Rapid degradation of solid-phase bound peptides by the 20S proteasome

J Pept Sci. 2013 Sep;19(9):588-97. doi: 10.1002/psc.2536. Epub 2013 Jul 28.

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.

Keywords: cleavage specificity; in vitro proteolytic assay; peptide; proteasome; proteolytic fragments; solid-phase digest.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Toxins / chemistry
  • Heat-Shock Proteins / chemistry
  • Hemolysin Proteins / chemistry
  • Humans
  • Immediate-Early Proteins / chemistry
  • Immobilized Proteins / chemistry*
  • Molecular Sequence Data
  • Ovalbumin / chemistry
  • Peptide Fragments / chemistry
  • Proteasome Endopeptidase Complex / chemistry*
  • Proteolysis
  • Solid-Phase Synthesis Techniques

Substances

  • Bacterial Toxins
  • Heat-Shock Proteins
  • Hemolysin Proteins
  • Immediate-Early Proteins
  • Immobilized Proteins
  • Peptide Fragments
  • cytomegalovirus immediate early phosphoprotein pp89
  • Ovalbumin
  • Proteasome Endopeptidase Complex
  • hlyA protein, Listeria monocytogenes