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Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc.

Gertz M, Fischer F, Leipelt M, Wolters D, Steegborn C - Aging (Albany NY) (2009)

Bottom Line: Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain.The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture.Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, 44801 Bochum, Germany.

ABSTRACT
Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

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Functional characterization of the p66Shc/Prx1 interaction. (a) The                                            dimer is the peroxidase-active form of Prx1. Changes in fluorescence of 10                                            μM H2DFFDA were recorded after addition of 0.005 % H2O2                                            in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser                                            and Prx1-Cys83Ser. (b) p66CH2CB has an additional copper-independent                                            activity which is inhibited by Prx1. Changes in fluorescence of 10 μM H2DFFDA                                            were recorded after addition of 20 μM p66CH2CB and 0.005 % H2O2                                            in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser                                            and Prx1-Cys83Ser. (c) p66CH2CB and Prx1 perform a disulfide                                            exchange reaction with each other. 10 μg p66CH2CB were incubated with Prx1                                            for 1 hour at room temperature and subjected to non-reducing SDS-PAGE.                                            Reduced p66CH2CB is formed, and the reduced Prx1 is concurrently oxidized. (d)                                            Trx1 does not prevent formation of the major p66CH2CB/Prx1 complex,                                            indicating separate binding sites for Prx1 and Trx1. 15 μg p66CH2CB were                                            incubated with 30 μg decameric Prx1-Cys52Ser and different amounts of Trx1                                            (5/10/20 μg) in the presence of 3 mM EDTA and subjected to BN-PAGE.
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Figure 3: Functional characterization of the p66Shc/Prx1 interaction. (a) The dimer is the peroxidase-active form of Prx1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 0.005 % H2O2 in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (b) p66CH2CB has an additional copper-independent activity which is inhibited by Prx1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 20 μM p66CH2CB and 0.005 % H2O2 in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (c) p66CH2CB and Prx1 perform a disulfide exchange reaction with each other. 10 μg p66CH2CB were incubated with Prx1 for 1 hour at room temperature and subjected to non-reducing SDS-PAGE. Reduced p66CH2CB is formed, and the reduced Prx1 is concurrently oxidized. (d) Trx1 does not prevent formation of the major p66CH2CB/Prx1 complex, indicating separate binding sites for Prx1 and Trx1. 15 μg p66CH2CB were incubated with 30 μg decameric Prx1-Cys52Ser and different amounts of Trx1 (5/10/20 μg) in the presence of 3 mM EDTA and subjected to BN-PAGE.

Mentions: p66Shc is known to generate H2O2 whereas peroxiredoxins exhibit weak but significant peroxidase activity. We therefore hypothized that Prx1 might degrade p66Shc-generated H2O2 and thereby inhibit p66Shc-dependent apoptosis. The other way round, p66Shc might inactivate Prx1 so that formed H2O2 does not get inactivated. In order to test this hypothesis, we analyzed the Prx1 forms in degrading H2O2 in a fluorescence-based ROS assay. While Prx1 WT decreased the fluorescence signal of 0.005 % H2O2 only slightly, the dimeric Prx1-Cys83Ser mutant significantly degraded H2O2 (Figure 3a). This result is consistent with published findings that dimeric Prx1 exhibits significant peroxidase activity, while the decameric form acts as a chaperone with decreased peroxidase activity [36,37]. As a control we generated a peroxidase-inactive Prx1 mutant by exchanging the peroxidasic cysteine residue Cys52 against Ser. As expected, this inactive mutant Prx1-Cys52Ser had no effect in the fluorescence-based ROS assay (Figure 3a).


Identification of Peroxiredoxin 1 as a novel interaction partner for the lifespan regulator protein p66Shc.

Gertz M, Fischer F, Leipelt M, Wolters D, Steegborn C - Aging (Albany NY) (2009)

