Characterization of Changes in the Apo Form of Enzyme Cu, Zn Superoxide Dismutase Promoted by...
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Oxidation of Macromolecules 322 Characterization of Changes in the Apo Form of Enzyme Cu, Zn Superoxide Dismutase Promoted by Docosahexaenoic Acid and their Hydroperoxides Patricia P. Appolinário 1 , Danilo Medinas 1 , Thiago C. Genaro-Mattos 1 , Rafaella M. Kazaoka 1 , José R. R. Cussiol 1 , Luis E. S. Netto 1 , Ohara Augusto 1 , and Sayuri Miyamoto 1 1 Universidade de São Paulo, Brazil ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain, brainstem and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Recent studies show the involvement of unsaturated fatty acids in neurodegenerative diseases, such as ALS. Docosahexaenoic acid (DHA) is one of the main fatty acids in the cerebral gray matter. It is highly unsaturated, therefore susceptible to ROS oxidation forming DHAOOH as primary products. The aim of this study was to characterize the changes in the apo form of SOD1 promoted by DHA and its hydroperoxides that may be involved in fALS. SDS- PAGE analyses detected high-molecular weight species under non-reducing conditions when DHA is incubated with either apo- SOD1WT or G93A. DHAOOH had a minor effect compared to DHA, but presented a SOD1 covalent dimer. This dimer did not occur when SOD1 was incubated with DHA or DHAOH. CD analysis shows changes in the secondary structure of apo-SOD1 promoted by DHA resulting in an increase in the surface hydrophobicity, which was also confirmed by bis-ANS assay. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Size- exclusion chromatography indicates that aggregation is dependent on both fatty acid unsaturation and double bond cis- conformation. Aggregation is also dependent on Cys residues in their thiolate form, since at acidic pH aggregation was not observed. Experiments with C111S and C6S mutants of both apo forms of SOD1 revealed that apo-SOD1WT aggregation is dependent on both free cysteines (Cys 6 and 111), whereas in the apo-SOD1G93A the aggregation is mainly dependent on the Cys6. Overall, our results point towards a mechanism of SOD1 fALS mutant aggregation dependent on the interaction between cis-unsaturated fatty acids and thiolates form SOD1 free Cys residues. Supported by: FAPESP, CNPq, INCT de Processos Redox em Biomedicina (Redoxoma) and Pró-Reitoria de Pesquisa-USP. 323 Site-Specific Detection and Imaging of Free Radicals in DNA Induced by Cu(11)-H 2 O 2 Oxidizing System and Its Repair Using ESR, Immuno-Spin Trapping, Confocal Microscopy, LC-MS and MS/MS Suchandra Bhattacharjee 1 , Jinjie Jiang 1 , Saurabh Chatterjee 2 , Leesa Deterding 1 , Birandra Sinha 1 , and Ron Mason 1 1 NIEHS/NIH, 2 USC Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death and aging. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC- MS, HPLC-MS and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. Measurement of DNA damage has been a matter of debate as most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H 2 O 2 oxidizing system using immuno spin-trapping (IST) with EPR, MS and MS/MS and visualized by confocal microscopy. In this investigation the short-lived radical generated is trapped by the spin trap DMPO immediately upon formation. The DMPO adduct formed is initially EPR active, but is subsequently oxidized to the stable nitrone adduct, which can be detected and visualized by immuno-spin trapping and has the potential to be further characterized by other analytical techniques. ESR and MS/MS confirmed the radical to be located on the 2ƍ-deoxyadenosine (dAdo) moiety of DNA. The nitrone adduct was repaired on a time scale consistent with DNA repair. In vivo experiments for the purpose of detecting DMPO-DNA nitrone adducts should be conducted over a range of time in order to avoid missing adducts due to the repair processes. 