172 results on '"LAMBEIR, AM"'
Search Results
2. A prolyl oligopeptidase inhibitor, KYP-2047, reduces α-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease
- Author
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Myöhänen, TT, primary, Hannula, MJ, additional, Van Elzen, R, additional, Gerard, M, additional, Van Der Veken, P, additional, García-Horsman, JA, additional, Baekelandt, V, additional, Männistö, PT, additional, and Lambeir, AM, additional
- Published
- 2012
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3. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES
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LAMBEIR, AM, LOISEAU, AM, KUNTZ, DA, VELLIEUX, FM, MICHELS, PAM, and OPPERDOES, FR
- Subjects
PENTALENOLACTONE ,INHIBITION ,COMPARTMENTATION ,ISOENZYMES ,MICROBODY ,GLYCOLYTIC-ENZYMES ,PHOSPHATE DEHYDROGENASE ,GOSSYPOL ,AFRICAN TRYPANOSOMES ,PHOSPHOGLYCERATE KINASES - Abstract
The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5 - 10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on K(m) and V(max). The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.
- Published
- 1991
4. IN VIVO INHIBITION OF DIPEPTIDYL PEPTIDASE IV (DPP IV) BY PRO-PRO-DIPHENYL PHOSPHONATE (DIPROFEN)
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De Meester, I., primary, Lambeir, AM., additional, Belyaev, A., additional, Borloo, M., additional, De Meyer, GRY., additional, Vanhoof, G., additional, Haemers, A., additional, and Scharpé, S., additional
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- 1996
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5. A prolyl oligopeptidase inhibitor, KYP-2047, reduces [alpha]-synuclein protein levels and aggregates in cellular and animal models of Parkinson's disease.
- Author
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Myöhänen TT, Hannula MJ, Van Elzen R, Gerard M, Van Der Veken P, García-Horsman JA, Baekelandt V, Männistö PT, and Lambeir AM
- Published
- 2012
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6. Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power.
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Williams, JC, Zeelen, JP, Neubauer, G, Vriend, G, Backmann, J, Michels, PAM, Lambeir, AM, and Wierenga, RK
- Published
- 1999
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7. KINETIC-PROPERTIES OF TRIOSE-PHOSPHATE ISOMERASE FROM TRYPANOSOMA-BRUCEI-BRUCEI - A COMPARISON WITH THE RABBIT MUSCLE AND YEAST ENZYMES
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LAMBEIR, AM, OPPERDOES, FR, and WIERENGA, RK
- Published
- 1987
8. Topogenic Signals for Import of Glycolytic-enzymes in Glycosomes (microbodies) of Trypanosoma-brucei
- Author
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UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Michels, Paulus, Lambeir, AM., Hart, D., Opperdoes, Frederik, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Michels, Paulus, Lambeir, AM., Hart, D., and Opperdoes, Frederik
- Published
- 1987
9. Kinetic-properties of Trypanosoma-brucei Glycolytic-enzymes
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UCL, Lambeir, AM., Kooystra, PJU., Kuntz, DA., Opperdoes, Frederik, UCL, Lambeir, AM., Kooystra, PJU., Kuntz, DA., and Opperdoes, Frederik
- Published
- 1988
10. Isolation and identification of naturally modified C-C chemokines MCP-1, MCP-2 and RANTES: Effects of posttranslational modifications on receptor usage, chemotactic and anti-HIV-1 activity
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Proost, P., Struyf, S., Wuyts, A., Menten, P., Meester, I., Lambeir, Am, Scharpe, S., Dominique Schols, Clercq, E., and Damme, J.
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Anti-HIV Agents ,Chemokines, CC ,HIV-1 ,Animals ,Chemokine CCL8 ,Humans ,Chemokine CCL5 ,Protein Processing, Post-Translational ,Chemokine CCL2 ,Monocyte Chemoattractant Proteins
11. The unique properties of dipeptidyl-peptidase IV (DPP IV/CD26) and the therapeutic potential of DPP IV inhibitors
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Koen Augustyns, Bal, G., Thonus, G., Belyaev, A., Zhang, Xm, Bollaert, W., Lambeir, Am, Durinx, C., Goossens, F., and Haemers, A.
