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Peroxynitrite Formation from Re-Oxidation of Zinc-Deficient Superoxide Dismutase: Implications in Amyotrophic Lateral Sclerosis
on ROS accumulation for 24 and 48 hours of incubation, while the congener BDE-209 was only able to affect ROS accumulation after 48 hours of exposure, in all tested concentrations. Our results demonstrate that PBDEs induces ROS accumulations and mitochondrial depolarization, and it was also observed an influence of the bromination pattern on the final effect.
doi:10.1016/j.freeradbiomed.2012.10.043
17 Peroxynitrite Formation from Re-Oxidation of ZincDeficient Superoxide Dismutase: Implications in Amyotrophic Lateral Sclerosis 1
Derek A Drechsel , Jacek Zielonka2, Balaraman Kalyanaraman2, and Joseph Beckman1 1 2 Oregon State University, Medical College of Wisconsin Mutations to copper zinc superoxide dismutase (Cu,Zn-SOD) are found in approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS). Studies in transgenic mice established that ALS-associated mutations of Cu,Zn-SOD confer a toxic gain-offunction. We have previously shown that loss of zinc results in the copper-containing SOD becoming toxic to motor neurons through a mechanism requiring nitric oxide (NO). Zinc-deficient SOD is readily reduced by ascorbate and its slow re-oxidation in the presence of NO generated peroxynitrite as indirectly measured by dihydrodicholorofluorescein (DCDHF (A Estevez, et al. 1999). However, DCDHF only detects radical decomposition products Ɣ Ɣ ( NO2, HO ) from peroxynitrite and could potentially be oxidized via different mechanisms. Recently developed boronatefluorogenic probes are oxidized directly and specifically by peroxynitrite anion (J Zielonka, et al. 2010) when catalase is present to remove hydrogen peroxide. Using boronate probes, we demonstrated the production of peroxynitrite from re-oxidation of 1+ reduced (Cu ) zinc-deficient SOD by oxygen in the presence of NO at the rate of 0.75 ± 0.40 μmol per μmol SOD per h. A 40-fold excess of wild-type Cu,Zn-SOD only slightly reduced peroxynitrite production from zinc-deficient SOD. With steady-state superoxide concentrations in the picomolar range in the cytoplasm of motor neurons, these data suggest that re-oxidation of SOD can be a relevant source of superoxide formation to react with NO even in the presence of high concentrations Cu,Zn-SOD. This may contribute to the dominant gain-of-function observed with SOD mutations. Using mass spectrometry, we are measuring 0.5 to 1 μmolar zinc-deficient SOD in ventral spinal cord of transgenic mouse models of ALS. These results show that such concentrations of zinc-deficient SOD can generate sufficient peroxynitrite to induce death in cultured motor neurons over a 2448 h period.
doi:10.1016/j.freeradbiomed.2012.10.044
18 Cytochrome C Modifications Promoted by Cholesterol Aldehydes – Implications to Electron Transport and Apoptosis 1
Thiago C. Genaro-Mattos , Patricia P. Appolinário1, Iseli L. Nantes2, Paolo Di Mascio1, and Sayuri Miyamoto1 1 2 Universidade de São Paulo, Brazil, Universidade Federal do ABC, Brazil. Cholesterol is a lipid poorly found in the mitochondria under normal conditions. Under certain pathological conditions, on the other hand, its levels have been reported elevated, which
S20
increased cells resistance in triggering apoptosis. Under these circumstances, excessive amounts of ROS are known to be produced, increasing lipid peroxidation and generating reactive aldehydes. Lipid-derived aldehydes are known to react with proteins leading to conformational changes, malfunction and diseases development. Among them, cholesterol carboxyaldehyde (ChAld) is of major interest, since it was found increased in atherosclerotic plaques and brain tissue of patients with neurodegenerative diseases. Therefore, we aimed to characterize the mechanisms of the reaction between ChAld and cytochrome c (cytc), a heme-protein involved in the electrons transport and in the apoptosis signaling. In order to mimic biological membranes, incubations were conducted in the presence of SDS micelles or liposomes. MALDI-TOF MS showed up to 6 covalent additions of ChAld to cytc, each addition occurring with a loss of one water molecule. This type of addition is consistent with Schiff base formations between the aldehyde and amino groups of Lys and Arg residues. Cytc incubated with ChAld in the absence of SDS micelles had its structure altered and underwent aggregation. This behavior was not observed in the presence of micelles. Tryptic digestions of the modified protein revealed K8 and K22 as main modified sites, being K22 a residue assumed to participate in protein-membrane interactions. In this sense, ultracentrifugation analyses showed that, upon incubation with ChAld containing liposomes, cytc binding to the membrane was improved and was not disrupted by increasing the ionic strength. Overall, our results suggest that ChAld modifies cytc at important sites with possible key biological implications. For instance, these modifications could prevent cytc movement along the membrane and its release to the cytosol, influencing, therefore, the electrons transport and the apoptosis signaling. Supported by: FAPESP, INCT de Processos Redox em Biomedicina-Redoxoma and CNPq.
