The Human 26 S and 20 S Proteasomes Generate Overlapping but Different Sets of Peptide Fragments from a Model Protein Substrate

Abstract

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate ␤-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions. Cells present foreign, altered self, and self antigens to cytotoxic T lymphocytes (CTL) 1 on the cell surface in the context of major histocompatibility (MHC) class I molecules. Following recognition and activation, the CTL then initiate target cell destruction. The antigenic peptides essential for target cell recognition are generated in the cytosol by proteolytic degradation of predominantly endogenous proteins. The resulting pool of peptides is a possible source for translocation of 7-15-mers by the transporter associated with antigen processing into the lumen of the endoplasmic reticulum. Translocated peptides that fulfill the criteria for binding to the available MHC class I allelic products stabilize membrane-bound, empty MHC class I heavy chains in association with ␤ 2

The contributions of mass spectrometry to understanding of immune recognition by T lymphocytes

“…Subsequently, mass spectrometry has been used to demonstrate how variations in the structure and activity of proteasomes and TAP transporters affect the repertoire of peptides displayed on cells by class I MHC molecules [83][84][85], and to analyze the products of proteasome proteolysis in vitro [86][87][88][89]. The latter efforts have led to the creation of algorithms that predict proteasome cleavage sites [90,91].…”

“…In one case this was due to the failure of the negative peptide to be efficiently transported by TAP [55], while in another, the amino acid substitutions in the negative peptide enhanced its destruction by the proteasome [56]. This work helped to establish the importance of the class I MHC antigen processing pathway in controlling the display of structurally similar peptides.Subsequently, mass spectrometry has been used to demonstrate how variations in the structure and activity of proteasomes and TAP transporters affect the repertoire of peptides displayed on cells by class I MHC molecules [83][84][85], and to analyze the products of proteasome proteolysis in vitro [86][87][88][89]. The latter efforts have led to the creation of algorithms that predict proteasome cleavage sites [90,91].…”

Pathway for Degradation of Peptides Generated by Proteasomes

“…Peak fractions containing 26 S proteasomes were concentrated and stored at Ϫ20°C. The TPP-II fractions from the Uno Q-12 column were concentrated by ultrafiltration and 0.2 mg were loaded on top of a 10-ml glycerol density gradient (25-40% glycerol in 50 mM K 2 /K-PO 4 buffer, pH 7.6, containing 1 mM DTT) and centrifuged as above. Active fractions were combined and concentrated by ultrafiltration.…”

“…This structure generates diverse peptides, most of which range in length from 2 to 24 residues, with approximately two-thirds less than 8 residues long (2)(3)(4)(5). Nearly all of these peptides are then rapidly degraded to amino acids in the cytosol or nucleus.…”

Ubiquitylation of BAG-1 Suggests a Novel Regulatory Mechanism during the Sorting of Chaperone Substrates to the Proteasome

“…CHIP-containing fractions were pooled and stored at Ϫ80°C. 26 S proteasomes were purified from human red blood cells as described previously (24). Protein concentrations were determined using the Bio-Rad Bradford reagent with purified IgG (Sigma) as the standard.…”