12. NH2-terminal processing of chemokines (RANTES, SDF1-alpha) by CD26/dipeptidylpeptidase IV generates inhibitors of chemotaxis and HIV-1 infection
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Damme, J., Proost, P., Struyf, S., Wuyts, A., Lambeir, Am, Ingrid De Meester, Schols, D., Scharpe, S., and Clercq, E.
13. Structural determinants for ligand binding and catalysis of triosephosphate isomerase
- Author
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Kursula, I., Partanen, S., Lambeir, Am, Antonov, Dm, Koen Augustyns, and Wierenga, Rk
14. Development of caspase-3-selective activity-based probes for PET imaging of apoptosis.
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Lauwerys L, Beroske L, Solania A, Vangestel C, Miranda A, Van Giel N, Adhikari K, Lambeir AM, Wyffels L, Wolan D, Van der Veken P, and Elvas F
- Abstract
Background: The cysteine-aspartic acid protease caspase-3 is recognized as the main executioner of apoptosis in cells responding to specific extrinsic and intrinsic stimuli. Caspase-3 represents an interesting biomarker to evaluate treatment response, as many cancer therapies exert their effect by inducing tumour cell death. Previously developed caspase-3 PET tracers were unable to reach routine clinical use due to low tumour uptake or lack of target selectivity, which are two important requirements for effective treatment response evaluation in cancer patients. Therefore, the goal of this study was to develop and preclinically evaluate novel caspase-3-selective activity-based probes (ABPs) for apoptosis imaging., Results: A library of caspase-3-selective ABPs was developed for tumour apoptosis detection. In a first attempt, the inhibitor Ac-DW3-KE (Ac-3Pal-Asp-βhLeu-Phe-Asp-KE) was
18 F-labelled on the N-terminus to generate a radiotracer that was incapable of adequately detecting an increase in apoptosis in vivo. The inability to effectively detect active caspase-3 in vivo was likely attributable to slow binding, as demonstrated with in vitro inhibition kinetics. Hence, a second generation of caspase-3 selective ABPs was developed based on the Ac-ATS010-KE (Ac-3Pal-Asp-Phe(F5 )-Phe-Asp-KE) with greatly improved binding kinetics over Ac-DW3-KE. Our probes based on Ac-ATS010-KE were made by modifying the N-terminus with 6 different linkers. All the linker modifications had limited effect on the binding kinetics, target selectivity, and pharmacokinetic profile in healthy mice. In an in vitro apoptosis model, the least hydrophilic tracer [18 F]MICA-316 showed an increased uptake in apoptotic cells in comparison to the control group. Finally, [18 F]MICA-316 was tested in an in vivo colorectal cancer model, where it showed a limited tumour uptake and was unable to discriminate treated tumours from the untreated group, despite demonstrating that the radiotracer was able to bind caspase-3 in complex mixtures in vitro. In contrast, the phosphatidylethanolamine (PE)-binding radiotracer [99m Tc]Tc-duramycin was able to recognize the increased cell death in the disease model, making it the best performing treatment response assessment tracer developed thus far., Conclusions: In conclusion, a novel library of caspase-3-binding PET tracers retaining similar binding kinetics as the original inhibitor was developed. The most promising tracer, [18 F]MICA-316, showed an increase uptake in an in vitro apoptosis model and was able to selectively bind caspase-3 in apoptotic tumour cells. In order to distinguish therapy-responsive from non-responsive tumours, the next generation of caspase-3-selective ABPs will be developed with higher tumour accumulation and in vivo stability., (© 2024. The Author(s).)- Published
- 2024
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15. Study of the Conformational Dynamics of Prolyl Oligopeptidase by Mass Spectrometry: Lessons Learned.
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Van Elzen R, Konijnenberg A, Van der Veken P, Edgeworth MJ, Scrivens JH, Fülöp V, Sobott F, and Lambeir AM
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- Ligands, Ion Mobility Spectrometry, Models, Molecular, Mass Spectrometry methods, Catalytic Domain, Humans, Prolyl Oligopeptidases metabolism, Serine Endopeptidases chemistry, Serine Endopeptidases metabolism, Protein Conformation
- Abstract
Ion mobility mass spectrometry (IM-MS) can be used to analyze native proteins according to their size and shape. By sampling individual molecules, it allows us to study mixtures of conformations, as long as they have different collision cross sections and maintain their native conformation after dehydration and vaporization in the mass spectrometer. Even though conformational heterogeneity of prolyl oligopeptidase has been demonstrated in solution, it is not detectable in IM-MS. Factors that affect the conformation in solution, binding of an active site ligand, the stabilizing Ser554Ala mutation, and acidification do not qualitatively affect the collision-induced unfolding pattern. However, measuring the protection of accessible cysteines upon ligand binding provides a principle for the development of MS-based ligand screening methods.