doi:10.1016/j.freeradbiomed.2012.10.045
19 Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1Į Deficiency Accelerates Endothelial Dysfunction during Chronic Angiotensin II Treatment by Increasing Mitochondrial Oxidative Stress and Vascular Aging 1
Jasmin Ghaemi Kerahrodi , Swenja Kröller-Schön1, Anna Gottschlich1, Thomas Jansen1, Matthias Oelze1, Philip Wenzel1, Michael Hausding1, Maike Beisele1, Andreas Daiber1, Thomas Münzel1, and Eberhard Schulz1 1 Universitätsmedizin Mainz, Germany Objective: Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is an important mediator of mitochondrial biogenesis and function. We have previously shown that the AMP-activated protein kinase (AMPK) protects endothelial cells against oxidative stress by preservation of mitochondrial function in a PGC-1α dependent manner. Since dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, the current study was designed to investigate the effects of in vivo PGC-1α deficiency during chronic angiotensin II (ATII) treatment. Methods and Results: Deletion of PGC-1α had no effect on endothelial function or mitochondrial mass in aortic tissue under basal conditions. However, chronic ATII infusion at subpressor doses (0.1mg/kg/d) resulted in mild endothelial dysfunction, which was significantly aggravated in PGC-1α knockout mice. In parallel, oxidative stress was increased in aortic rings from ATIItreated PGC-1α knockout mice while serum antioxidative capacity was decreased. By using the mitochondrial specific superoxide
SFRBM 2012
doi:10.1016/j.freeradbiomed.2012.10.043
17 Peroxynitrite Formation from Re-Oxidation of ZincDeficient Superoxide Dismutase: Implications in Amyotrophic Lateral Sclerosis 1
Derek A Drechsel , Jacek Zielonka2, Balaraman Kalyanaraman2, and Joseph Beckman1 1 2 Oregon State University, Medical College of Wisconsin Mutations to copper zinc superoxide dismutase (Cu,Zn-SOD) are found in approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS). Studies in transgenic mice established that ALS-associated mutations of Cu,Zn-SOD confer a toxic gain-offunction. We have previously shown that loss of zinc results in the copper-containing SOD becoming toxic to motor neurons through a mechanism requiring nitric oxide (NO). Zinc-deficient SOD is readily reduced by ascorbate and its slow re-oxidation in the presence of NO generated peroxynitrite as indirectly measured by dihydrodicholorofluorescein (DCDHF (A Estevez, et al. 1999). However, DCDHF only detects radical decomposition products Ɣ Ɣ ( NO2, HO ) from peroxynitrite and could potentially be oxidized via different mechanisms. Recently developed boronatefluorogenic probes are oxidized directly and specifically by peroxynitrite anion (J Zielonka, et al. 2010) when catalase is present to remove hydrogen peroxide. Using boronate probes, we demonstrated the production of peroxynitrite from re-oxidation of 1+ reduced (Cu ) zinc-deficient SOD by oxygen in the presence of NO at the rate of 0.75 ± 0.40 μmol per μmol SOD per h. A 40-fold excess of wild-type Cu,Zn-SOD only slightly reduced peroxynitrite production from zinc-deficient SOD. With steady-state superoxide concentrations in the picomolar range in the cytoplasm of motor neurons, these data suggest that re-oxidation of SOD can be a relevant source of superoxide formation to react with NO even in the presence of high concentrations Cu,Zn-SOD. This may contribute to the dominant gain-of-function observed with SOD mutations. Using mass spectrometry, we are measuring 0.5 to 1 μmolar zinc-deficient SOD in ventral spinal cord of transgenic mouse models of ALS. These results show that such concentrations of zinc-deficient SOD can generate sufficient peroxynitrite to induce death in cultured motor neurons over a 2448 h period.
