The Cytoplasmic Peptidase DPP9 Is Rate-limiting for Degradation of Proline-containing Peptides

Ruth Geiss-Friedlander,Nicolas Parmentier,Ulrike Möller,Henning Urlaub,Benoit J Van Den Eynde,Frauke Melchior

doi:10.1074/jbc.m109.041871

Abstract

Protein degradation is an essential process that continuously takes place in all living cells. Regulated degradation of most cellular proteins is initiated by proteasomes, which produce peptides of varying length. These peptides are rapidly cleaved to single amino acids by cytoplasmic peptidases. Proline-containing peptides pose a specific problem due to structural constrains imposed by the pyrrolidine ring that prevents most peptidases from cleavage. Here we show that DPP9, a poorly characterized cytoplasmic prolyl-peptidase, is rate-limiting for destruction of proline-containing substrates both in cell extracts and in intact cells. We identified the first natural substrate for DPP9, the RU1(34-42) antigenic peptide (VPYGSFKHV). RU1(34-42) is degraded in vitro by DPP9, and down-regulation of DPP9 in intact cells results in increased presentation of this antigen. Together our findings demonstrate an important role for DPP9 in peptide turnover and antigen presentation.

Highlights

Protein turn-over is an essential process that continuously occurs in all living cells
Few peptidases are known to cleave after prolines, including the cytoplasmic peptidases prolyl oligopeptidase (POP) and cytoplasmic members of the S9B/DPPIV family (DPP8 and DPP9)
Three post-proline peptidases are known to localize to the cytosol, POP, DPP8, and DPP9

Summary

Introduction

Protein turn-over is an essential process that continuously occurs in all living cells. The addition of the DPP8/9 inhibitor to cytosolic extracts strongly inhibits GP-AMC degradation, suggesting that these peptidases and not POP are responsible for GP-AMC cleavage. To ensure that the stabilizing effect of DPP9 silencing on XP-AMC substrates was not due to off-target effects, we repeated the experiment using two additional DPP9 siRNAs. Each of the three siRNAs led to strong reduction of post-proline cleavage activity in cell lysates, and the extent of reduction correlated well with the reduction in DPP9 levels (compare activity assays in Fig. 1C with the corresponding Western blot).

Results

Conclusion