- Published
- 2024
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16. 5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.
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Pätsi HT, Kilpeläinen TP, Jumppanen M, Uhari-Väänänen J, Wielendaele PV, De Lorenzo F, Cui H, Auno S, Saharinen J, Seppälä E, Sipari N, Savinainen J, De Meester I, Lambeir AM, Lahtela-Kakkonen M, Myöhänen TT, and Wallén EAA
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- Ligands, Binding Sites, Prolyl Oligopeptidases metabolism, Serine Endopeptidases metabolism, Thiazoles
- Abstract
A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.
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- 2024
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17. Chemically diverse activity-based probes with unexpected inhibitory mechanisms targeting trypsin-like serine proteases.
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Ramos-Llorca A, Decraecker L, Cacheux VMY, Zeiburlina I, De Bruyn M, Battut L, Moreno-Cinos C, Ceradini D, Espinosa E, Dietrich G, Berg M, De Meester I, Van Der Veken P, Boeckxstaens G, Lambeir AM, Denadai-Souza A, and Augustyns K
- Abstract
Activity-based probes (ABP) are molecules that bind covalently to the active form of an enzyme family, making them an attractive tool for target and biomarker identification and drug discovery. The present study describes the synthesis and biochemical characterization of novel activity-based probes targeting trypsin-like serine proteases. We developed an extensive library of activity-based probes with "clickable" affinity tags and a diaryl phosphonate warhead. A wide diversity was achieved by including natural amino acid analogs as well as basic polar residues as side chains. A detailed enzymatic characterization was performed in a panel of trypsin-like serine proteases. Their inhibitory potencies and kinetic profile were examined, and their IC
50 values, mechanism of inhibition, and kinetic constants were determined. The activity-based probes with a benzyl guanidine side chain showed the highest inhibitory effects in the panel. Surprisingly, some of the high-affinity probes presented a reversible inhibitory mechanism. On the other hand, probes with different side chains exhibited the expected irreversible mechanism. For the first time, we demonstrate that not only irreversible probes but also reversible probes can tightly label recombinant proteases and proteases released from human mast cells. Even under denaturing SDS-PAGE conditions, reversible slow-tight-binding probes can label proteases due to the formation of high-affinity complexes and slow dissociation rates. This unexpected finding will transform the view on the required irreversible nature of activity-based probes. The diversity of this library of activity-based probes combined with a detailed enzyme kinetic characterization will advance their applications in proteomic studies and drug discovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ramos-Llorca, Decraecker, Cacheux, Zeiburlina, De bruyn, Battut, Moreno-Cinos, Ceradini, Espinosa, Dietrich, Berg, De Meester, Van Der Veken, Boeckxstaens, Lambeir, Denadai-Souza and Augustyns.)- Published
- 2023
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18. Proteolytic Cleavage of Bioactive Peptides and Protease-Activated Receptors in Acute and Post-Colitis.
