The 20S Proteasome Splicing Activity Discovered by SpliceMet

Juliane Liepe,Christin Keller,Katharina Janek,Michele Mishto,Alexey Zaikin,Petra Henklein,Peter Michael Kloetzel,Kathrin Textoris-Taube,Rob J De Boer

doi:10.1371/journal.pcbi.1000830

Abstract

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.

Highlights

The multiple subunit 20S proteasome is the central catalytic unit of the ubiquitin proteasome system (UPS) and catalytic core of the 26S proteasome that is built by the association of the two 19S regulator complexes with the catalytic 20S core [19S-20S-19S]
With its N-terminal threonine residues as the single active site of the b-subunits b1, b2 and b5, the 20S proteasome is a N-terminal nucleophilic hydrolase responsible for the generation of the vast majority of virus- or tumor-derived peptides presented by MHC class I molecules at the cell surface for recognition by peptidespecific cytotoxic T lymphocytes (CTL) [1,2]
MHC class I molecules present antigenic peptides derived from endogenously expressed foreign or aberrant protein molecules to the outside world so that they can be recognised by cytotoxic T lymphocytes (CTLs) at the cell surface

Summary

Introduction

The multiple subunit 20S proteasome is the central catalytic unit of the ubiquitin proteasome system (UPS) and catalytic core of the 26S proteasome that is built by the association of the two 19S regulator complexes with the catalytic 20S core [19S-20S-19S]. With its N-terminal threonine residues as the single active site of the b-subunits b1, b2 and b5, the 20S proteasome is a N-terminal nucleophilic hydrolase responsible for the generation of the vast majority of virus- or tumor-derived peptides presented by MHC class I molecules at the cell surface for recognition by peptidespecific cytotoxic T lymphocytes (CTL) [1,2]. This function is generally aided by the interferon-c- (IFN-c)-induced synthesis of the alternative catalytic subunits b1i, b2i, b5i, with concomitant formation of immunoproteasome subtypes possessing altered proteolytic properties as well as by the IFN-c-induced upregulation of the proteasome activator subunits PA28-a and PA28-b [3,4].

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