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.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

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 Photo-Crosslinking Approach to Identify Class II SUMO-1 Binders.

The small ubiquitin-like modifier (SUMO) is involved in various cellular processes and mediates known non-covalent protein-protein interactions by three distinct binding surfaces, whose interactions are termed class I to class III. While interactors for the class I interaction, which involves binding of a SUMO-interacting motif (SIM) to a hydrophobic groove in SUMO-1 and SUMO-2/3, are widely abundant, only a couple of examples have been reported for the other two types of interactions. Class II binding is conveyed by the E67 loop region on SUMO-1. Many previous studies to identify SUMO binders using pull-down or microarray approaches did not strategize on the SUMO binding mode. Identification of SUMO binding partners is further complicated due to the typically transient and low affinity interactions with the modifier. Here we aimed to identify SUMO-1 binders selectively enriched for class II binding. Using a genetically encoded photo-crosslinker approach, we have designed SUMO-1 probes to covalently capture class II SUMO-1 interactors by strategically positioning the photo-crosslinking moiety on the SUMO-1 surface. The probes were validated using known class II and class I binding partners. We utilized the probe with p-benzoyl-phenylalanine (BzF, also termed BpF or Bpa) at the position of Gln69 to identify binding proteins from mammalian cell extracts using mass spectrometry. By comparison with results obtained with a similarly designed SUMO-1 probe to target SIM-mediated binders of the class I type, we identified 192 and 96 proteins specifically enriched by either probe, respectively. The implicated preferential class I or class II binding modes of these proteins will further contribute to unveiling the complex interplay of SUMO-1-mediated interactions.

Aerosol-based ligand soaking of reservoir-free protein crystals.

Soaking of macromolecular crystals allows the formation of complexes via diffusion of molecules into a preformed crystal for structural analysis. Soaking offers various advantages over co-crystallization, e.g. small samples and high-throughput experimentation. However, this method has disadvantages, such as inducing mechanical stress on crystals and reduced success rate caused by low affinity/solubility of the ligand. To bypass these issues, the Picodropper was previously developed in the authors' laboratory. This technique aimed to deliver small volumes of compound solution in response to crystal dehydration supported by the Free Mounting System humidity control or by IR-laser-induced protein crystal transformation. Herein, a new related soaking development, the Aerosol-Generator, is introduced. This device delivers compounds onto the solution-free surface of protein crystals using an ultrasonic technique. The produced aerosol stream enables an easier and more accurate control of solution volumes, reduced crystal handling, and crystal-size-independent soaking. The Aerosol-Generator has been used to produce complexes of DPP8 crystals, where otherwise regular soaking did not achieve complex formation. These results demonstrate the potential of this device in challenging ligand-binding scenarios and contribute to further understanding of DPP8 inhibitor binding.

Challenges for Targeting SARS-CoV-2 Proteases as a Therapeutic Strategy for COVID-19.

Two proteases produced by the SARS-CoV-2 virus, the main protease and papain-like protease, are essential for viral replication and have become the focus of drug development programs for treatment of COVID-19. We screened a highly focused library of compounds containing covalent warheads designed to target cysteine proteases to identify new lead scaffolds for both Mpro and PLpro proteases. These efforts identified a small number of hits for the Mpro protease and no viable hits for the PLpro protease. Of the Mpro hits identified as inhibitors of the purified recombinant protease, only two compounds inhibited viral infectivity in cellular infection assays. However, we observed a substantial drop in antiviral potency upon expression of TMPRSS2, a transmembrane serine protease that acts in an alternative viral entry pathway to the lysosomal cathepsins. This loss of potency is explained by the fact that our lead Mpro inhibitors are also potent inhibitors of host cell cysteine cathepsins. To determine if this is a general property of Mpro inhibitors, we evaluated several recently reported compounds and found that they are also effective inhibitors of purified human cathepsins L and B and showed similar loss in activity in cells expressing TMPRSS2. Our results highlight the challenges of targeting Mpro and PLpro proteases and demonstrate the need to carefully assess selectivity of SARS-CoV-2 protease inhibitors to prevent clinical advancement of compounds that function through inhibition of a redundant viral entry pathway.

Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).

Fibroblast activation protein (FAP) is a proline-selective serine protease. It is hardly expressed in healthy adult tissue but upregulated in tissue remodeling sites associated with several diseases including epithelial cancer types, atherosclerosis, arthritis and fibrosis. Ongoing research aims at clinical implementation of FAP as a biomarker for these diseases. Several immunochemical methods that quantify FAP expression have been reported. An alternative/complementary approach focuses on quantification of FAP's enzymatic activity. Developing an activity-based assay for FAP has nonetheless proven challenging because of selectivity issues with respect to prolyl oligopeptidase (PREP). Here, we present substrate-type FAP probes that are structurally derived from a FAP-inhibitor (UAMC1110) that we published earlier. Both cleavage efficiency and FAP-selectivity of the best compounds in the series equal or surpass the most advanced peptide-based FAP substrates reported to date. Finally, proof-of-concept is provided that 4-aminonaphthol containing probes can spatially localize FAP activity in biological samples.

DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.

Activating germline mutations in the human inflammasome sensor NLRP1 causes palmoplantar dyskeratosis and susceptibility to Mendelian autoinflammatory diseases. Recent studies have shown that the cytosolic serine dipeptidyl peptidases DPP8 and DPP9 suppress inflammasome activation upstream of NLRP1 and CARD8 in human keratinocytes and peripheral blood mononuclear cells. Moreover, pharmacological inhibition of DPP8/DPP9 protease activity was shown to induce pyroptosis in murine C57BL/6 macrophages without eliciting other inflammasome hallmark responses. Here, we show that DPP8/DPP9 inhibition in macrophages that express a Bacillus anthracis lethal toxin (LeTx)-sensitive Nlrp1b allele triggered significantly accelerated pyroptosis concomitant with caspase-1 maturation, ASC speck assembly, and secretion of mature IL-1ß and IL-18. Genetic ablation of ASC prevented DPP8/DPP9 inhibition-induced caspase-1 maturation and partially hampered pyroptosis and inflammasome-dependent cytokine release, whereas deletion of caspase-1 or gasdermin D triggered apoptosis in the absence of IL-1ß and IL-18 secretion. In conclusion, blockade of DPP8/DPP9 protease activity triggers rapid pyroptosis and canonical inflammasome hallmarks in primary macrophages that express a LeTx-responsive Nlrp1b allele.

Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer.

Dipeptidyl peptidases 8 and 9 are intracellular N-terminal dipeptidyl peptidases (preferentially postproline) associated with pathophysiological roles in immune response and cancer biology. While the DPP family member DPP4 is extensively characterized in molecular terms as a validated therapeutic target of type II diabetes, experimental 3D structures and ligand-/substrate-binding modes of DPP8 and DPP9 have not been reported. In this study we describe crystal and molecular structures of human DPP8 (2.5 Å) and DPP9 (3.0 Å) unliganded and complexed with a noncanonical substrate and a small molecule inhibitor, respectively. Similar to DPP4, DPP8 and DPP9 molecules consist of one ß-propeller and α/ß hydrolase domain, forming a functional homodimer. However, they differ extensively in the ligand binding site structure. In intriguing contrast to DPP4, where liganded and unliganded forms are closely similar, ligand binding to DPP8/9 induces an extensive rearrangement at the active site through a disorder-order transition of a 26-residue loop segment, which partially folds into an α-helix (R-helix), including R160/133, a key residue for substrate binding. As vestiges of this helix are also seen in one of the copies of the unliganded form, conformational selection may contributes to ligand binding. Molecular dynamics simulations support increased flexibility of the R-helix in the unliganded state. Consistently, enzyme kinetics assays reveal a cooperative allosteric mechanism. DPP8 and DPP9 are closely similar and display few opportunities for targeted ligand design. However, extensive differences from DPP4 provide multiple cues for specific inhibitor design and development of the DPP family members as therapeutic targets or antitargets.

Hair cell synaptic dysfunction, auditory fatigue and thermal sensitivity in otoferlin Ile515Thr mutants.

The multi-C2 domain protein otoferlin is required for hearing and mutated in human deafness. Some OTOF mutations cause a mild elevation of auditory thresholds but strong impairment of speech perception. At elevated body temperature, hearing is lost. Mice homozygous for one of these mutations, OtofI515T/I515T, exhibit a moderate hearing impairment involving enhanced adaptation to continuous or repetitive sound stimulation. In OtofI515T/I515T inner hair cells (IHCs), otoferlin levels are diminished by 65%, and synaptic vesicles are enlarged. Exocytosis during prolonged stimulation is strongly reduced. This indicates that otoferlin is critical for the reformation of properly sized and fusion-competent synaptic vesicles. Moreover, we found sustained exocytosis and sound encoding to scale with the amount of otoferlin at the plasma membrane. We identified a 20 amino acid motif including an RXR motif, presumably present in human but not in mouse otoferlin, which reduces the plasma membrane abundance of Ile515Thr-otoferlin. Together, this likely explains the auditory synaptopathy at normal temperature and the temperature-sensitive deafness in humans carrying the Ile515Thr mutation.

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.

Identification of novel dipeptidyl peptidase 9 substrates by two-dimensional differential in-gel electrophoresis.