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De Bruyn M, Ceuleers H, Hanning N, Berg M, De Man JG, Hulpiau P, Hermans C, Stenman UH, Koistinen H, Lambeir AM, De Winter BY, and De Meester I
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- Amino Acid Sequence, Animals, Biomarkers, Colitis etiology, Colitis pathology, Disease Models, Animal, Disease Susceptibility, Inflammatory Bowel Diseases etiology, Inflammatory Bowel Diseases metabolism, Inflammatory Bowel Diseases pathology, Male, Peptides chemistry, Proteolysis, Rats, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Colitis metabolism, Peptides metabolism, Receptors, Proteinase-Activated metabolism
- Abstract
The protease activity in inflammatory bowel disease (IBD) and irritable bowel syndrome has been studied extensively using synthetic fluorogenic substrates targeting specific sets of proteases. We explored activities in colonic tissue from a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat model by investigating the cleavage of bioactive peptides. Pure trypsin- and elastase-like proteases on the one hand and colonic tissue from rats with TNBS-induced colitis in the acute or post-inflammatory phase on the other, were incubated with relevant peptides to identify their cleavage pattern by mass spectrometry. An increased cleavage of several peptides was observed in the colon from acute colitis rats. The tethered ligand (TL) sequences of peptides mimicking the N-terminus of protease-activated receptors (PAR) 1 and 4 were significantly unmasked by acute colitis samples and these cleavages were positively correlated with thrombin activity. Increased cleavage of β-endorphin and disarming of the TL-sequence of the PAR3-based peptide were observed in acute colitis and linked to chymotrypsin-like activity. Increased processing of the enkephalins points to the involvement of proteases with specificities different from trypsin- or chymotrypsin-like enzymes. In conclusion, our results suggest thrombin, chymotrypsin-like proteases and a set of proteases with different specificities as potential therapeutic targets in IBD.
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- 2021
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19. The C-terminal cleavage of angiotensin II and III is mediated by prolyl carboxypeptidase in human umbilical vein and aortic endothelial cells.
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De Hert E, Bracke A, Lambeir AM, Van der Veken P, and De Meester I
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- Angiotensin III antagonists & inhibitors, Aorta cytology, Aorta drug effects, Carboxypeptidases antagonists & inhibitors, Cells, Cultured, Human Umbilical Vein Endothelial Cells drug effects, Humans, Peptides pharmacology, Angiotensin II metabolism, Angiotensin III metabolism, Angiotensin-Converting Enzyme Inhibitors pharmacology, Aorta metabolism, Carboxypeptidases metabolism, Human Umbilical Vein Endothelial Cells metabolism
- Abstract
The renin-angiotensin system, with the octapeptide angiotensin II as key player, is important in the renal, cardiac and vascular physiology. Prolyl carboxypeptidase (PRCP), prolyl endopeptidase (PREP) and angiotensin converting enzyme 2 (ACE2) are reported to be involved in the conversion of angiotensin II to angiotensin (1-7). Previous investigations showed that the processing of angiotensin II is cell- and species-specific and little is known about its conversion in human endothelial cells. Therefore, we aimed to investigate the C-terminal processing of angiotensin II and III in comparison to the processing of des-Arg
9 -bradykinin in human endothelial cells. To this end, human umbilical vein and aortic endothelial cells (HUVEC and HAoEC) were incubated with the peptides for different time periods. Mass spectrometry analysis was performed on the supernatants to check for cleavage products. Contribution of PRCP, ACE2 and PREP to the peptide cleavage was evaluated by use of the selective inhibitors compound 8o, DX600 and KYP-2047. The use of these selective inhibitors revealed that the C-terminal cleavage of angiotensin II and III was PRCP-dependent in HUVEC and HAoEC. In contrast, the C-terminal cleavage of des-Arg9 -bradykinin was PRCP-dependent in HUVEC and PRCP- and ACE2-dependent in HAoEC. With this study, we contribute to a better understanding of the processing of peptides involved in the alternative renin-angiotensin system. We conclude that PRCP is the main enzyme for the C-terminal processing of angiotensin peptides in human umbilical vein and aortic endothelial cells. For the first time the contribution of PRCP was investigated by use of a selective PRCP-inhibitor., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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20. The Effect of a Novel Serine Protease Inhibitor on Inflammation and Intestinal Permeability in a Murine Colitis Transfer Model.
