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Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening.

ABSTRACT: In eukaryotic cells, ubiquitination of proteins leads to their degradation by the 26S proteasome. We tested if the ubiquitin (Ub) chain also regulates the proteasome's capacity for proteolysis. After incubation with polyubiquitinated proteins, 26S proteasomes hydrolyzed peptides and proteins 2- to 7-fold faster. Ub conjugates enhanced peptide hydrolysis by stimulating gate opening in the 20S proteasome. This stimulation was seen when this gate was closed or transiently open, but not maximally open. Gate opening requires conjugate association with Usp14/Ubp6 and also occurs if Ub aldehyde occupies this isopeptidase's active site. No stimulation was observed with 26S from Ubp6Delta mutants, but this effect was restored upon addition of Usp14/Ubp6 (even an inactive Ubp6). The stimulation of gate opening by Ub conjugates through Usp14/Ubp6 requires nucleotide binding to the gate-regulatory ATPases. This activation enhances the selectivity of the 26S proteasome for ubiquitinated proteins and links their deubiquitination to their degradation.

SUBMITTER: Peth A

PROVIDER: S-EPMC2796264 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening.

Peth Andreas A Besche Henrike C HC Goldberg Alfred L AL

Molecular cell 20091201 5

In eukaryotic cells, ubiquitination of proteins leads to their degradation by the 26S proteasome. We tested if the ubiquitin (Ub) chain also regulates the proteasome's capacity for proteolysis. After incubation with polyubiquitinated proteins, 26S proteasomes hydrolyzed peptides and proteins 2- to 7-fold faster. Ub conjugates enhanced peptide hydrolysis by stimulating gate opening in the 20S proteasome. This stimulation was seen when this gate was closed or transiently open, but not maximally op ...[more]

PMID: 20005843

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Project description:USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or ?-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated ?-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect ?-catenin, demonstrating USP14 but not UCHL5 has a specific effect on ?-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides ?-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

Dataset Information

Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening.

Publications

Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening.

Similar Datasets

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