324 Effect of Oxidation on Viral Protein Macrostructures Assemblies Ricardo M. Castro-Acosta 1 , Brenda Valderrama 1 , Octavio T. Ramírez 1 , and Laura A. Palomares 1 1 Biotechnology Institute UNAM, Mexico There are several modifications on proteins capable of producing negative effects in their function, as they reduce their stability and integrity. One of the most important is oxidation, which is principally caused by reactive oxygen species (ROS). Oxidation starts as proteins accumulate inside cells, and continues during downstream processing and storage. Protein oxidation has been extensively studied, but the effect of oxidation in multimeric protein assemblies, such as virus-like particles (VLP), has not. In this work, rotavirus VP6 was used as a model to investigate the effect of ROS on self-assembling proteins and determine if ROS affect protein assembly and assembled macrostructures. Rotavirus VP6 self-assembles into nanotubes of 50-75 nm of diameter with micrometers of length or icosahedra of ~50 nm. VP6 was oxidized with H 2 O 2 and Ɣ OH at concentrations from 50 to 10,000 μM. Oxidation with H 2 O 2 did not produce alterations in the structure or stability of VP6. However, Ɣ OH at concentrations above 5 mM severely damaged VP6 monomers (VP6 U ) but not VP6 nanotubes (VP6 NT ), as evidenced by SDS-PAGE. Carbonyl concentration increased exponentially during the oxidation of both VP6 configurations. Carbonyl appearance was faster during the oxidation of VP6u. Oxidation reduced the length of VP6NT, as determined by dynamic light scattering. Interestingly, oxidation reduced VP6 fluorescence (λ ex 280/ λ em 350 nm) and provoked a change in the spectral center of mass, probably due to the oxidation of aromatic amino acids or a change in the protein tertiary structure. Oxidized VP6u was capable of self-assembling into VP6 NT , but the assembly efficiency was low. It was shown that assembled VP6 is less susceptible to oxidation than the monomeric protein. However, oxidation did affect protein assemblies and reduced the self-assembling ability of VP6. To our knowledge, this is the first study of the effect of ROS in viral proteins and their assembly capacity. The results obtained here can be extrapolated to other macromolecular protein assemblies with application as vaccines, gene therapy vectors or SFRBM 2012 S133 doi:10.1016/j.freeradbiomed.2012.10.360 doi:10.1016/j.freeradbiomed.2012.10.361
Transcript of Characterization of Changes in the Apo Form of Enzyme Cu, Zn Superoxide Dismutase Promoted by...
Oxidation of Macromolecules 322 Characterization of Changes in the Apo Form of Enzyme Cu, Zn Superoxide Dismutase Promoted by Docosahexaenoic Acid and their Hydroperoxides Patricia P. Appolinário1, Danilo Medinas1, Thiago C. Genaro-Mattos1, Rafaella M. Kazaoka1, José R. R. Cussiol1, Luis E. S. Netto1, Ohara Augusto1, and Sayuri Miyamoto1 1Universidade de São Paulo, Brazil ALS is a progressive and fatal disease caused by selective degeneration of motor neurons in the brain, brainstem and spinal cord. Twenty percent of familial ALS (fALS) cases are caused mainly by point mutations in the sod1 gene. Recent studies show the involvement of unsaturated fatty acids in neurodegenerative diseases, such as ALS. Docosahexaenoic acid (DHA) is one of the main fatty acids in the cerebral gray matter. It is highly unsaturated, therefore susceptible to ROS oxidation forming DHAOOH as primary products. The aim of this study was to characterize the changes in the apo form of SOD1 promoted by DHA and its hydroperoxides that may be involved in fALS. SDS-PAGE analyses detected high-molecular weight species under non-reducing conditions when DHA is incubated with either apo-SOD1WT or G93A. DHAOOH had a minor effect compared to DHA, but presented a SOD1 covalent dimer. This dimer did not occur when SOD1 was incubated with DHA or DHAOH. CD analysis shows changes in the secondary structure of apo-SOD1 promoted by DHA resulting in an increase in the surface hydrophobicity, which was also confirmed by bis-ANS assay. These changes enhance the interaction of SOD1 and DHA, leading to amorphous aggregates as revealed by FESEM. Size-exclusion chromatography indicates that aggregation is dependent on both fatty acid unsaturation and double bond cis-conformation. Aggregation is also dependent on Cys residues in their thiolate form, since at acidic pH aggregation was not observed. Experiments with C111S and C6S mutants of both apo forms of SOD1 revealed that apo-SOD1WT aggregation is dependent on both free cysteines (Cys 6 and 111), whereas in the apo-SOD1G93A the aggregation is mainly dependent on the Cys6. Overall, our results point towards a mechanism of SOD1 fALS mutant aggregation dependent on the interaction between cis-unsaturated fatty acids and thiolates form SOD1 free Cys residues. Supported by: FAPESP, CNPq, INCT de Processos Redox em Biomedicina (Redoxoma) and Pró-Reitoria de Pesquisa-USP.
