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Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

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Reconstitution with phosphorylation resistant CcO Vb subunit attenuates hypoxia induced CcO dysfunction and ROS production.siRNA resistant (SiR) - wild type (WT) or S40A mutant CcO Vb were expressed in RAW 264.7 macrophages with CcO Vb knockdown (VbKD). A) Immunoblot of cell lysates showing CcO Vb levels in control, CcO Vb knockdown (VbKD) and si-RNA resistant CcO Vb expressing VbKD cells. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. Blots were stained with CcO Vb and SDHA antibodies. B) Blue Native PAGE performed with mitochondrial proteins from all cell types. 150µg of mitochondria from each sample was solubilized with Lauryl maltoside as described in Materials and Methods. Electrophoresis was carried out in a 6-13% gradient gel. Gel was destained to remove excess Coomassie stain and the bands were imaged in a scanner. C&D) Control, VbKD and SiR-wild type and S40A mutant cells were maintained under either normoxia or hypoxia. Mitochondria were isolated and proteins (25µg each) were used to measure CcO activity (C) and ROS production by Amplex red oxidation (D). n=4. **, p<0.005; ***, p<0.001.
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pone-0077129-g005: Reconstitution with phosphorylation resistant CcO Vb subunit attenuates hypoxia induced CcO dysfunction and ROS production.siRNA resistant (SiR) - wild type (WT) or S40A mutant CcO Vb were expressed in RAW 264.7 macrophages with CcO Vb knockdown (VbKD). A) Immunoblot of cell lysates showing CcO Vb levels in control, CcO Vb knockdown (VbKD) and si-RNA resistant CcO Vb expressing VbKD cells. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. Blots were stained with CcO Vb and SDHA antibodies. B) Blue Native PAGE performed with mitochondrial proteins from all cell types. 150µg of mitochondria from each sample was solubilized with Lauryl maltoside as described in Materials and Methods. Electrophoresis was carried out in a 6-13% gradient gel. Gel was destained to remove excess Coomassie stain and the bands were imaged in a scanner. C&D) Control, VbKD and SiR-wild type and S40A mutant cells were maintained under either normoxia or hypoxia. Mitochondria were isolated and proteins (25µg each) were used to measure CcO activity (C) and ROS production by Amplex red oxidation (D). n=4. **, p<0.005; ***, p<0.001.

Mentions: Our previous studies showed that under both hypoxia and alcohol toxicity CcO subunit Vb was one of the three subunits reduced as a function of time [25,46]. To assess the role of phosphorylation on the selective degradation of Vb, we first generated a macrophage cell line stably expressing siRNA against CcO subunit Vb (VbKD). We then expressed either wild type (WT CcO Vb) or phosphorylation resistant mutant (S40A-CcO Vb) of the subunit in the VbKD cells. The CcO Vb cDNA contained degenerate codons in the siRNA target region to prevent siRNA mediated degradation in VbKD cells. Immunoblot in Figure 5A shows that the level of subunit Vb was markedly reduced in siRNA expressing cells (VbKD), which was restored in cells expressing the siRNA resistant wild type or S40A mutant cDNA. Figure 5B shows the Blue Native gel profiles of control, Vb knocked down cells and cells reconstituted with WT or S40A mutant CcO Vb subunits. It is seen that CcO complex was markedly reduced in Vb knocked down cells which was restored in cells reconstituted with WT or S40A mutant CcO Vb subunits. The effect of hypoxia on CcO activities of mitochondrial preparations from these cells is presented in Figure 5C. The CcO activity was reduced by about 45% in control cells exposed to hypoxia. In Vb knocked down cells, the basal activity was reduced by more than 95%. Reconstitution with either wild type or S40A mutant CcO Vb nearly completely restores CcO activity in these cells. Notably, in cells reconstituted with WT subunit Vb, hypoxia reduced CcO activity by 42%, while cells reconstituted with phosphorylation resistant S40A subunit showed only 21% loss of CcO activity under hypoxia (Figure 5C). It is also seen that under hypoxic conditions, the ROS production in cells reconstituted with WT CcO Vb was similar to the control cells. In cells reconstituted with S40A mutant subunit, however, there was a significant decrease in ROS production which was constituent with the extent of resistance to hypoxia induced subunit degradation (Figure 5D). These results suggest that the S40A mutant protein is relatively more resistant to hypoxia mediated damage and support our hypothesis that phosphorylation of subunits is responsible for hypoxia mediated loss of CcO activity.


Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.