Dipeptidyl peptidase 9 (DPP9) is a member of the S9B/DPPIV (DPP4) serine protease family, which cleaves N-terminal dipeptides at an Xaa-Pro consensus motif. Cytoplasmic DPP9 has roles in epidermal growth factor signalling and in antigen processing, whilst the role of the recently discovered nuclear form of DPP9 is unknown. Mice lacking DPP9 proteolytic activity die as neonates. We applied a modified 2D differential in-gel electrophoresis approach to identify novel DPP9 substrates, using mouse embryonic fibroblasts lacking endogenous DPP9 activity. A total of 111 potential new DPP9 substrates were identified, with nine proteins/peptides confirmed as DPP9 substrates by MALDI-TOF or immunoblotting. Moreover, we also identified the dipeptide Val-Ala as a consensus site for DPP9 cleavage that was not recognized by DPP8, suggesting different in vivo roles for these closely related enzymes. The relative kinetics for the cleavage of these nine candidate substrates by DPP9, DPP8 and DPP4 were determined. This is the first identification of DPP9 substrates from cells lacking endogenous DPP9 activity. These data greatly expand the potential roles of DPP9 and suggest different in vivo roles for DPP9 and DPP8.

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.

The amino terminus extension in the long dipeptidyl peptidase 9 isoform contains a nuclear localization signal targeting the active peptidase to the nucleus.

The intracellular prolyl peptidase DPP9 is implied to be involved in various cellular pathways including amino acid recycling, antigen maturation, cellular homeostasis, and viability. Interestingly, the major RNA transcript of DPP9 contains two possible translation initiation sites, which could potentially generate a longer (892 aa) and a shorter version (863 aa) of DPP9. Although the endogenous expression of the shorter DPP9 form has been previously verified, it is unknown whether the longer version is expressed, and what is its biological significance. By developing specific antibodies against the amino-terminal extension of the putative DPP9-long form, we demonstrate for the first time the endogenous expression of this longer isoform within cells. Furthermore, we show that DPP9-long represents a significant fraction of total DPP9 in cells, under steady-state conditions. Using biochemical cell fractionation assays in combination with immunofluorescence studies, we find the two isoforms localize to separate subcellular compartments. Whereas DPP9-short is present in the cytosol, DPP9-long localizes preferentially to the nucleus. This differential localization is attributed to a classical monopartite nuclear localization signal (K(K/R)X(K/R)) in the N-terminal extension of DPP9-long. Furthermore, we detect prolyl peptidase activity in nuclear fractions, which can be inhibited by specific DPP8/9 inhibitors. In conclusion, a considerable fraction of DPP9, which was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors.

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.

The cytoplasmic peptidase DPP9 is rate-limiting for degradation of proline-containing peptides.

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.

Concepts in sumoylation: a decade on.

A decade has passed since SUMO (small ubiquitin-related modifier) was discovered to be a reversible post-translational protein modifier. During this time many enzymes that participate in regulated SUMO-conjugation and -deconjugation pathways have been identified and characterized. In parallel, the search for SUMO substrates has produced a long list of targets, which appear to be involved in most cellular functions. Sumoylation is a highly dynamic process and its outcomes are extremely diverse, ranging from changes in localization to altered activity and, in some cases, stability of the modified protein. At first glance, these effects have nothing in common; however, it seems that they all result from changes in the molecular interactions of the sumoylated proteins.

Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins.

Steroidogenic acute regulatory protein (StAR) is a nuclear encoded mitochondrial protein that enhances steroid synthesis by facilitating the transfer of cholesterol to the inner membranes of mitochondria in hormonally regulated steroidogenic cells. It is currently assumed that StAR activity commences before or during StAR import into the mitochondrial matrix. The present study was designed to demonstrate that, once imported and becoming physiologically irrelevant, exhaustive accumulation of StAR must be limited by a rapid degradation of the protein to prevent potential damage to the organelles. The use of uncouplers and manipulation of the interior mitochondrial pH in hormone-induced ovarian granulosa cells and StAR-expressing COS cells suggests that StAR degradation is biphasic and involves two classes of proteases. During phase I, which normally lasts for the first approximately 2 h following import, StAR is rapidly degraded by a protease, or proteases, that can be arrested by a nonclassical action of proteasome inhibitors such as MG132. StAR molecules that evade phase I are subjected to a second class of protease(s), which is slower and MG132 resistant. A third proteolytic entity was revealed in studies with C-28 StAR, a loss-of-function mutant of StAR. Upon initiation of its import, C-28 StAR dissipates the inner membrane potential and causes swelling of the mitochondria. Degradation of C-28 StAR, probably by an intermembrane space protease, is extremely rapid and MG132 insensitive. Collectively, this study defines StAR as the first naturally occurring mitochondrial protein that can serve as a substrate to probe multiple proteolytic activities in mammalian mitochondria.