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Regeneration of activity for oxidized protein kinase C by thioredoxin reductase, a selenoprotein
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1
LOSS OF METAL ION BINDING SPECIFICITY IN MUTANT CUZNSOD ASSOCIATED WITH FALS Jov J. Goto’, James A. Roe2, Edith Butler GraIla’, Diane E. Cabelli”, and Joan Selverstone Valentine” ‘UCLA Department of Chemistry and Biochemistry. Los Angeles, California 90095-1569; 2Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, California, 90045; %hemistry Department Brookhaven National Laboratory, 90095-1569.
0F ACTIVITY FOR OXIDIZED PROTEIN KINASE c BY THIOREDOXN REDUCTASE, A SELENOPROTEIN
REGENERATION
Ravudu Gopalakrishna, and Usha Gundimeda Dept. of Cell & Neurobiol. USC Sch. of Med., Los Angeles, CA 90033 Although protein kinase C (PKC) is oxidatively modified in cells, this modification is readily reversible by an endogenous reductase. Thioredoxin reductase (TR) may be well qualified for this regulation. A calf tbymus TR system (purified TR, thioredoxin, NADPH) regenerated both the kinase activity and phorbol binding for PKC which was inactivated by sulfhydryl oxidation with benzoyl peroxide (BPO). Unexpectedly, TR induced this reduction to an appreciable extent even in the absence of thioredoxin. Nevertheless, thioredoxin was needed for the TR-mediated regeneration of kinase activity for the proteolytically derived catalytic domain of PKC, but was not required for the regeneration of the regulatory domain having four zincfinger thiolates. Furthermore, TR regenerated the phorbol binding for the oxidized recombinant fragment of PKC having two zinc-fingers. In the presence of NADP, TR acted as an oxidase and directly inactivated PKC. Furthermore, modified TR, in which selenocysteine was either selectively alkylated or removed by carboxypeptidase treatment, was weaker in inducing the redox modification of PKC. Similarly, E. coli TR, which is not a selenoprotein, was not effective. Conceivably, although PKC has no homology to thioredoxin, it has four zinc-fingers having the thioredoxintype redox motif (C-X-X-C) and a flexible protein-binding region in the regulatory domain, enabling it to directly bind and react with the seleno-sulfur redox center present in TR.
Copper-zinc superoxide dismutase (CuZnSOD) is a dimeric, type 2 copper protein found in the cytoplasm of most eukaryotic cells. In 1993, familial amyotrophic lateral sclerosis (FALS) was linked to single point mutations in the gene coding for CuZnSOD (sod]). ALS or Lou Gehrig’s disease involves progressive weakness of extremities, paralysis, and inevitable death in 2-5 years of onset. In an effort to identify the characteristic(s) of these mutant proteins, we have studied the kinetic and spectroscopic features of WT human CuZnSOD and the FALS mutant CuZnSODs A4V, L38V, and G93A using pulse radiolysis, W-vis absorption spectroscopy. and EPR spin-trapping. In contrast with the WT enzyme, the FALS mutants have lost the ability to segregate and bind copper and zinc ions in their proper location in vitro. Furthermore, the rates of deactivation, and therefore peroxidative ability, for the CuCu derivatives of both WT and mutant enzyme are greater than the as-isolated enzymes. The mutants also show differences in their peroxidative ability in in vitro and in-viva EPR spintrapping experiments. The FALS mutants and WT enzymes both have an abnormally low copper-to-zinc ratio when isolated from yeast presumably due to the interaction with the copper chaperone Lys7p (yeast CCS). Understanding these differences between FALS mutant CuZnSODs and WT CuZnSOD may be clues to help solve the puzzle behind SOD-associated FALS.
I 23 I
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PEROXIDASE ACTIVITY OF THE EXTRACELLULAR SUPEROXIDE DISMUTASE Ulrlch Tokru Fukni, Marin Wendt, Sampatk Partkasaratky, Dnmd Harrison, HOSpitld
Emory
University
Cnrdiology
Dimsion
and Veterans
this property
INVOLVED
x
Y
RESPONSE
A large family of mononuclear iron proteins, widespread among anaerobic archaea and bacteria, has been recently found to be involved in oxygen utilization and/or detoxification. The data so far gathered will be presented using the “strict” anaerobe sulfate reducing bacterium Desulfovibrio gigas as a prototype. The bacterium was found to be capable of surviving in the presence of oxygen, regenerating NAD+ through utilization of internal reserves of polyglucose: electrons are driven from NADH to oxygen through a soluble redox chain of three proteins: NADH: rubredoxin oxidoreductase (NRO), Rubredoxin (Rd), and Rubredoxin: oxygen oxido-reductase (ROO). Rd and ROO are located in the same polycistronic unit of D. gigas genome. ROO is a homodimer with 86 kDa, containing flavin, mesoheme M and Fe-uroporphyrin I. Neelaredoxin is a small iron proteins, containing a unique highspin iron center with a His&ys square pyramidal structure, responsible for its superoxide dismutase activity. The gene locci encoding for neelaredoxin includes also ORFs coding for chemosensory proteins. Finally, the same organism also contains a “classical” hemecontaining catalase and an Fe-SOD. (Supported by NIH and PRAXIS).