doi:10.1016/j.freeradbiomed.2012.10.044
18 Cytochrome C Modifications Promoted by Cholesterol Aldehydes – Implications to Electron Transport and Apoptosis 1
Thiago C. Genaro-Mattos , Patricia P. Appolinário1, Iseli L. Nantes2, Paolo Di Mascio1, and Sayuri Miyamoto1 1 2 Universidade de São Paulo, Brazil, Universidade Federal do ABC, Brazil. Cholesterol is a lipid poorly found in the mitochondria under normal conditions. Under certain pathological conditions, on the other hand, its levels have been reported elevated, which
S20
increased cells resistance in triggering apoptosis. Under these circumstances, excessive amounts of ROS are known to be produced, increasing lipid peroxidation and generating reactive aldehydes. Lipid-derived aldehydes are known to react with proteins leading to conformational changes, malfunction and diseases development. Among them, cholesterol carboxyaldehyde (ChAld) is of major interest, since it was found increased in atherosclerotic plaques and brain tissue of patients with neurodegenerative diseases. Therefore, we aimed to characterize the mechanisms of the reaction between ChAld and cytochrome c (cytc), a heme-protein involved in the electrons transport and in the apoptosis signaling. In order to mimic biological membranes, incubations were conducted in the presence of SDS micelles or liposomes. MALDI-TOF MS showed up to 6 covalent additions of ChAld to cytc, each addition occurring with a loss of one water molecule. This type of addition is consistent with Schiff base formations between the aldehyde and amino groups of Lys and Arg residues. Cytc incubated with ChAld in the absence of SDS micelles had its structure altered and underwent aggregation. This behavior was not observed in the presence of micelles. Tryptic digestions of the modified protein revealed K8 and K22 as main modified sites, being K22 a residue assumed to participate in protein-membrane interactions. In this sense, ultracentrifugation analyses showed that, upon incubation with ChAld containing liposomes, cytc binding to the membrane was improved and was not disrupted by increasing the ionic strength. Overall, our results suggest that ChAld modifies cytc at important sites with possible key biological implications. For instance, these modifications could prevent cytc movement along the membrane and its release to the cytosol, influencing, therefore, the electrons transport and the apoptosis signaling. Supported by: FAPESP, INCT de Processos Redox em Biomedicina-Redoxoma and CNPq.
doi:10.1016/j.freeradbiomed.2012.10.045
19 Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1Į Deficiency Accelerates Endothelial Dysfunction during Chronic Angiotensin II Treatment by Increasing Mitochondrial Oxidative Stress and Vascular Aging 1
Jasmin Ghaemi Kerahrodi , Swenja Kröller-Schön1, Anna Gottschlich1, Thomas Jansen1, Matthias Oelze1, Philip Wenzel1, Michael Hausding1, Maike Beisele1, Andreas Daiber1, Thomas Münzel1, and Eberhard Schulz1 1 Universitätsmedizin Mainz, Germany Objective: Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) is an important mediator of mitochondrial biogenesis and function. We have previously shown that the AMP-activated protein kinase (AMPK) protects endothelial cells against oxidative stress by preservation of mitochondrial function in a PGC-1α dependent manner. Since dysfunctional mitochondria might be involved in the pathogenesis of vascular disease, the current study was designed to investigate the effects of in vivo PGC-1α deficiency during chronic angiotensin II (ATII) treatment. Methods and Results: Deletion of PGC-1α had no effect on endothelial function or mitochondrial mass in aortic tissue under basal conditions. However, chronic ATII infusion at subpressor doses (0.1mg/kg/d) resulted in mild endothelial dysfunction, which was significantly aggravated in PGC-1α knockout mice. In parallel, oxidative stress was increased in aortic rings from ATIItreated PGC-1α knockout mice while serum antioxidative capacity was decreased. By using the mitochondrial specific superoxide
SFRBM 2012