Dipeptidyl peptidases in atherosclerosis: expression and role in macrophage differentiation, activation and apoptosis.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 ± 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 ± 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 ± 16 vs. 146 ± 19 pg/ml; TNFα 3.8 ± 1.0 vs. 6.6 ± 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFα and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 ± 4 vs. 7 ± 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.

Dipeptidyl peptidase 9 (DPP9) from bovine testes: identification and characterization as the short form by mass spectrometry.

The dipeptidyl peptidases (DPP) 8 and 9 belong to the DPP4 activity and/or structure homologues (DASH). Recently, a DPP9-like protein was purified from bovine testes. The aim of the present study was to prove its identity and to investigate the characteristics of this natural enzyme. We report the identification and N-terminal sequence analysis by MALDI-TOF/TOF MS, of the purified bovine enzyme as DPP9. The tryptic peptides after in-gel digestion covered 41% and 38% of the short and full-length variants of bovine DPP9, respectively. Using Asp-N digestion combined with a very recently described mass spectrometric method using DITC glass beads, the N-terminal peptide (XTGALTSERG) was isolated. It corresponds to the N-terminus of the short form of bovine DPP9. There was no evidence for glycosylation of purified bovine DPP9. The purified DPP9 was activated and stabilized by DTT. Bovine DPP9 lost its activity almost completely after alkylation with N-ethylmaleimide. Also alkylation with iodoacetamide inhibited DPP9, albeit only 70%. Other properties of bovine DPP9 are reported, including functional stability and sensitivity towards metal ions. Our results indicate that the short form of DPP9 can be isolated from bovine testes and that it behaves as a stable enzyme suitable for further functional and biochemical characterization as well as for inhibitor screening and characterization.

Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries.

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ, DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.

The role of tryptophan catabolism along the kynurenine pathway in acute ischemic stroke.

Post-stroke inflammation may induce upregulation of the kynurenine (KYN) pathway for tryptophan (TRP) oxidation, resulting in neuroprotective (kynurenic acid, KA) and neurotoxic metabolites (3-hydroxyanthranillic acid, 3-HAA). We investigated whether activity of the kynurenine pathway in acute ischemic stroke is related to initial stroke severity, long-term stroke outcome and the ischemia-induced inflammatory response. Plasma concentrations of TRP and its metabolites were measured in 149 stroke patients at admission, at 24 h, at 72 h and at day 7 after stroke onset. We evaluated the relation between the KYN/TRP ratio, the KA/3-HAA ratio and stroke severity, outcome and inflammatory parameters (C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and neutrophil/lymphocyte ratio (NLR)). KYN/TRP but not KA/3-HAA correlated with the NIHSS score and with the infarct volume. Patients with poor outcome had higher mean KYN/TRP ratios than patients with more favourable outcome. The KYN/TRP ratio at admission correlated with CRP levels, ESR and NLR. The activity of the kynurenine pathway for tryptophan degradation in acute ischemic stroke correlates with stroke severity and long-term stroke outcome. Tryptophan oxidation is related to the stroke-induced inflammatory response.

Enzyme activity and immunohistochemical localization of dipeptidyl peptidase 8 and 9 in male reproductive tissues.

The mRNA expression pattern of dipeptidyl peptidase (DPP) 8 and DPP9, two DPP4 homologs, was studied previously and showed a broad tissue distribution. In this study, protein expression and activity of DPP8 and DPP9 were investigated in male reproductive tissues of different mammals. Based on specific DPP activities and inhibition profiles, the proline-selective DPP activity in the bovine and rat testis could predominantly be attributed to DPP8/9 and not to DPP4. This is in contrast to the epididymis, where most of the activity was caused by DPP4. Bovine sperm preparations had very low or undetectable DPP8/9 activity. After characterization of polyclonal antibodies specific for DPP8 or DPP9, we could localize both enzymes in seminiferous tubules of the testis. A specific staining for DPP9 was found associated with spermatozoids embedded in the epithelium, just before their release into the lumen, and in spermatids. DPP8 was localized in spermatozoids in an earlier stage of maturation. These findings help to provide insight into the physiological role of DPP4-like enzymes in the male reproductive system. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Cytokine changes and tryptophan metabolites in medication-naïve and medication-free schizophrenic patients.

