Abstract
When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells.
Highlights
When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of a-subunits
Our results indicate that enhancing proteasome activity through opening of the CP gate might be beneficial in protecting cells under oxidative stress conditions during neurodegeneration
Opening CP gate enhanced the activity of both free CP and proteasome holoenzymes with translocation-competent conformations, implicating that the gating system may function as a critical regulator of the substrate translocation rates from the RP to the catalytic core
Summary
When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of a-subunits. The 26S proteasome, a B2.5-MDa holoenzyme complex, is the sole adenosine triphosphate (ATP)-dependent protease in the eukaryotic cytosol and nucleus, and mediates the irreversible degradation of target substrates conjugated to ubiquitin It controls intracellular protein levels on a global scale and in particular plays a key role in protein quality control[1,2]. Previous studies using the yeast proteasome indicated that, among the key components of the gate, such as a2, a3 and a4, deletion of the N-terminal tail of the a3 subunit resulted in conformational destabilization of other N-terminal residues and opening of the CP channel into the proteolytically active interior chamber[16,23]. The gating of mammalian proteasomes and the consequences of gate opening in mammalian cells are essentially uncharacterized
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