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Van Spaendonk H, Ceuleers H, Smet A, Berg M, Joossens J, Van der Veken P, Francque SM, Lambeir AM, De Man JG, De Meester I, Augustyns K, and De Winter BY
- Abstract
Background: A protease/antiprotease disbalance is observed in inflammatory bowel diseases (IBD). We therefore studied the effect of the novel serine protease inhibitor UAMC-00050 on intestinal inflammation and permeability in a chronic colitis T cell transfer mouse model to get further insight into the regulation of T cell-mediated immunopathology. Methods: Colitis was induced in severe combined immunodeficient (SCID) mice, by the adoptive transfer of CD4
+ CD25- CD62L+ T cells. Animals were treated intraperitoneally (i.p.) 2x/day with vehicle or UAMC-00050 (5 mg/kg) from week 2 onwards. Colonic inflammation was assessed by clinical parameters, colonoscopy, macroscopy, microscopy, myeloperoxidase activity and cytokine expression levels. At week 4, 4 kDa FITC-dextran intestinal permeability was evaluated and T helper transcription factors, protease-activated receptors and junctional proteins were quantified by RT-qPCR. Results: Adoptive transfer of CD4+ CD25- CD62L+ T cells resulted in colonic inflammation and an altered intestinal permeability. The serine protease inhibitor UAMC-00050 ameliorated both the inflammatory parameters and the intestinal barrier function. Furthermore, a decrease in colonic mRNA expression of Tbet and PAR4 was observed in colitis mice after UAMC-00050 treatment. Conclusion: The beneficial effect of UAMC-00050 on inflammation was apparent via a reduction of Tbet, IFN-γ, TNF-α, IL-1β and IL-6. Based on these results, we hypothesize a pivotal effect of serine protease inhibition on the Th1 inflammatory profile potentially mediated via PAR4., Competing Interests: KA, JJ, PVdV, BDW, HC, and HVS are inventors of the patent application WO/2017/198753. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van Spaendonk, Ceuleers, Smet, Berg, Joossens, Van der Veken, Francque, Lambeir, De Man, De Meester, Augustyns and De Winter.)- Published
- 2021
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21. Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells.
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De Hert E, Bracke A, Pintelon I, Janssens E, Lambeir AM, Van Der Veken P, and De Meester I
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- Angiotensin-Converting Enzyme 2 metabolism, Cell Line, Cytokines metabolism, Endothelial Cells drug effects, Human Umbilical Vein Endothelial Cells drug effects, Humans, Inflammation Mediators metabolism, Peptides blood, Proteolysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Aorta cytology, Carboxypeptidases metabolism, Endothelial Cells metabolism, Human Umbilical Vein Endothelial Cells metabolism, Intercellular Signaling Peptides and Proteins metabolism
- Abstract
The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1β-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.
- Published
- 2021
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22. In Vitro and In Situ Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes.
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Van Rymenant Y, Tanc M, Van Elzen R, Bracke A, De Wever O, Augustyns K, Lambeir AM, Kockx M, De Meester I, and Van Der Veken P
- Abstract
Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for in situ FAP activity staining in patient-derived cryosections of urothelial tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Van Rymenant, Tanc, Van Elzen, Bracke, De Wever, Augustyns, Lambeir, Kockx, De Meester and Van Der Veken.)
- Published
- 2021
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23. Selective inhibition of carboxypeptidase U may reduce microvascular thrombosis in rat experimental stroke.
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Mertens JC, Boisseau W, Leenaerts D, Di Meglio L, Loyau S, Lambeir AM, Ducroux C, Jandrot-Perrus M, Michel JB, Mazighi M, Hendriks D, and Desilles JP
- Subjects
- Animals, Fibrinolysis, Rats, Tissue Plasminogen Activator, Brain Ischemia, Carboxypeptidase B2, Stroke drug therapy, Thrombosis drug therapy
- Abstract
Background: Carboxypeptidase U (CPU, CPB2, TAFIa) is a potent attenuator of fibrinolysis. The inhibition of CPU is thus an interesting strategy for improving thrombolysis., Objectives: The time course of CPU generation and proCPU consumption were assessed in an experimental rat model of acute ischemic stroke (AIS). In addition, the effects of the selective CPU inhibitor AZD9684 on CPU kinetics, microvascular thrombosis (MT), and AIS outcome were evaluated., Methods: Rats were subjected to transient middle cerebral artery occlusion (tMCAO) and received recombinant tissue-type plasminogen activator (tPA), a specific CPU inhibitor (AZD9684), combination therapy of tPA and AZD9684, or saline for 1 hour using a randomized treatment regime. CPU and proCPU levels were determined at five time points and assessed in light of outcome parameters (a.o.: infarct volume and fibrin[ogen] deposition as a measure for MT)., Results: Clear activation of the CPU system was observed after AIS induction, in both saline- and tPA-treated rats. Maximal CPU activities were observed at treatment cessation and were higher in tPA-treated animals compared to the saline group. Concomitant proCPU consumption was more pronounced in tPA-treated rats. AZD9684 suppressed the CPU activity and reduced fibrin(ogen) deposition, suggesting a reduction of MT. Nonetheless, a significant decrease in infarct volume was not observed., Conclusions: A pronounced activation of the CPU system was observed during tMCAO in rats. Selective inhibition of CPU with AZD9684 was able to reduce fibrin(ogen) deposition and brain edema, suggesting a reduction of MT but without a significant effect on final infarct volume., (© 2020 International Society on Thrombosis and Haemostasis.)
