N-terminal processing by dipeptidyl peptidase 9: Cut and Go!

Dipeptidyl peptidase 9 (DPP9) is an intracellular amino-dipeptidase with physiological roles in the immune system, DNA repair and mitochondria homeostasis, while its deregulation is linked to cancer progression and immune-associated defects. Through its rare ability to cleave a peptide bond following the imino-acid proline, DPP9 acts as a molecular switch that regulates key proteins, such as the tumor-suppressor BRCA2. In this review we will discuss key concepts underlying the outcomes of protein processing by DPP9, including substrate turn-over by the N-degron pathway. Additionally, we will review non-enzymatic roles and the regulation of DPP9 by discussing the interactome of this protease, which includes SUMO1, Filamin A, NLRP1 and CARD8.

The amino-dipeptidyl peptidases DPP8 and DPP9: Purification and enzymatic assays.

Proline residues highly impact protein stability when present either in the first or second N-terminal position. While the human genome encodes for more than 500 proteases, only few proteases are capable of hydrolyzing a proline-containing peptide bond. The two intra-cellular amino-dipeptidyl peptidases DPP8 and DPP9 are exceptional as they possess the rare ability to cleave post-proline. By removing N-terminal Xaa-Pro dipeptides, DPP8 and DPP9 expose a neo N-terminus of their substates, which can consequently alter inter- or intra-molecular interactions of the modified protein. Both DPP8 and DPP9 play key roles in the immune response and are linked to cancer progression, emerging as attractive drug targets. DPP9 is more abundant than DPP8 and is rate limiting for cleavage of cytosolic proline-containing peptides. Only few DPP9 substrates have been characterized; these include Syk, a central kinase for B-cell receptor mediated signaling; Adenylate Kinase 2 (AK2) which is important for cellular energy homeostasis; and the tumor suppressor Breast cancer type 2 susceptibility protein (BRCA2) that is critical for repair of DNA double strand breaks. N-terminal processing of these proteins by DPP9 triggers their rapid turn-over by the proteasome, highlighting a role for DPP9 as upstream components of the N-degron pathway. Whether N-terminal processing by DPP9 leads to substrate-degradation in all cases, or whether additional outcomes are possible, remains to be tested. In this chapter we will describe methods for purification of DPP8 and DPP9 as well as protocols for biochemical and enzymatic characterization of these proteases.

Evidence for Prepulse Inhibition of Visually Evoked Motor Response in Drosophila melanogaster.

Prepulse inhibition (PPI) is a widely investigated behavior to study the mechanisms of disorders such as anxiety, schizophrenia, and bipolar mania. PPI has been observed across various vertebrate and invertebrate species; however, it has not yet been reported in adult Drosophila melanogaster. In this study, we describe the first detection of PPI of visually evoked locomotor arousal in flies. To validate our findings, we demonstrate that PPI in Drosophila can be partially reverted by the N-methyl D-aspartate (NMDA) receptor antagonist MK-801, known for inducing sensorimotor gating deficits in rodent models. Additionally, we show that the visually evoked response can be inhibited by multiple stimuli presentation, which can also be affected by MK-801. Given the versatility of Drosophila as a model organism for genetic screening and analysis, our results suggest that high-throughput behavioral screenings of adult flies can become a valuable tool for investigating the mechanisms behind PPI.

Effects of Transient Administration of the NMDA Receptor Antagonist MK-801 in Drosophila melanogaster Activity, Sleep, and Negative Geotaxis.

MK-801, also called dizocilpine, is an N-methyl-D-aspartate (NMDA) receptor antagonist widely used in animal research to model schizophrenia-like phenotypes. Although its effects in rodents are well characterised, little is known about the outcomes of this drug in other organisms. In this study, we characterise the effects of MK-801 on the locomotion, sleep, and negative geotaxis of the fruit fly Drosophila melanogaster. We observed that acute (24 h) and chronic (7 days) administration of MK-801 enhanced negative geotaxis activity in the forced climbing assay for all tested concentrations (0.15 mM, 0.3 mM, and 0.6 mM). Moreover, acute administration, but not chronic, increased the flies' locomotion in a dose-dependent matter. Finally, average sleep duration was not affected by any concentration or administration protocol. Our results indicate that acute MK-801 could be used to model hyperactivity phenotypes in Drosophila melanogaster. Overall, this study provides further evidence that the NMDA receptor system is functionally conserved in flies, suggesting the usefulness of this model to investigate several phenotypes as a complement and replacement of the rodent models within drug discovery.

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.

A ligand-based system for receptor-specific delivery of proteins.

Gene delivery using vector or viral-based methods is often limited by technical and safety barriers. A promising alternative that circumvents these shortcomings is the direct delivery of proteins into cells. Here we introduce a non-viral, ligand-mediated protein delivery system capable of selectively targeting primary skin cells in-vivo. Using orthologous self-labelling tags and chemical cross-linkers, we conjugate large proteins to ligands that bind their natural receptors on the surface of keratinocytes. Targeted CRE-mediated recombination was achieved by delivery of ligand cross-linked CRE protein to the skin of transgenic reporter mice, but was absent in mice lacking the ligand's cell surface receptor. We further show that ligands mediate the intracellular delivery of Cas9 allowing for CRISPR-mediated gene editing in the skin more efficiently than adeno-associated viral gene delivery. Thus, a ligand-based system enables the effective and receptor-specific delivery of large proteins and may be applied to the treatment of skin-related genetic diseases.