title: Rapid degradation of solid-phase bound peptides by the 20S proteasome.
abstract: Proteasomes are cellular proteases involved in the degradation of numerous cellular proteins. The 20S proteasome is a cylindrical 28-mer protein complex composed of two outer heptameric α-rings forming the entrance for the protein substrate and two inner heptameric β-rings carrying the catalytic sites. Numerous in vitro studies have provided evidence that the 20S proteasome may degrade peptides of various lengths and even unfolded full-length polypeptide chains. However, a direct demonstration that the 20S proteasome may also cleave surface-attached immobilized peptides is lacking so far. To this end, we used a model system by coupling peptides from different source proteins covalently to the surface of glass beads and applied nanoLC/MS analysis to monitor the generation of proteolytic fragments in the presence of the 20S proteasome. Detectable amounts of cleavage products occurred within a few minutes indicating a much higher cleavage rate than observed with the same substrates in solution. Our finding lends support to the idea that proteasomes may directly degrade segments of membrane-bound proteins protruding into the aqueous phase.
authors.affiliation: Mathematical Systems Biochemistry Group, Institute of Biochemistry, Charité-Universitätsmedizin Berlin, Berlin, Germany.
authors.forename: Marc
authors.initials: M
authors.lastname: Hovestädt
authors.forename: Ulrike
authors.initials: U
authors.lastname: Kuckelkorn
authors.forename: Agathe
authors.initials: A
authors.lastname: Niewienda
authors.forename: Christin
authors.initials: C
authors.lastname: Keller
authors.forename: Andrean
authors.initials: A
authors.lastname: Goede
authors.forename: Bernhard
authors.initials: B
authors.lastname: Ay
authors.forename: Stefan
authors.initials: S
authors.lastname: Günther
authors.forename: Katharina
authors.initials: K
authors.lastname: Janek
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer
authors.forename: Hermann-Georg
authors.initials: HG
authors.lastname: Holzhütter
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry; D006360:Heat-Shock Proteins / Q000737:chemistry; D006460:Hemolysin Proteins / Q000737:chemistry; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry; D055767:Immobilized Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry*; D059748:Proteolysis; D060327:Solid-Phase Synthesis Techniques
chemical_list: D001427:Bacterial Toxins; D006360:Heat-Shock Proteins; D006460:Hemolysin Proteins; D017874:Immediate-Early Proteins; D055767:Immobilized Proteins; D010446:Peptide Fragments; C052612:cytomegalovirus immediate early phosphoprotein pp89; D010047:Ovalbumin; D046988:Proteasome Endopeptidase Complex; C038705:hlyA protein, Listeria monocytogenes
keywords: cleavage specificity; in vitro proteolytic assay; peptide; proteasome; proteolytic fragments; solid-phase digest