Cytokine imbalances especially between T helper type (Th) 1 and Th2 and tryptophan breakdown were reported to be involved in the pathophysiology of schizophrenia. The hyperactive inflammatory response system could induce enhanced tryptophan breakdown. This study aimed to investigate the relationship between cytokine changes, tryptophan breakdown parameter changes and clinical parameters in patients with schizophrenia in comparison with normal controls. In the plasma of schizophrenic patients, Th1-specific interferon-gamma was significantly higher (F = 7.485, p = 0.007) and Th2-specific interleukin (IL)-4 was significantly lower (F = 126.327, p < 0.0001). The Th1-related cytokine IL-2 was lower (F = 5.409, p = 0.021) but tumor necrosis factor-alpha (TNF-alpha) and Th2-related IL-6 were higher (F = 95.004, p < 0.0001 and F = 408.176, p < 0.0001, respectively) in the plasma of schizophrenic patients. After 6 weeks of treatment, IL-6 and TNF-alpha were significantly reduced (t = -3.762, p < 0.0001 and z = -2.668, p = 0.008). At the time of admission, plasma tryptophan concentrations were lower (F = 6.339, p = 0.012) in schizophrenic patients and were negatively correlated with the total positive symptoms score (r(2) = -0.343, p = 0.004). After 6 weeks of medication, both plasma tryptophan and kynurenine concentrations were increased (t = -2.937, p = 0.005 and t = -3.214, p = 0.002, respectively). The findings of this study indicate a hyperactive pro-inflammatory response inducing a change in tryptophan metabolism that might be related to the development of positive symptoms in schizophrenia.

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over.

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.

Purification and characterization of dipeptidyl peptidase IV-like enzymes from bovine testes.

Until now, only recombinant forms of dipeptidyl peptidase (DPP) 8 and 9 have been characterized. We purified non DPPII-non DPPIV enzymes from a natural source. A first DPP8/9-like enzyme was enriched 1160-fold from bovine testes and identified as 'DPP9-like enzyme' by using an anti-DPP9 antibody. A second 576-fold enriched preparation ('DPP enriched peak 3') also showed DPP8/9-like activity. SDS-PAGE analysis showed that the DPP9-like enzyme had a monomeric molecular mass of approx. 100 kDa. Size exclusion chromatography generated a native molecular mass of 164 kDa for the DPP9-like enzyme and one of 234 kDa for the DPP enriched peak 3, suggesting that both proteins appeared to be dimeric. Both enriched preparations and rDPP8 showed roughly similar substrate specificity and inhibitor profiles. The DPP9-like enzyme and the DPP enriched peak 3 possessed a neutral pH optimum and were stable at -80 degrees C. We can conclude that the natural DPP9-like enzyme and the DPP enriched peak 3 are closely related to the recombinant forms of human DPP9 and DPP8.

Prolyl oligopeptidase stimulates the aggregation of alpha-synuclein.

Despite its thorough enzymological and biochemical characterization the exact function of prolyl oligopeptidase (PO, E.C. 3.4.21.26) remains unclear. The positive effect of PO inhibitors on learning and memory in animal models for amnesia, enzyme activity measurements in patient samples and (neuro)peptide degradation studies link the enzyme with neurodegenerative disorders. The brain protein alpha-synuclein currently attracts much attention because of its proposed role in the pathology of Parkinson's disease. A fundamental question concerns how the essentially disordered protein is transformed into the highly organized fibrils that are found in Lewy bodies, the hallmarks of Parkinson's disease. Using gel electrophoresis and MALDI TOF/TOF mass spectrometry we investigated the possibility of alpha-synuclein as a PO substrate. We found that in vitro incubation of the protein with PO did not result in truncation of full-length alpha-synuclein. Surprisingly, however, we found an acceleration of the aggregation process of alpha-synuclein using turbidity measurements that was reversed by specific inhibitors of PO enzymatic activity. If PO displays this activity also in vivo, PO inhibitors might have an effect on neurodegenerative disorders through a decrease in the aggregation of alpha-synuclein.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors.

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads.

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure-activity-relationship data obtained in this way led to the preparation of a series of alpha-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.

Dipeptidyl peptidase 8/9-like activity in human leukocytes.

The proline-specific dipeptidyl peptidases (DPPs) are emerging as a protease family with important roles in the regulation of signaling by peptide hormones. Inhibitors of DPPs have an intriguing, therapeutic potential, with clinical efficacy seen in patients with diabetes. Until now, only recombinant forms of DPP8 and DPP9 have been characterized. Their enzymatic activities have not been demonstrated in or purified from any natural source. Using several selective DPP inhibitors, we show that DPP activity, attributable to DPP8/9 is present in human PBMC. All leukocyte types tested (lymphocytes, monocytes, Jurkat, and U937 cells) were shown to contain similar DPP8/9-specific activities, and DPPII- and DPPIV-specific activities varied considerably. The results were confirmed by DPPIV/CD26 immunocapture experiments. Subcellular fractionation localized the preponderance of DPP8/9 activity to the cytosol and DPPIV in the membrane fractions. Using Jurkat cell cytosol as a source, a 30-fold, enriched DPP preparation was obtained, which had enzymatic characteristics closely related to the ones of DPP8 and/or -9, including inhibition by allo-Ile-isoindoline and affinity for immobilized Lys-isoindoline.

