Abstract
Although the 20S core particle (CP) of the proteasome is an important component of the 26S holoenzyme, the stand-alone 20S CP acts directly on intrinsically disordered and oxidized/damaged proteins to degrade them in a ubiquitin-independent manner. It has been postulated that some structural features of substrate proteins are recognized by the 20S CP to promote substrate uptake, but the mechanism of substrate recognition has not been fully elucidated. In this study, we screened peptides that bind to the 20S CP from a random eight-residue pool of amino acid sequences using complementary DNA display an in vitro molecular evolution technique. The identified 20S CP-binding amino acid sequence was chemically synthesized and its effects on the 20S CP were investigated. The 20S CP-binding peptide stimulated the proteolytic activity of the inactive form of 20S CP. The peptide bound directly to one of the α-subunits, opening a gate for substrate entry on the α-ring. Furthermore, the attachment of this peptide sequence to α-synuclein enhanced its degradation by the 20S CP in vitro. In addition to these results, docking simulations indicated that this peptide binds to the top surface of the α-ring. These peptides could function as a key to control the opening of the α-ring gate.