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KIAA0368-deficiency affects disassembly of 26S proteasome under oxidative stress condition.

ABSTRACT: Many cellular stresses cause damages of intracellular proteins, which are eventually degraded by the ubiquitin and proteasome system. The proteasome is a multicatalytic protease complex composed of 20S core particle and the proteasome activators that regulate the proteasome activity. Extracellular mutants 29 (Ecm29) is a 200 kDa protein encoded by KIAA0368 gene, associates with the proteasome, but its role is largely unknown. Here, we generated KIAA0368-deficient mice and investigated the function of Ecm29 in stress response. KIAA0368-deficient mice showed normal peptidase activity and proteasome formation at normal condition. Under stressed condition, 26S proteasome dissociates in wild-type cells, but not in KIAA0368(-/-) cells. This response was correlated with efficient degradation of damaged proteins and resistance to oxidative stress of KIAA0368(-/-) cells. Thus, Ecm29 is involved in the dissociation process of 26S proteasome, providing clue to analyse the mechanism of proteasomal degradation under various stress condition.

SUBMITTER: Haratake K

PROVIDER: S-EPMC4892395 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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KIAA0368-deficiency affects disassembly of 26S proteasome under oxidative stress condition.

Haratake Kousuke K Sato Akitsugu A Tsuruta Fuminori F Chiba Tomoki T

Journal of biochemistry 20160122 6

Many cellular stresses cause damages of intracellular proteins, which are eventually degraded by the ubiquitin and proteasome system. The proteasome is a multicatalytic protease complex composed of 20S core particle and the proteasome activators that regulate the proteasome activity. Extracellular mutants 29 (Ecm29) is a 200 kDa protein encoded by KIAA0368 gene, associates with the proteasome, but its role is largely unknown. Here, we generated KIAA0368-deficient mice and investigated the functi ...[more]

PMID: 26802743

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Project description:Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.

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KIAA0368-deficiency affects disassembly of 26S proteasome under oxidative stress condition.

Publications

KIAA0368-deficiency affects disassembly of 26S proteasome under oxidative stress condition.

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