Acceleration of Protein Degradation by 20S Proteasome-Binding Peptides Generated by In Vitro Artificial Evolution

Author:

Zhu Yunhao1,Shigeyoshi Kaishin1,Hayakawa Yumiko1,Fujiwara Sae1,Kishida Masamichi2,Ohki Hitoshi2ORCID,Horibe Tomohisa1,Shionyu Masafumi1ORCID,Mizukami Tamio13,Hasegawa Makoto1

Affiliation:

1. Graduate School of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama 526-0829, Japan

2. Modality Research Laboratories, Biologics Division, Daiichi Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan

3. Frontier Pharma Inc., 1281-8 Tamura, Nagahama 526-0829, Japan

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.

Funder

JSPS KAKENHI

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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