Dipeptidyl peptidase 9 triggers BRCA2 degradation and promotes DNA damage repair.

N-terminal sequences are important sites for post-translational modifications that alter protein localization, activity, and stability. Dipeptidyl peptidase 9 (DPP9) is a serine aminopeptidase with the rare ability to cleave off N-terminal dipeptides with imino acid proline in the second position. Here, we identify the tumor-suppressor BRCA2 as a DPP9 substrate and show this interaction to be induced by DNA damage. We present crystallographic structures documenting intracrystalline enzymatic activity of DPP9, with the N-terminal Met1-Pro2 of a BRCA21-40 peptide captured in its active site. Intriguingly, DPP9-depleted cells are hypersensitive to genotoxic agents and are impaired in the repair of DNA double-strand breaks by homologous recombination. Mechanistically, DPP9 targets BRCA2 for degradation and promotes the formation of RAD51 foci, the downstream function of BRCA2. N-terminal truncation mutants of BRCA2 that mimic a DPP9 product phenocopy reduced BRCA2 stability and rescue RAD51 foci formation in DPP9-deficient cells. Taken together, we present DPP9 as a regulator of BRCA2 stability and propose that by fine-tuning the cellular concentrations of BRCA2, DPP9 alters the BRCA2 interactome, providing a possible explanation for DPP9's role in cancer.

Coping with Protein Quality Control Failure.

Cells and organisms have evolved numerous mechanisms to cope with and to adapt to unexpected challenges and harsh conditions. Proteins are essential to perform the vast majority of cellular and organismal functions. To maintain a healthy proteome, cells rely on a network of factors and pathways collectively known as protein quality control (PQC) systems, which not only ensure that newly synthesized proteins reach a functional conformation but also are essential for surveillance, prevention, and rescue of protein defects. The main players of PQC systems are chaperones and protein degradation systems: the ubiquitin-proteasome system and autophagy. Here we provide an integrated overview of the diverse PQC systems in eukaryotic cells in health and diseases, with an emphasis on the key regulatory aspects and their cross talks. We also highlight how PQC regulation may be exploited for potential therapeutic benefit.

DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk.

The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway.

Decoding the Protein Destruction Code: A Panoramic View.

Proteasome degradation is essential, but the intrinsic features of a protein that signals its destruction remain incompletely understood. In this issue of Molecular Cell, Geffen et al. (2016) report an unbiased and proteome-wide method that provided insights into the protein destruction signals and pathways.

The SUMO1-E67 interacting loop peptide is an allosteric inhibitor of the dipeptidyl peptidases 8 and 9.

The intracellular peptidases dipeptidyl peptidase (DPP) 8 and DPP9 are involved in multiple cellular pathways including antigen maturation, cellular homeostasis, energy metabolism, and cell viability. Previously we showed that the small ubiquitin-like protein modifier SUMO1 interacts with an armlike structure in DPP9, leading to allosteric activation of the peptidase. Here we demonstrate that the E67-interacting loop (EIL) peptide, which corresponds to the interaction surface of SUMO1 with DPP9, acts as a noncompetitive inhibitor of DPP9. Moreover, by analyzing the sensitivity of DPP9 arm mutants to the EIL peptide, we mapped specific residues in the arm that are important for inhibition by the EIL, suggesting that the peptide acts as an allosteric inhibitor of DPP9. By modifying the EIL peptide, we constructed peptide variants with more than a 1,000-fold selectivity toward DPP8 (147 nM) and DPP9 (170 nM) over DPPIV (200 µM). Furthermore, application of these peptides to cells leads to a clear inhibition of cellular prolyl peptidase activity. Importantly, in line with previous publications, inhibition of DPP9 with these novel allosteric peptide inhibitors leads to an increase in EGF-mediated phosphorylation of Akt. This work highlights the potential use of peptides that mimic interaction surfaces for modulating enzyme activity.

A novel SUMO1-specific interacting motif in dipeptidyl peptidase 9 (DPP9) that is important for enzymatic regulation.

Sumoylation affects many cellular processes by regulating the interactions of modified targets with downstream effectors. Here we identified the cytosolic dipeptidyl peptidase 9 (DPP9) as a SUMO1 interacting protein. Surprisingly, DPP9 binds to SUMO1 independent of the well known SUMO interacting motif, but instead interacts with a loop involving Glu(67) of SUMO1. Intriguingly, DPP9 selectively associates with SUMO1 and not SUMO2, due to a more positive charge in the SUMO1-loop. We mapped the SUMO-binding site of DPP9 to an extended arm structure, predicted to directly flank the substrate entry site. Importantly, whereas mutants in the SUMO1-binding arm are less active compared with wild-type DPP9, SUMO1 stimulates DPP9 activity. Consistent with this, silencing of SUMO1 leads to a reduced cytosolic prolyl-peptidase activity. Taken together, these results suggest that SUMO1, or more likely, a sumoylated protein, acts as an allosteric regulator of DPP9.