- Published
- 2020
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24. Effects of Detergent on α-Synuclein Structure. A Native MS-Ion Mobility Study.
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Moons R, van der Wekken-de Bruijne R, Maudsley S, Lemière F, Lambeir AM, and Sobott F
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- Humans, Models, Biological, Models, Molecular, Nanotechnology, Protein Binding, Protein Conformation, Protein Multimerization, Spectrometry, Mass, Electrospray Ionization, alpha-Synuclein drug effects, Detergents pharmacology, alpha-Synuclein chemistry, alpha-Synuclein metabolism
- Abstract
The intrinsically disordered protein α-synuclein plays a major role in Parkinson's disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca
2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.- Published
- 2020
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25. A novel serine protease inhibitor as potential treatment for dry eye syndrome and ocular inflammation.
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Joossen C, Baán A, Moreno-Cinos C, Joossens J, Cools N, Lanckacker E, Moons L, Lemmens K, Lambeir AM, Fransen E, Delputte P, Caljon G, Van Der Veken P, Maes L, De Meester I, Kiekens F, Augustyns K, and Cos P
- Subjects
- Animals, Conjunctiva drug effects, Conjunctiva immunology, Disease Models, Animal, Dry Eye Syndromes genetics, Humans, Interleukin-1alpha genetics, Interleukin-1alpha immunology, Male, Matrix Metalloproteinase 9 genetics, Matrix Metalloproteinase 9 immunology, Rats, Rats, Wistar, Serine Proteinase Inhibitors chemistry, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha immunology, Dry Eye Syndromes drug therapy, Dry Eye Syndromes immunology, Serine Proteinase Inhibitors administration & dosage
- Abstract
Dry eye syndrome (DES), a multifactorial disorder which leads to ocular discomfort, visual disturbance and tear film instability, has a rising prevalence and limited treatment options. In this study, a newly developed trypsin-like serine protease inhibitor (UAMC-00050) in a tear drop formulation was evaluated to treat ocular inflammation. A surgical animal model of dry eye was employed to investigate the potential of UAMC-00050 on dry eye pathology. Animals treated with UAMC-00050 displayed a significant reduction in ocular surface damage after evaluation with sodium fluorescein, compared to untreated, vehicle treated and cyclosporine-treated animals. The concentrations of IL-1α and TNF-α were also significantly reduced in tear fluid from UAMC-00050-treated rats. Additionally, inflammatory cell infiltration in the palpebral conjunctiva (CD3 and CD45), was substantially reduced. An accumulation of pro-MMP-9 and a decrease in active MMP-9 were found in tear fluid from animals treated with UAMC-00050, suggesting that trypsin-like serine proteases play a role in activating MMP-9 in ocular inflammation in this animal model. Comparative qRT-PCR analyses on ocular tissue indicated the upregulation of tryptase, urokinase plasminogen activator receptor (uPAR) and protease-activated receptor 2 (PAR2). The developed UAMC-00050 formulation was stable up to 6 months at room temperature in the absence of light, non-irritating and sterile with compatible pH and osmolarity. These results provide a proof-of-concept for the in vivo modifying potential of UAMC-00050 on dry eye pathology and suggest a central role of trypsin-like serine proteases and PAR2 in dry eye derived ocular inflammation.
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- 2020
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26. Metal ions shape α-synuclein.