of ecSOD may
0
STRESS
Jean Le Gall’.‘, Ming-Yih Liu’, Wagner Santos’.*, M. Teixeira*, COlia Romb’, ClAudio Games*, and Ant6nio V. Xavie? ‘Dept. of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602. * Institute de Tecnologia Quimica e Biol6gia. Apt. 127, Rua da Quinta Grande 6,2780-156 Oeiras, Portugal
Administration
presence of hydrogen peroxide and nitrite produced a dose-dependent increase in dien formation from L-a-lecithin, reflecting lipid peroxidation (OD234 increments: background 0.06+0.01, with 2U ecSOD 0.15, and with 4U ecSOD 0.42?0.03). Thus, ecSOD exhibits not only superoxide dismutating activity, but also peroxidase-like activity. Since ecSOD exists in the interstitial space, where other hydrogen peroxide disposal systems are absent, it may serve as a major route for hydrogen peroxide metabolism. Because peroxidase
activity may be proatherogenic,
IN OXYGEN IN ANAEROBES
PROTEINS
Apart from its SOD activity the cytoplasmic copper zinc SOD (Cu/ZnSOD) exhibits peroxidase activity, utilizing hydrogen peroxide as a substrate to oxidize other molecules. The extracellular SOD (ecSOD) is also a Cu/ZnSOD and a major vascular form of SOD. We sought to determine if the ecSOD might exhibit peroxidase activity in a fashion similar to Cu/ZnSOD. Murine ecSOD was expressed in yeast and semi-purified using a Con A/sepharose column. The recombinant protein exhibited potent SOD activity. Peroxidase activity was examined using 3 approaches: Inhibition of SOD activity by hydrogen peroxide; detection of hydroxyl radical derived from the reaction of ecSOD with hydrogen peroxide; and oxidation of L-a-lecithin. After three hours incubation of 0.25 U/ml ecSOD with 1 mM hydrogen peroxide at 37C, ecSOD activity was inhibited by 72+10%. This effect was partially reversed by additon of 1 n&l nitrite, as observed for peroxidase activity of the Cu/ZnSOD. A characteristic 8 line ESR spectra typical for hydroxyl radical formation was detected after adding 10 mh4 hydrogen peroxide to 4 U/ml ecSOD and the spin trap DEPMPO. Finally, a 5 hour incubation of ecSOD in the
s20
I
G
E
N
’ 9
9
1
LOSS OF METAL ION BINDING SPECIFICITY IN MUTANT CUZNSOD ASSOCIATED WITH FALS Jov J. Goto’, James A. Roe2, Edith Butler GraIla’, Diane E. Cabelli”, and Joan Selverstone Valentine” ‘UCLA Department of Chemistry and Biochemistry. Los Angeles, California 90095-1569; 2Department of Chemistry and Biochemistry, Loyola Marymount University, Los Angeles, California, 90045; %hemistry Department Brookhaven National Laboratory, 90095-1569.
0F ACTIVITY FOR OXIDIZED PROTEIN KINASE c BY THIOREDOXN REDUCTASE, A SELENOPROTEIN
REGENERATION
Ravudu Gopalakrishna, and Usha Gundimeda Dept. of Cell & Neurobiol. USC Sch. of Med., Los Angeles, CA 90033 Although protein kinase C (PKC) is oxidatively modified in cells, this modification is readily reversible by an endogenous reductase. Thioredoxin reductase (TR) may be well qualified for this regulation. A calf tbymus TR system (purified TR, thioredoxin, NADPH) regenerated both the kinase activity and phorbol binding for PKC which was inactivated by sulfhydryl oxidation with benzoyl peroxide (BPO). Unexpectedly, TR induced this reduction to an appreciable extent even in the absence of thioredoxin. Nevertheless, thioredoxin was needed for the TR-mediated regeneration of kinase activity for the proteolytically derived catalytic domain of PKC, but was not required for the regeneration of the regulatory domain having four zincfinger thiolates. Furthermore, TR regenerated the phorbol binding for the oxidized recombinant fragment of PKC having two zinc-fingers. In the presence of NADP, TR acted as an oxidase and directly inactivated PKC. Furthermore, modified TR, in which selenocysteine was either selectively alkylated or removed by carboxypeptidase treatment, was weaker in inducing the redox modification of PKC. Similarly, E. coli TR, which is not a selenoprotein, was not effective. Conceivably, although PKC has no homology to thioredoxin, it has four zinc-fingers having the thioredoxintype redox motif (C-X-X-C) and a flexible protein-binding region in the regulatory domain, enabling it to directly bind and react with the seleno-sulfur redox center present in TR.
