Abstract
The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.
Highlights
The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like
Hydrophobic Peptides Appear to Stimulate Peptide Hydrolysis by Opening the Channel in the ␣-Rings—This study has uncovered the following two new regulatory properties of the 20 S proteasome: 1) that the binding of hydrophobic peptides to multiple non-catalytic sites stimulates the hydrolysis of peptides by all of its active sites, and 2) that this stimulation occurs most probably by peptide-induced opening of the channel in the ␣-rings through which substrates enter the 20 S particle
This latter conclusion is based on our finding that the hydrophobic peptides did not stimulate peptide hydrolysis under various conditions where the entrance channels are primarily in an open state, including the ⌬N␣3 mutant of yeast proteasomes, 20 S-PA26 complexes, 26 S proteasomes, and SDS-activated 20 S proteasomes
Summary
Substrates and Inhibitors—Ac1-nLPnLD-amc and Ac-GPLD-amc were kindly provided by Dr Mike Pennington (Bachem, King of Prussia, PA). The proteasomes were eluted with a 0 – 0.5 M NaCl gradient in 250 ml of buffer A. 20 S and 26 S proteasomes were eluted as a single peak by a 0.10 – 0.35 M gradient of NaCl in 150 ml of buffer A and separated on a 6-ml UnoQ column (Bio-Rad) as described [16]. 26 S proteasomes were purified to homogeneity by a glycerol gradient as described [16]. 20 S proteasomes were purified on a 5-ml hydroxylapatite (CHTII EconPack cartridge, Bio-Rad) column as described by Groll et al [12], except that 10% glycerol was present in all buffers to maintain the particles in Suc-LLVY-amc cleaving fractions were pooled and loaded on a 6-ml Resource Q (Amersham Biosciences) column. 20 S and 26 S proteasomes were eluted as a single peak by a 0.10 – 0.35 M gradient of NaCl in 150 ml of buffer A and separated on a 6-ml UnoQ column (Bio-Rad) as described [16]. 26 S proteasomes were purified to homogeneity by a glycerol gradient as described [16]. 20 S proteasomes were purified on a 5-ml hydroxylapatite (CHTII EconPack cartridge, Bio-Rad) column as described by Groll et al [12], except that 10% glycerol was present in all buffers to maintain the particles in
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