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Moons R, Konijnenberg A, Mensch C, Van Elzen R, Johannessen C, Maudsley S, Lambeir AM, and Sobott F
- Subjects
- Calcium chemistry, Copper chemistry, Intrinsically Disordered Proteins chemistry, Ion Mobility Spectrometry, Mass Spectrometry, Metals chemistry, Potassium chemistry, Protein Conformation, Sodium chemistry, Zinc chemistry, alpha-Synuclein chemistry
- Abstract
α-Synuclein is an intrinsically disordered protein that can self-aggregate and plays a major role in Parkinson's disease (PD). Elevated levels of certain metal ions are found in protein aggregates in neurons of people suffering from PD, and environmental exposure has also been linked with neurodegeneration. Importantly, cellular interactions with metal ions, particularly Ca
2+ , have recently been reported as key for α-synuclein's physiological function at the pre-synapse. Here we study effects of metal ion interaction with α-synuclein at the molecular level, observing changes in the conformational behaviour of monomers, with a possible link to aggregation pathways and toxicity. Using native nano-electrospray ionisation ion mobility-mass spectrometry (nESI-IM-MS), we characterize the heterogeneous interactions of alkali, alkaline earth, transition and other metal ions and their global structural effects on α-synuclein. Different binding stoichiometries found upon titration with metal ions correlate with their specific binding affinity and capacity. Subtle conformational effects seen for singly charged metals differ profoundly from binding of multiply charged ions, often leading to overall compaction of the protein depending on the preferred binding sites. This study illustrates specific effects of metal coordination, and the associated electrostatic charge patterns, on the complex structural space of the intrinsically disordered protein α-synuclein.- Published
- 2020
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27. Small molecule 3PO inhibits glycolysis but does not bind to 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3).
- Author
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Emini Veseli B, Perrotta P, Van Wielendaele P, Lambeir AM, Abdali A, Bellosta S, Monaco G, Bultynck G, Martinet W, and De Meyer GRY
- Subjects
- Humans, Protein Binding, Glycolysis drug effects, Human Umbilical Vein Endothelial Cells metabolism, Phosphofructokinase-2 metabolism, Pyridines pharmacology
- Abstract
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2020
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28. The effect of prolyl oligopeptidase inhibitors on alpha-synuclein aggregation and autophagy cannot be predicted by their inhibitory efficacy.
- Author
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Kilpeläinen TP, Hellinen L, Vrijdag J, Yan X, Svarcbahs R, Vellonen KS, Lambeir AM, Huttunen H, Urtti A, Wallen EAA, and Myöhänen TT
- Subjects
- Dose-Response Relationship, Drug, HEK293 Cells, Humans, Microtubule-Associated Proteins genetics, Microtubule-Associated Proteins metabolism, Proline pharmacology, Prolyl Oligopeptidases genetics, Prolyl Oligopeptidases metabolism, Protein Aggregates, Protein Multimerization, Antiparkinson Agents pharmacology, Autophagy drug effects, Proline analogs & derivatives, Prolyl Oligopeptidases antagonists & inhibitors, Serine Proteinase Inhibitors pharmacology, alpha-Synuclein metabolism
- Abstract
Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (αSyn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of αSyn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC
50 -values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess αSyn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free αSyn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased αSyn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and αSyn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the current study., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)- Published
- 2020
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29. Dysregulated activities of proline-specific enzymes in septic shock patients (sepsis-2).
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Vliegen G, Kehoe K, Bracke A, De Hert E, Verkerk R, Fransen E, Jongers B', Peters E, Lambeir AM, Kumar-Singh S, Pickkers P, Jorens PG, and De Meester I
- Subjects
- Area Under Curve, Biomarkers blood, Critical Care, Endopeptidases, Female, Humans, Longitudinal Studies, Male, Middle Aged, Proline metabolism, Prolyl Oligopeptidases, Prospective Studies, ROC Curve, Shock, Septic mortality, Shock, Septic therapy, Survival Analysis, Carboxypeptidases blood, Dipeptidyl Peptidase 4 blood, Gelatinases blood, Membrane Proteins blood, Serine Endopeptidases blood, Shock, Septic blood, Shock, Septic enzymology
- Abstract
The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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30. Efforts towards an On-Target Version of the Groebke-Blackburn-Bienaymé (GBB) Reaction for Discovery of Druglike Urokinase (uPA) Inhibitors.