323 Site-Specific Detection and Imaging of Free Radicals in DNA Induced by Cu(11)-H2O2 Oxidizing System and Its Repair Using ESR, Immuno-Spin Trapping, Confocal Microscopy, LC-MS and MS/MS Suchandra Bhattacharjee1, Jinjie Jiang1, Saurabh Chatterjee2, Leesa Deterding1, Birandra Sinha1, and Ron Mason1 1NIEHS/NIH, 2USC Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death and aging. To understand damage to DNA, it is important to study the free radical reactions causing the damage. Many of these lesions have
been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. Measurement of DNA damage has been a matter of debate as most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)-H2O2 oxidizing system using immuno spin-trapping (IST) with EPR, MS and MS/MS and visualized by confocal microscopy. In this investigation the short-lived radical generated is trapped by the spin trap DMPO immediately upon formation. The DMPO adduct formed is initially EPR active, but is subsequently oxidized to the stable nitrone adduct, which can be detected and visualized by immuno-spin trapping and has the potential to be further characterized by other analytical techniques. ESR and MS/MS confirmed the radical to be located on the 2 -deoxyadenosine (dAdo) moiety of DNA. The nitrone adduct was repaired on a time scale consistent with DNA repair. In vivo experiments for the purpose of detecting DMPO-DNA nitrone adducts should be conducted over a range of time in order to avoid missing adducts due to the repair processes.
324 Effect of Oxidation on Viral Protein Macrostructures Assemblies Ricardo M. Castro-Acosta1, Brenda Valderrama1, Octavio T. Ramírez1, and Laura A. Palomares1 1Biotechnology Institute UNAM, Mexico There are several modifications on proteins capable of producing negative effects in their function, as they reduce their stability and integrity. One of the most important is oxidation, which is principally caused by reactive oxygen species (ROS). Oxidation starts as proteins accumulate inside cells, and continues during downstream processing and storage. Protein oxidation has been extensively studied, but the effect of oxidation in multimeric protein assemblies, such as virus-like particles (VLP), has not. In this work, rotavirus VP6 was used as a model to investigate the effect of ROS on self-assembling proteins and determine if ROS affect protein assembly and assembled macrostructures. Rotavirus VP6 self-assembles into nanotubes of 50-75 nm of diameter with micrometers of length or icosahedra of ~50 nm. VP6 was oxidized with H2O2 and OH at concentrations from 50 to 10,000 μM. Oxidation with H2O2 did not produce alterations in the structure or stability of VP6. However, OH at concentrations above 5 mM severely damaged VP6 monomers (VP6U) but not VP6 nanotubes (VP6NT), as evidenced by SDS-PAGE. Carbonyl concentration increased exponentially during the oxidation of both VP6 configurations. Carbonyl appearance was faster during the oxidation of VP6u. Oxidation reduced the length of VP6NT, as determined by dynamic light scattering. Interestingly, oxidation reduced VP6 fluorescence (λex280/ λem350 nm) and provoked a change in the spectral center of mass, probably due to the oxidation of aromatic amino acids or a change in the protein tertiary structure. Oxidized VP6u was capable of self-assembling into VP6NT, but the assembly efficiency was low. It was shown that assembled VP6 is less susceptible to oxidation than the monomeric protein. However, oxidation did affect protein assemblies and reduced the self-assembling ability of VP6. To our knowledge, this is the first study of the effect of ROS in viral proteins and their assembly capacity. The results obtained here can be extrapolated to other macromolecular protein assemblies with application as vaccines, gene therapy vectors or
SFRBM 2012 S133
doi:10.1016/j.freeradbiomed.2012.10.360
doi:10.1016/j.freeradbiomed.2012.10.361
