Ubiquitinated Proteins Activate the Proteasome by Binding to Usp14/Ubp6, which Causes 20S Gate Opening

Abstract

Summary 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 2–7 fold faster. Ub conjugates enhanced peptide hydrolysis by stimulating gate opening in the 20S proteasome, since this stimulation was seen when this gate was closed or transiently open, but not maximally open. Gate opening requires conjugate association with Usp14/Ubp6, since it is also mediated by occupancy of the Ubp6 active site with Ub aldehyde. No stimulation was observed with 26S from Ubp6Δ mutants, but was restored by addition of Usp14/Ubp6 or an inactive Ubp6 mutant. 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.

Deubiquitylases From Genes to Organism

“…Binding of ubiquitin chains to USP14/Ubp6 or UCH37 opens the gate of the 20S channel, and in combination with an unfolded substrate domain stimulates proteasomal ATPase activity. Although this offers the possibility of coupling ubiquitin recycling with degradation, gate opening or ATPase stimulation does not require catalytic activity (197,198). Expression of catalytically inactive USP14/ Ubp6 has been shown to have either positive or negative effects on proteasomal degradative activity, and these observations remain to be fully reconciled (84,143,197).…”

“…Although this offers the possibility of coupling ubiquitin recycling with degradation, gate opening or ATPase stimulation does not require catalytic activity (197,198). Expression of catalytically inactive USP14/ Ubp6 has been shown to have either positive or negative effects on proteasomal degradative activity, and these observations remain to be fully reconciled (84,143,197). Deletion of 31 amino acids from the COOH terminus of yeast Rpn11 leads to cell cycle defects and altered mitochondrial morphology.…”

Role of the ubiquitin–proteasome system in brain ischemia: Friend or foe?

“…The overall function of DUBs is to cleave ubiquitinlinked molecules after the C-terminus of the last residue of ubiquitin (Gly76), being essential to: (i) the maintenance of monomeric ubiquitin pool, either by cleaving the ubiquitin precursor or by trimming polyubiquitin chains; (ii) rescue proteins targeted for degradation, allowing the cell to adapt quickly to physiological changes, and (iii) prevent ubiquitinproteasome dependent protein degradation (Guterman and Glickman, 2004;Komander et al, 2009). Activation of the DUBs Usp14 and Uch37, either by polyubiquitin chains or their chemical mimetics, stimulates the 19S associated ATPases thereby opening the 20S closed gate, and thus promoting peptide hydrolysis (Peth et al, 2009(Peth et al, , 2013.…”

“…At the end, small peptides ranging from 2 to 20 amino acids and free ubiquitin are regenerated (Glickman and Ciechanover, 2002). The catalytic activity of the 26S proteasome is significantly enhanced in the presence of ubiquitinated substrates whereas the 20S activity remains unchanged (Peth et al, 2009). This supports a role for the 26S proteasome in the selective degradation of ubiquitinated substrates.…”

“…In the free CP this pore is closed by the Nterminus of the a-subunits, namely a 2 , a 3 and a 4 (Xie, 2010). However, the truncation of the a3 N-terminal is enough to keep the pore open and increases the degradation of small peptides (Peth et al, 2009). Besides closing the pore, a-subunits are also responsible for compartmentalizing the catalytic b-subunits, thereby preventing uncontrolled cleavage of cytosolic proteins.…”