Functional characterization of the p66Shc/Prx1 interaction. (a) The                                            dimer is the peroxidase-active form of Prx1. Changes in fluorescence of 10                                            μM H2DFFDA were recorded after addition of 0.005 % H2O2                                            in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser                                            and Prx1-Cys83Ser. (b) p66CH2CB has an additional copper-independent                                            activity which is inhibited by Prx1. Changes in fluorescence of 10 μM H2DFFDA                                            were recorded after addition of 20 μM p66CH2CB and 0.005 % H2O2                                            in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser                                            and Prx1-Cys83Ser. (c) p66CH2CB and Prx1 perform a disulfide                                            exchange reaction with each other. 10 μg p66CH2CB were incubated with Prx1                                            for 1 hour at room temperature and subjected to non-reducing SDS-PAGE.                                            Reduced p66CH2CB is formed, and the reduced Prx1 is concurrently oxidized. (d)                                            Trx1 does not prevent formation of the major p66CH2CB/Prx1 complex,                                            indicating separate binding sites for Prx1 and Trx1. 15 μg p66CH2CB were                                            incubated with 30 μg decameric Prx1-Cys52Ser and different amounts of Trx1                                            (5/10/20 μg) in the presence of 3 mM EDTA and subjected to BN-PAGE.
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Figure 3: Functional characterization of the p66Shc/Prx1 interaction. (a) The dimer is the peroxidase-active form of Prx1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 0.005 % H2O2 in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (b) p66CH2CB has an additional copper-independent activity which is inhibited by Prx1. Changes in fluorescence of 10 μM H2DFFDA were recorded after addition of 20 μM p66CH2CB and 0.005 % H2O2 in the absence and presence of 20 μM Prx1 WT or the mutants Prx1-Cys52Ser and Prx1-Cys83Ser. (c) p66CH2CB and Prx1 perform a disulfide exchange reaction with each other. 10 μg p66CH2CB were incubated with Prx1 for 1 hour at room temperature and subjected to non-reducing SDS-PAGE. Reduced p66CH2CB is formed, and the reduced Prx1 is concurrently oxidized. (d) Trx1 does not prevent formation of the major p66CH2CB/Prx1 complex, indicating separate binding sites for Prx1 and Trx1. 15 μg p66CH2CB were incubated with 30 μg decameric Prx1-Cys52Ser and different amounts of Trx1 (5/10/20 μg) in the presence of 3 mM EDTA and subjected to BN-PAGE.
Mentions: p66Shc is known to generate H2O2 whereas peroxiredoxins exhibit weak but significant peroxidase activity. We therefore hypothized that Prx1 might degrade p66Shc-generated H2O2 and thereby inhibit p66Shc-dependent apoptosis. The other way round, p66Shc might inactivate Prx1 so that formed H2O2 does not get inactivated. In order to test this hypothesis, we analyzed the Prx1 forms in degrading H2O2 in a fluorescence-based ROS assay. While Prx1 WT decreased the fluorescence signal of 0.005 % H2O2 only slightly, the dimeric Prx1-Cys83Ser mutant significantly degraded H2O2 (Figure 3a). This result is consistent with published findings that dimeric Prx1 exhibits significant peroxidase activity, while the decameric form acts as a chaperone with decreased peroxidase activity [36,37]. As a control we generated a peroxidase-inactive Prx1 mutant by exchanging the peroxidasic cysteine residue Cys52 against Ser. As expected, this inactive mutant Prx1-Cys52Ser had no effect in the fluorescence-based ROS assay (Figure 3a).

Bottom Line: Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain.The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture.Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Chemistry, Ruhr-University Bochum, 44801 Bochum, Germany.

ABSTRACT
Damage caused by reactive oxygen species (ROS) contributes to many aging processes and accompanying diseases. ROS are toxic side products of cellular respiration, but also function as signal, e.g. in the mitochondrial apoptosis pathway. The protein p66Shc, which has been implicated in life-span regulation and aging-related diseases, is a central player in stress-induced apoptosis and the associated ROS burst. Stress signals, such as UV radiation or ROS themselves, activate p66Shc, which was proposed to stimulate its H(2)O(2) forming activity, ultimately triggering mitochondrial disintegration. However, mechanistic details of H(2)O(2) formation and apoptosis induction by p66Shc and regulation of these activities remain to be revealed. Here, we describe the effects of Ser36 phosphorylation and Pin1 binding on p66Shc activity, and the identification of Peroxiredoxin 1 (Prx1) as a novel interaction partner for the unique p66Shc N-terminal domain. Prx1 was identified in affinity experiments as dominant interaction partner. Complex formation leads to disassembly of Prx1 decamers, which is known to increase its peroxidase activity. The interaction leads to reduction of the p66CH2CB tetramer, which reduces its ability to induce mitochondrial rupture. Our results indicate that p66CH2CB and Prx1 form a stress-sensing complex that keeps p66Shc inactive at moderate stress levels.

Show MeSH
Related in: MedlinePlus