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Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease.

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems.Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

ABSTRACT
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

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Aggregation of model proteins during oxidative stress. (A, B) Isolated mitochondria from WT, sod1Δ and sod2Δ strains were treated at 25°C with the indicated amounts of oxidative stressor ranging from 0 to 20 mM H2O2 (A) or 0 to 2 mM menadione (B), and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (C) Mitochondria from wild-type (WT) or sod2Δ yeast strains were treated at the indicated temperatures, and aggregates separated by ultracentrifugation. Pellets were analyzed by SDS–PAGE and Western blotting. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.
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Figure 4: Aggregation of model proteins during oxidative stress. (A, B) Isolated mitochondria from WT, sod1Δ and sod2Δ strains were treated at 25°C with the indicated amounts of oxidative stressor ranging from 0 to 20 mM H2O2 (A) or 0 to 2 mM menadione (B), and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (C) Mitochondria from wild-type (WT) or sod2Δ yeast strains were treated at the indicated temperatures, and aggregates separated by ultracentrifugation. Pellets were analyzed by SDS–PAGE and Western blotting. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.

Mentions: Stress through ROS has been described as another major threat to protein integrity aside from heat stress (Cabiscol et al., 2000). In fact, it should be noted that in vivo heat treatment at 42°C might also cause an elevated level of ROS due to increased radical production within the respiratory chain during those conditions. Because mitochondria are the site of oxidative metabolism, it has been discussed that mitochondrial proteins especially are at risk of being damaged by ROS. Consequently, we examined protein aggregation in mitochondria also under oxidative stress conditions. To induce oxidative stress, isolated mitochondria were treated with either hydrogen peroxide (H2O2) or menadione, the latter being a vitamin K3 metabolite leading to the formation of superoxide radicals (Chaput et al., 1983). One of the model proteins examined in the context of heat stress, Aco1, contains an oxidant-sensitive Fe/S cluster as a prosthetic group, making it also an interesting candidate to investigate its aggregation after ROS treatments. However, both Aco1 and Ilv2 remained completely soluble in wild-type mitochondria at concentrations of up to 20 mM H2O2 or 2 mM menadione at 25°C, with the exception of a marginal amount of Ilv2 in the pellet with menadione treatment (Figure 4, A and B; WT).


Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease.

Bender T, Lewrenz I, Franken S, Baitzel C, Voos W - Mol. Biol. Cell (2011)

Aggregation of model proteins during oxidative stress. (A, B) Isolated mitochondria from WT, sod1Δ and sod2Δ strains were treated at 25°C with the indicated amounts of oxidative stressor ranging from 0 to 20 mM H2O2 (A) or 0 to 2 mM menadione (B), and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (C) Mitochondria from wild-type (WT) or sod2Δ yeast strains were treated at the indicated temperatures, and aggregates separated by ultracentrifugation. Pellets were analyzed by SDS–PAGE and Western blotting. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: Aggregation of model proteins during oxidative stress. (A, B) Isolated mitochondria from WT, sod1Δ and sod2Δ strains were treated at 25°C with the indicated amounts of oxidative stressor ranging from 0 to 20 mM H2O2 (A) or 0 to 2 mM menadione (B), and aggregates were separated by ultracentrifugation at 125,000 × g. Total lysates (T), supernatants (Sup), and pellets (Pel) were analyzed by SDS–PAGE, Western blotting, and immunodecoration with the indicated specific antisera against mitochondrial proteins. (C) Mitochondria from wild-type (WT) or sod2Δ yeast strains were treated at the indicated temperatures, and aggregates separated by ultracentrifugation. Pellets were analyzed by SDS–PAGE and Western blotting. Values shown are means ± SEM of the ratio of protein amount in the pellet compared with total mitochondrial lysate.
Mentions: Stress through ROS has been described as another major threat to protein integrity aside from heat stress (Cabiscol et al., 2000). In fact, it should be noted that in vivo heat treatment at 42°C might also cause an elevated level of ROS due to increased radical production within the respiratory chain during those conditions. Because mitochondria are the site of oxidative metabolism, it has been discussed that mitochondrial proteins especially are at risk of being damaged by ROS. Consequently, we examined protein aggregation in mitochondria also under oxidative stress conditions. To induce oxidative stress, isolated mitochondria were treated with either hydrogen peroxide (H2O2) or menadione, the latter being a vitamin K3 metabolite leading to the formation of superoxide radicals (Chaput et al., 1983). One of the model proteins examined in the context of heat stress, Aco1, contains an oxidant-sensitive Fe/S cluster as a prosthetic group, making it also an interesting candidate to investigate its aggregation after ROS treatments. However, both Aco1 and Ilv2 remained completely soluble in wild-type mitochondria at concentrations of up to 20 mM H2O2 or 2 mM menadione at 25°C, with the exception of a marginal amount of Ilv2 in the pellet with menadione treatment (Figure 4, A and B; WT).

Bottom Line: To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins.Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems.Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie (IBMB), Universität Bonn, Nussallee 11, D-53115 Bonn, Germany.

ABSTRACT
Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria.

Show MeSH
Related in: MedlinePlus