHHQWKH75395HFHSWRU DQG3XULQH5HODWHG5HDFWLYH2[\JHQ6SHFLHV 3URGXFWLRQRQ'2063RVW(FFHQWULF([HUFLVHLQ5DW
Leandro Retamoso1, Mauro Silveira Junior1, Frederico Diniz Lima1, Guilherme Busanello1, Guilherme Bresciani2, Leandro Ribeiro1, Pietro Chagas1, Cristina Nogueira1, Ana Claudia Braga1, Ana Flávia Furian1, Mauro Oliveira1, Michele Fighera1, and Luiz Fernando Royes1 1 Universidade Federal de Santa Maria, Brazil, 2Universidad Autonoma de Chile, Chile It is well-known that unaccustomed exercise with a great eccentric component is highly related to the delayed onset muscle soreness (DOMS), which has also been described to be associated with increased reactive oxygen species (ROS) production. Although excessive ROS production has been reported to activate pain receptors, whether eccentric exercise-related DOMS may be associated to ROS-mediated activation of pain receptors is still an open question. Thus, the aim of this study was to investigate the ROS production effects on the transient receptor potential vanilloid 1 (TRPV1) receptor and its relationship with DOMS after an eccentric exercise bout. For this purpose, Wistar male rats performed a downhill running protocol on a treadmill (-16º tilt) at a constant speed of 16m/min during 90 min (5 min/bout separated by 2 min of rest). Mechanical allodynia (von Frey hair) were analyzed before and 1, 3, 6, 9, 12, 24, 48 and 72 h after the eccentric exercise. Biochemical assays for protein carbonyl, purine degradation, xanthine oxidase and TRPV1 protein content were performed in the gastrocnemius muscle at 12, 24, and 48h after the exercise. The statistical analysis showed an increase in the mechanical allodynia at 24-48h after the exercise. Similarly, an increase on the protein carbonyl, xanthine oxidase activity, uric acid levels and TRPV1 protein content was found 12 h post exercise. These results suggest that there may be a relationship between purine-related ROS and the TRPV1 receptor activation on the events preceding the appearance of eccentric exerciserelated DOMS. These biochemical modulations may be the early trigger of exercise-related DOMS. Moreover, these data reinforces the hypothesis that purine-related ROS could be also an important source of ROS during exercise, which corroborates the current rationale that exercise-related free radical production is not restricted to mitochondrial sources. However, future studies are needed in order to determine the transition between
SFRBM 2015
S109
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.284 239 ,QKLELWLRQRI3HUR[LGDVLQ0HGLDWHG&ROODJHQ,9 &URVVOLQNLQJ Bee Bathish1, Anthony Kettle1, and Christine Winterbourn1 1 University of Otago, Christchurch, New Zealand
Cross-linking of collagen IV is necessary for formation of extracellular matrix. Strong evidence suggest that the crosslinking reaction is catalysed by peroxidasin, a recently discovered human peroxidase. The crosslink forms between juxtaposed methionine and lysine residues on two collagen IV protomers. This linkage was shown to be a sulfilimine bond that forms when hypohalous acid (HOX) reacts with sulfur atom on methionine. HOX is generated by peroxidase-catalysed reaction between hydrogen peroxide and halide. Peroxidasin catalyses the same reaction with higher affinity for Br- than Cl- as a substrate. We investigated the effect of several endogenous and exogenous substrates and inhibitors of other peroxidases on the activity of peroxidasin and the cross-linking of collagen IV in cell culture. Our results show that peroxidasin is secreted from the cells and able to form the crosslink when supplied with H2O2 and Br- as substrates. Endogenous and exogenous peroxidase substrates and inhibitors interfered with the crosslinking. Catalase and methionine, which scavenge H2O2 and HOX, respectively, interfered with the activity of peroxidasin. Moreover, cross-linking of collagen IV is susceptible to inhibition when treated with thiocyanate and acetaminophen. Thiocyanate inhibited the activity of peroxidasin at physiological concentrations. Acetaminophen, a widely used pharmacological analgesic, also inhibited peroxidasin at therapeutic concentrations. Given the essential role of collagen IV for structural integrity of extracellular matrix, this data suggests that pathological consequences of physiological levels of thiocyanate or therapeutic uses of acetaminophen should be considered.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.285 240 373%2[LGDWLRQ3URPRWHVD1RYHO,QWHUDFWLRQZLWK ȗ