Neurotoxic and neuroprotective metabolites of kynurenine in patients with renal cell carcinoma treated with interferon-alpha: course and relationship with psychiatric status.

AIMS: Immunotherapy with interferon-alpha (IFN-alpha) is associated with psychiatric side-effects, including depression. One of the putative pathways underlying these psychiatric side-effects involves tryptophan (TRP) metabolism. Cytokines including IFN-alpha induce the enzyme indoleamine 2,3-dioxygenase (IDO), which converts TRP to kynurenine (KYN), leading to a shortage of serotonin (5-HT). In addition, the production of neurotoxic metabolites of KYN such as 3-hydroxykynurenine and quinolinic acid (QA) might increase and contribute to IFN-alpha-induced psychopathology. In contrast, other catabolites of KYN, such as kynurenic acid (KA), are thought to have neuroprotective properties. METHODS: In a group of 24 patients treated with standard IFN-alpha for metastatic renal cell carcinoma (RCC), combined psychiatric and laboratory assessments were performed at baseline, 4 and 8 weeks, and at 6 months. RESULTS: No psychopathology was observed, despite an increase in neurotoxic challenge as reflected in indices for the balance between neurotoxic and neuroprotective metabolites of KYN. CONCLUSIONS: The present hypothesis that a shift in the balance between neurotoxic and neuroprotective metabolites of KYN underlies the neuropsychiatric side-effects of IFN-alpha-based immunotherapy, is neither supported nor rejected.

Faulty serotonin--DHEA interactions in autism: results of the 5-hydroxytryptophan challenge test.

BACKGROUND: Autism is accompanied by peripheral and central disorders in the metabolism of serotonin (5-HT). The present study examines plasma dehydroepiandrosterone-sulphate (DHEA-S) and the cortisol/DHEA-S ratio following administration of L-5-hydroxytryptophan (5-HTP), the direct precursor of 5-HT, to autistic patients. METHODS: Plasma DHEA-S levels were determined both before and after administration of 5-HTP or placebo, on two consecutive days in a single blind order in 18 male autistic patients and 22 matched healthy controls. RESULTS: The 5-HTP-induced DHEA-S responses were significantly higher in autistic patients than in controls. In baseline conditions, the cortisol/DHEA-S ratio was significantly higher in autistic patients than in controls. DISCUSSION: The results suggest that autism is accompanied by a major disequilibrium in the serotonergic system. The increased Cortisol (neurotoxic) versus DHEA-S (neuroprotective) ratio suggests that an increased neurotoxic potential occurs in autism. CONCLUSIONS: It is concluded that a disequilibrium in the peripheral and central turnover of serotonin and an increased neurotoxic capacity by glucocorticoids are important pathways in autism.

Irreversible inhibition of dipeptidyl peptidase 8 by dipeptide-derived diaryl phosphonates.

Dipeptide-derived compounds, bearing various P2 residues and a diaryl pyrrolidin-2-yl phosphonate at the P1 position, were evaluated as dipeptidyl peptidase 8 (DPP8) inhibitors. With these products, irreversible inhibition of DPP8 was observed. To obtain inhibitors with an improved activity and selectivity profile, a set of selected analogues containing a diaryl isoindolin-1-ylphosphonate at P1 was synthesized and evaluated. Within this latter series, compound 2e was shown to be a potent, irreversible inhibitor of DPP8, demonstrating very low affinity for DPP IV and DPP II.

Suggested functions for prolyl oligopeptidase: a puzzling paradox.

Prolyl oligopeptidase (PO, E.C. 3.4.21.26) is a post-proline cleaving enzyme with endopeptidase activity towards peptides not longer than 30 amino acids. It has been purified and characterized from various mammalian and bacterial sources, but despite its thorough enzymological and structural characterization, the exact function of PO remains obscure. Many investigations have addressed the physiological role of this enzyme, mainly by the use of specific PO inhibitors, activity measurements in clinical samples and (neuro)peptide degradation studies. From the combined results emerges a puzzling paradox: how can an intracellular, cytoplasmatic oligopeptidase affect not only the amount of extracellular neuropeptides but also signal transduction and secretion? This report provides a review of the literature on the suggested functions for PO, highlighting possible pitfalls and contradictions.

Dipeptidyl peptidase II (DPPII), a review.