Srinivasan S, Spear J, Chandran K, Joseph J, Kalyanaraman B, Avadhani NG - PLoS ONE (2013)

Reconstitution with phosphorylation resistant CcO Vb subunit attenuates hypoxia induced CcO dysfunction and ROS production.siRNA resistant (SiR) - wild type (WT) or S40A mutant CcO Vb were expressed in RAW 264.7 macrophages with CcO Vb knockdown (VbKD). A) Immunoblot of cell lysates showing CcO Vb levels in control, CcO Vb knockdown (VbKD) and si-RNA resistant CcO Vb expressing VbKD cells. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. Blots were stained with CcO Vb and SDHA antibodies. B) Blue Native PAGE performed with mitochondrial proteins from all cell types. 150µg of mitochondria from each sample was solubilized with Lauryl maltoside as described in Materials and Methods. Electrophoresis was carried out in a 6-13% gradient gel. Gel was destained to remove excess Coomassie stain and the bands were imaged in a scanner. C&D) Control, VbKD and SiR-wild type and S40A mutant cells were maintained under either normoxia or hypoxia. Mitochondria were isolated and proteins (25µg each) were used to measure CcO activity (C) and ROS production by Amplex red oxidation (D). n=4. **, p<0.005; ***, p<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795003&req=5

pone-0077129-g005: Reconstitution with phosphorylation resistant CcO Vb subunit attenuates hypoxia induced CcO dysfunction and ROS production.siRNA resistant (SiR) - wild type (WT) or S40A mutant CcO Vb were expressed in RAW 264.7 macrophages with CcO Vb knockdown (VbKD). A) Immunoblot of cell lysates showing CcO Vb levels in control, CcO Vb knockdown (VbKD) and si-RNA resistant CcO Vb expressing VbKD cells. 30µg of mitochondrial protein was separated on SDS PAGE and transferred to nitrocellulose membrane. Blots were stained with CcO Vb and SDHA antibodies. B) Blue Native PAGE performed with mitochondrial proteins from all cell types. 150µg of mitochondria from each sample was solubilized with Lauryl maltoside as described in Materials and Methods. Electrophoresis was carried out in a 6-13% gradient gel. Gel was destained to remove excess Coomassie stain and the bands were imaged in a scanner. C&D) Control, VbKD and SiR-wild type and S40A mutant cells were maintained under either normoxia or hypoxia. Mitochondria were isolated and proteins (25µg each) were used to measure CcO activity (C) and ROS production by Amplex red oxidation (D). n=4. **, p<0.005; ***, p<0.001.
Mentions: Our previous studies showed that under both hypoxia and alcohol toxicity CcO subunit Vb was one of the three subunits reduced as a function of time [25,46]. To assess the role of phosphorylation on the selective degradation of Vb, we first generated a macrophage cell line stably expressing siRNA against CcO subunit Vb (VbKD). We then expressed either wild type (WT CcO Vb) or phosphorylation resistant mutant (S40A-CcO Vb) of the subunit in the VbKD cells. The CcO Vb cDNA contained degenerate codons in the siRNA target region to prevent siRNA mediated degradation in VbKD cells. Immunoblot in Figure 5A shows that the level of subunit Vb was markedly reduced in siRNA expressing cells (VbKD), which was restored in cells expressing the siRNA resistant wild type or S40A mutant cDNA. Figure 5B shows the Blue Native gel profiles of control, Vb knocked down cells and cells reconstituted with WT or S40A mutant CcO Vb subunits. It is seen that CcO complex was markedly reduced in Vb knocked down cells which was restored in cells reconstituted with WT or S40A mutant CcO Vb subunits. The effect of hypoxia on CcO activities of mitochondrial preparations from these cells is presented in Figure 5C. The CcO activity was reduced by about 45% in control cells exposed to hypoxia. In Vb knocked down cells, the basal activity was reduced by more than 95%. Reconstitution with either wild type or S40A mutant CcO Vb nearly completely restores CcO activity in these cells. Notably, in cells reconstituted with WT subunit Vb, hypoxia reduced CcO activity by 42%, while cells reconstituted with phosphorylation resistant S40A subunit showed only 21% loss of CcO activity under hypoxia (Figure 5C). It is also seen that under hypoxic conditions, the ROS production in cells reconstituted with WT CcO Vb was similar to the control cells. In cells reconstituted with S40A mutant subunit, however, there was a significant decrease in ROS production which was constituent with the extent of resistance to hypoxia induced subunit degradation (Figure 5D). These results suggest that the S40A mutant protein is relatively more resistant to hypoxia mediated damage and support our hypothesis that phosphorylation of subunits is responsible for hypoxia mediated loss of CcO activity.

Bottom Line: Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS).Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production.The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.

Show MeSH
Related in: MedlinePlus