Alexandre Bergeron1,2, Gérald Coulis1,2, Seung Jun Kim3, Nicholas K. Tonks4, and Benoit Boivin1,2 1 Montreal Heart Institute, Canada, 2Université de Montréal, Canada, 3Korea Research Institute of Bioscience and Biotechnology, Republic of Korea, 4Cold Spring Harbor Laboratory, USA Despite the contribution of cardiac hypertrophy to heart failure, molecular mechanisms involved in this transition remain unclear. However, work from several groups point towards a critical role for reactive oxygen species (ROS) in this process. Importantly, the inactivation of the catalytic cysteine of Protein Tyrosine Phosphatases (PTPs) by ROS inhibits their ability for phosphotyrosyl hydrolysis, perturbs the delicate balance between the actions of PTPs and protein kinases, and potentially
integrates ROS signalling to control signal transduction in cardiac hypertrophy and heart failure. We explored whether PTPs were inactivated in hypertrophying hearts and we identified PTP1B as a target of ROS in these conditions. We modeled the tridimensional structure of the reversibly oxidized form of PTP1B and synthetized a peptide that is exposed to the cytosol in these conditions. We used this peptide to pull-down, purify and identify new interacting partners. Using this approach, we identified 14-33] by mass spectrometry as a major interacting partner. We then induced PTP1B oxidation in cells using EGF, and confirmed that 14-3-3] interacted with PTP1B in a transient manner. As expected, this interaction was prevented by treating cells with Nacetylcysteine. Moreover, a stable interaction was detected between 14-3-3] and PTP1B (C215A, S216A), a mutant known to adopt the oxidized conformation of the enzyme. Finally, GSTμ was also identified as a potential partner of the protein complex. We are currently exploring whether a protein complex formed of 14-3-3] and GSTμ participate in the regulated reactivation of PTP1B and how this affects cardiac hypertrophy.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.286 241 3RVVLEOH5HODWLRQVKLSEHWZHHQWKH75395HFHSWRU DQG3XULQH5HODWHG5HDFWLYH2[\JHQ6SHFLHV 3URGXFWLRQRQ'2063RVW(FFHQWULF([HUFLVHLQ5DW
Leandro Retamoso1, Mauro Silveira Junior1, Frederico Diniz Lima1, Guilherme Busanello1, Guilherme Bresciani2, Leandro Ribeiro1, Pietro Chagas1, Cristina Nogueira1, Ana Claudia Braga1, Ana Flávia Furian1, Mauro Oliveira1, Michele Fighera1, and Luiz Fernando Royes1 1 Universidade Federal de Santa Maria, Brazil, 2Universidad Autonoma de Chile, Chile It is well-known that unaccustomed exercise with a great eccentric component is highly related to the delayed onset muscle soreness (DOMS), which has also been described to be associated with increased reactive oxygen species (ROS) production. Although excessive ROS production has been reported to activate pain receptors, whether eccentric exercise-related DOMS may be associated to ROS-mediated activation of pain receptors is still an open question. Thus, the aim of this study was to investigate the ROS production effects on the transient receptor potential vanilloid 1 (TRPV1) receptor and its relationship with DOMS after an eccentric exercise bout. For this purpose, Wistar male rats performed a downhill running protocol on a treadmill (-16º tilt) at a constant speed of 16m/min during 90 min (5 min/bout separated by 2 min of rest). Mechanical allodynia (von Frey hair) were analyzed before and 1, 3, 6, 9, 12, 24, 48 and 72 h after the eccentric exercise. Biochemical assays for protein carbonyl, purine degradation, xanthine oxidase and TRPV1 protein content were performed in the gastrocnemius muscle at 12, 24, and 48h after the exercise. The statistical analysis showed an increase in the mechanical allodynia at 24-48h after the exercise. Similarly, an increase on the protein carbonyl, xanthine oxidase activity, uric acid levels and TRPV1 protein content was found 12 h post exercise. These results suggest that there may be a relationship between purine-related ROS and the TRPV1 receptor activation on the events preceding the appearance of eccentric exerciserelated DOMS. These biochemical modulations may be the early trigger of exercise-related DOMS. Moreover, these data reinforces the hypothesis that purine-related ROS could be also an important source of ROS during exercise, which corroborates the current rationale that exercise-related free radical production is not restricted to mitochondrial sources. However, future studies are needed in order to determine the transition between
SFRBM 2015
S109