An increasing number of biological processes appear to be regulated by Pro-specific N-terminal processing. The proline-specific dipeptidyl peptidases (DPPs) like DPPIV, fibroblast activation protein alpha (FAP), DPPII, DPP8 and DPP9, because of their preference for cleavage after X-Pro in vitro, are likely to be involved in many of these processes. These DPPs are emerging as an important protease family with roles in the regulation of signaling by peptide hormones. Dipeptidyl peptidase II (DPPII, E.C. 3.4.14.2) is an intracellular protease that localizes to the vesicular system. It releases, preferably at acidic pH, N-terminal dipeptides from oligopeptides with Pro or Ala in the penultimate position. Despite the fact that the physiological role of DPPII still has not been elucidated, several suggestions were made on possible functions of the enzyme depending on its localization in different cells, body fluids and organs. DPPII was a.o. suggested to be involved in the processes of cell differentiation and in the protection from cell death, and to have a role in the degradation of collagen fragments, myofibrillar proteins and short neuropeptides. Moreover, changes in the level and distribution of the enzyme provided clues indicating additional roles in disease-related processes. Here we review the DPPII literature, aiming to bring more clarity in the disperse data on this subject and give a state of the art on DPPII research.

Kynurenine pathway in major depression: evidence of impaired neuroprotection.

The neurodegeneration hypothesis proposed major depression as a consequence of the imbalance between neuroprotective and neurodegenerative metabolites in the kynurenine pathway. To test the hypothesis, plasma tryptophan and kynurenine pathway metabolites were studied in 58 patients with major depression and 189 normal controls. The mean tryptophan breakdown index was higher (p=0.036), and mean kynurenic acid concentration and mean neuroprotective ratios were lower, in depressed patients (p=0.003 and 0.003, respectively). In receiver operating characteristic analysis, the kynurenic acid concentrations and the neuroprotective ratio showed clear discrimination between depressed patients and controls with area under the curve 79% and 76.3% respectively. The neuroprotective ratio did not change after treatment in those with repeated episodes of depression but it increased significantly (p=0.044) in those with first episodes. The results suggested that the reduction in neuroprotective markers, which indicated an impaired neuroprotection, might play an important role in pathophysiology of major depression.

Tryptophan breakdown pathway in bipolar mania.

The upregulation of the initiating step of the kynurenine pathway was demonstrated in postmortem anterior cingulated cortex from individuals with schizophrenia and bipolar disorder. However, the tryptophan and kynurenine metabolism in bipolar mania patients especially in drug naïve state has not been clearly explored. This study explored the plasma tryptophan and its competing amino acids, kynurenine, kynurenic acid and 3-hydroxyanthranillic acid and their association with psychopathological scores in 39 drug naïve and drug-free bipolar manic patients in comparison with 80 healthy controls. When age and gender were controlled in multivariate analysis, bipolar manic patients have significantly lower tryptophan index than normal controls (f=9.779, p=0.004). The mean plasma tryptophan concentration and mean tryptophan index were reduced and mean tryptophan breakdown index was increased significantly after a 6-week treatment. The reduction in plasma tryptophan and reduction in tryptophan index showed significant negative correlation with reduction in YMRS score (r=-0.577, p=0.019 and r=-0.520, p=0.039 respectively). The reduction in YMRS also showed positive correlation with both plasma tryptophan concentration and tryptophan index both at the time of admission (r=0.464, p=0.019 and r=0.4, p=0.047 respectively) and discharged (r=0.529, p=0.035 and r=0.607, p=0.013 respectively). The reduction in BPRS score also showed positive correlation with tryptophan index at the time of discharge (r=0.406, p=0.044). These findings indicated the involvement of bi-directional tryptophan metabolism and kynurenine pathway in pathophysiology and response to medication in bipolar mania.

Central serotonergic hypofunction in autism: results of the 5-hydroxy-tryptophan challenge test.

Some studies have suggested that disorders in the central serotonergic function may play a role in the pathophysiology of autistic disorder. In order to assess the central serotonergic turnover in autism, this study examines the cortisol and prolactin responses to administration of L-5-hydroxy-tryptophan (5-HTP), the direct precursor of 5-HT in 18 male, post-pubertal, Caucasian autistic patients (age 13-19 y.; I.Q.>55) and 22 matched healthy volunteers. Serum cortisol and prolactin were determined 45 and 30 minutes before administration of 5-HTP (4 mg/kg in non enteric-coated tablets) or an identical placebo in a single blind order and, thereafter, every 30 minutes over a 3-hour period. The 5-HTP-induced increases in serum cortisol were significantly lower in autistic patients than in controls, whereas there were no significant differences in 5-HTP-induced prolactin responses between both study groups. In baseline conditions, no significant differences were found in serum cortisol and prolactin between autistic and normal children. The results suggest that autism is accompanied by a central serotonergic hypoactivity and that the latter could play a role in the pathophysiology of autism.