Updated publication reference for PubMed record(s): 26957043. When in the closed form, the axial substrate translocation channel of the proteasome core particle (CP) is topologically blocked by the convergent N-termini of  subunits. To probe the role of channel gating in mammalian proteasomes, we have deleted the N-terminal tail of 3. The resulting 3N proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and degradation of polyubiquitinated Sic1. Cells expressing the hyperactive proteasomes show markedly elevated degradation of established proteasome substrates and resistance to oxidative stress, and multiplexed quantitative proteomics reveal ~ 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.