Copper-zinc superoxide dismutase (CuZnSOD) is a dimeric, type 2 copper protein found in the cytoplasm of most eukaryotic cells. In 1993, familial amyotrophic lateral sclerosis (FALS) was linked to single point mutations in the gene coding for CuZnSOD (sod]). ALS or Lou Gehrig’s disease involves progressive weakness of extremities, paralysis, and inevitable death in 2-5 years of onset. In an effort to identify the characteristic(s) of these mutant proteins, we have studied the kinetic and spectroscopic features of WT human CuZnSOD and the FALS mutant CuZnSODs A4V, L38V, and G93A using pulse radiolysis, W-vis absorption spectroscopy. and EPR spin-trapping. In contrast with the WT enzyme, the FALS mutants have lost the ability to segregate and bind copper and zinc ions in their proper location in vitro. Furthermore, the rates of deactivation, and therefore peroxidative ability, for the CuCu derivatives of both WT and mutant enzyme are greater than the as-isolated enzymes. The mutants also show differences in their peroxidative ability in in vitro and in-viva EPR spintrapping experiments. The FALS mutants and WT enzymes both have an abnormally low copper-to-zinc ratio when isolated from yeast presumably due to the interaction with the copper chaperone Lys7p (yeast CCS). Understanding these differences between FALS mutant CuZnSODs and WT CuZnSOD may be clues to help solve the puzzle behind SOD-associated FALS.
I 23 I
I 24
PEROXIDASE ACTIVITY OF THE EXTRACELLULAR SUPEROXIDE DISMUTASE Ulrlch Tokru Fukni, Marin Wendt, Sampatk Partkasaratky, Dnmd Harrison, HOSpitld
Emory
University
Cnrdiology
Dimsion
and Veterans
this property
INVOLVED
x
Y
RESPONSE
A large family of mononuclear iron proteins, widespread among anaerobic archaea and bacteria, has been recently found to be involved in oxygen utilization and/or detoxification. The data so far gathered will be presented using the “strict” anaerobe sulfate reducing bacterium Desulfovibrio gigas as a prototype. The bacterium was found to be capable of surviving in the presence of oxygen, regenerating NAD+ through utilization of internal reserves of polyglucose: electrons are driven from NADH to oxygen through a soluble redox chain of three proteins: NADH: rubredoxin oxidoreductase (NRO), Rubredoxin (Rd), and Rubredoxin: oxygen oxido-reductase (ROO). Rd and ROO are located in the same polycistronic unit of D. gigas genome. ROO is a homodimer with 86 kDa, containing flavin, mesoheme M and Fe-uroporphyrin I. Neelaredoxin is a small iron proteins, containing a unique highspin iron center with a His&ys square pyramidal structure, responsible for its superoxide dismutase activity. The gene locci encoding for neelaredoxin includes also ORFs coding for chemosensory proteins. Finally, the same organism also contains a “classical” hemecontaining catalase and an Fe-SOD. (Supported by NIH and PRAXIS).
of ecSOD may
0
STRESS
Jean Le Gall’.‘, Ming-Yih Liu’, Wagner Santos’.*, M. Teixeira*, COlia Romb’, ClAudio Games*, and Ant6nio V. Xavie? ‘Dept. of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602. * Institute de Tecnologia Quimica e Biol6gia. Apt. 127, Rua da Quinta Grande 6,2780-156 Oeiras, Portugal
Administration
presence of hydrogen peroxide and nitrite produced a dose-dependent increase in dien formation from L-a-lecithin, reflecting lipid peroxidation (OD234 increments: background 0.06+0.01, with 2U ecSOD 0.15, and with 4U ecSOD 0.42?0.03). Thus, ecSOD exhibits not only superoxide dismutating activity, but also peroxidase-like activity. Since ecSOD exists in the interstitial space, where other hydrogen peroxide disposal systems are absent, it may serve as a major route for hydrogen peroxide metabolism. Because peroxidase
activity may be proatherogenic,
IN OXYGEN IN ANAEROBES
PROTEINS
Apart from its SOD activity the cytoplasmic copper zinc SOD (Cu/ZnSOD) exhibits peroxidase activity, utilizing hydrogen peroxide as a substrate to oxidize other molecules. The extracellular SOD (ecSOD) is also a Cu/ZnSOD and a major vascular form of SOD. We sought to determine if the ecSOD might exhibit peroxidase activity in a fashion similar to Cu/ZnSOD. Murine ecSOD was expressed in yeast and semi-purified using a Con A/sepharose column. The recombinant protein exhibited potent SOD activity. Peroxidase activity was examined using 3 approaches: Inhibition of SOD activity by hydrogen peroxide; detection of hydroxyl radical derived from the reaction of ecSOD with hydrogen peroxide; and oxidation of L-a-lecithin. After three hours incubation of 0.25 U/ml ecSOD with 1 mM hydrogen peroxide at 37C, ecSOD activity was inhibited by 72+10%. This effect was partially reversed by additon of 1 n&l nitrite, as observed for peroxidase activity of the Cu/ZnSOD. A characteristic 8 line ESR spectra typical for hydroxyl radical formation was detected after adding 10 mh4 hydrogen peroxide to 4 U/ml ecSOD and the spin trap DEPMPO. Finally, a 5 hour incubation of ecSOD in the
s20
I
G
E
N
’ 9
9