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Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

ABSTRACT: The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (?1-?2-?5), the immunoproteasome (?1i-?2i-?5i) and the two intermediate proteasomes (?1-?2-?5i and ?1i-?2-?5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observed that ubiquitinated p21 and c--myc were degraded at similar rates, indicating that the four 26S proteasomes degrade ubiquitinated proteins equally well. Under oxidative stress, we observed a partial dissociation of 26S into 20S proteasomes, which can degrade non-ubiquitinated oxidized proteins. Oxidized calmodulin and hemoglobin were best degraded in vitro by the three ?5i-containing 20S proteasomes, while their native forms were not degraded. Circular dichroism analyses indicated that ubiquitin-independent recognition of oxidized proteins by 20S proteasomes was triggered by the disruption of their structure. Accordingly, ?5i-containing 20S proteasomes degraded unoxidized naturally disordered protein tau, while 26S proteasomes did not. Our results suggest that the three ?5i-containing 20S proteasomes, namely the immunoproteasome and the two intermediate proteasomes, might help cells to eliminate proteins containing disordered domains, including those induced by oxidative stress.

SUBMITTER: Abi Habib J

PROVIDER: S-EPMC7519072 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

Abi Habib Joanna J De Plaen Etienne E Stroobant Vincent V Zivkovic Dusan D Bousquet Marie-Pierre MP Guillaume Benoît B Wahni Khadija K Messens Joris J Busse Antonia A Vigneron Nathalie N Van den Eynde Benoit J BJ

Scientific reports 20200925 1

The proteasome is responsible for selective degradation of proteins. It exists in mammalian cells under four main subtypes, which differ by the combination of their catalytic subunits: the standard proteasome (β1-β2-β5), the immunoproteasome (β1i-β2i-β5i) and the two intermediate proteasomes (β1-β2-β5i and β1i-β2-β5i). The efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins remains unclear. Using cells expressing exclusively one proteasome subtype, we observe ...[more]

PMID: 32978409

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Project description:BackgroundUbiquitin-proteasome-system-mediated clearance of misfolded proteins is essential for cells to maintain proteostasis and reduce the proteotoxicity caused by these aberrant proteins. When proteasome activity is inadequate, ubiquitinated proteins are sorted into perinuclear aggresomes, which is a significant defense mechanism employed by cells to combat insufficient proteasome activity, hence mitigating the proteotoxic crisis. It has been demonstrated that phosphorylation of SQSTM1 is crucial in regulating misfolded protein aggregation and autophagic degradation. Although SQSTM1 S403 phosphorylation is essential for the autophagic degradation of ubiquitinated proteins, its significance in proteasome inhibition-induced aggresome formation is yet unknown. Herein, we investigated the influence of SQSTM1 S403 phosphorylation on the aggresome production of ubiquitinated proteins during proteasome suppression.MethodsWe examined the phosphorylation levels of SQSTM1 S403 or T269/S272 in cells after treated with proteasome inhibitors or/and autophagy inhibitors, by western blot and immunofluorescence. We detected the accumulation and aggresome formation of ubiquitinated misfolded proteins in cells treated with proteasome inhibition by western blot and immunofluorescence. Furthermore, we used SQSTM1 phosphorylation-associated kinase inhibitors and mutant constructs to confirm the regulation of different SQSTM1 phosphorylation in aggresome formation. We examined the cell viability using CCK-8 assay.ResultsHerein, we ascertained that phosphorylation of SQSTM1 S403 did not enhance the autophagic degradation of ubiquitinated proteins during proteasome inhibition. Proteasome inhibition suppresses the phosphorylation of SQSTM1 S403, which facilitated the aggresome production of polyubiquitinated proteins. Interestingly, we found proteasome inhibition-induced SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation. Suppressing S403 phosphorylation rescues the defective aggresome formation and protects cells from cell death caused by unphosphorylated SQSTM1 (T269/S272).ConclusionsThis study shows that inhibition of SQSTM1 S403 phosphorylation facilitates the aggresome formation of ubiquitinated proteins during proteasome dysfunction. SQSTM1 T269/S272 phosphorylation inhibits the S403 phosphorylation, boosting the aggresome formation of ubiquitinated protein and shielding cells from proteotoxic crisis.

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Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

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Efficiency of the four proteasome subtypes to degrade ubiquitinated or oxidized proteins.

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