- Author
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Gladysz R, Vrijdag J, Van Rompaey D, Lambeir AM, Augustyns K, De Winter H, and Van der Veken P
- Subjects
- Imidazoles chemistry, Molecular Structure, Pyridines chemistry, Drug Discovery, Urokinase-Type Plasminogen Activator chemistry
- Abstract
Target-guided synthesis (TGS) has emerged as a promising strategy in drug discovery. Although reported examples of TGS generally involve two-component reactions, there is a strong case for developing target-guided versions of three-component reactions (3CRs) because of their potential to deliver highly diversified druglike molecules. To this end, the Groebke-Blackburn-Bienaymé reaction was selected as a model 3CR. We recently reported a series of druglike urokinase inhibitors, and these serve as reference compounds in the present study. Due to the limited number of literature reports on target-guided 3CRs, multiple experimental parameters were optimized here. Most challenging was the formation of imine intermediates under near-physiological conditions. This aspect was addressed by exploring chemical imine stabilization strategies. Notably, imines are also crucial intermediates of other 3CRs. Such systematic studies are strongly required for further development of the TGS domain but are largely absent in the literature. Hence, this work is intended as a reference for future multicomponent-based TGS studies., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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31. The development and validation of a combined kinetic fluorometric activity assay for fibroblast activation protein alpha and prolyl oligopeptidase in plasma.
- Author
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Bracke A, Van Elzen R, Van Der Veken P, Augustyns K, De Meester I, and Lambeir AM
- Subjects
- Blood Platelets chemistry, Endopeptidases, Hemolysis, Humans, Kinetics, Limit of Detection, Linear Models, Prolyl Oligopeptidases, Blood Chemical Analysis methods, Fluorometry methods, Gelatinases blood, Membrane Proteins blood, Serine Endopeptidases blood
- Abstract
Background: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma., Methods: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors., Results: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation., Conclusion: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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32. Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP).
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De Decker A, Vliegen G, Van Rompaey D, Peeraer A, Bracke A, Verckist L, Jansen K, Geiss-Friedlander R, Augustyns K, De Winter H, De Meester I, Lambeir AM, and Van der Veken P
- Abstract
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., Competing Interests: The authors declare no competing financial interest.
- Published
- 2019
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33. Inhibition of the procarboxypeptidase U (proCPU, TAFI, proCPB2) system due to hemolysis.
- Author
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Mertens JC, Claesen K, Leenaerts D, Sim Y, Lambeir AM, and Hendriks D
- Subjects
- Blood Coagulation Tests statistics & numerical data, Fibrin Clot Lysis Time methods, Fibrin Clot Lysis Time statistics & numerical data, Fibrinolysis physiology, Healthy Volunteers, Humans, In Vitro Techniques, Blood Coagulation Tests methods, Carboxypeptidase B2 antagonists & inhibitors, Carboxypeptidase B2 blood, Hemolysis physiology
- Abstract
Essentials Hemolytic influence on the (pro)carboxypeptidase U ((pro)CPU) system is not known. In the current manuscript, this was assessed by spiking pooled normal plasma with hemolysate. CPU activity, proCPU levels, and clot lysis times showed a dose-dependent hemolytic bias. The observed bias in the several CPU related parameters is due to inhibition of CPU activity., Introduction: Spurious hemolysis of samples is the leading cause of interference in coagulation testing and was described to interfere in fibrinolysis assays. The influence of hemolysis on the procarboxypeptidase U (proCPU) system is not known., Methods: By means of spiking of hemolysate in pooled normal plasma, the effect of hemolysis on CPU, proCPU, and functional clot lysis assays was assessed. The influence of hemolysis on CPU generation during in vitro clot lysis was also evaluated. Cutoffs corresponding to maximal acceptable bias were determined., Results and Discussion: When active CPU was added to pooled plasma, a severe decrease in activity - up to 97.2% inhibition - was seen with increasing plasma concentrations of oxyhemoglobin (oxyHb) and the 10% cutoff value was found to be 0.3 g/L oxyHb. Using an activity-based assay, proCPU levels appeared to decrease gradually with increased hemolysis (maximal reduction of 19.5%) with a 10% cutoff value of 4.2 g/L oxyHb. The relative clot lysis time (CLT) showed a maximal negative bias of 68.5%. The reduction in CLT paralleled a significant reduction of the first CPU activity peak during clot lysis. The cutoff value for the CLT was 0.4 g/L oxyHb. In presence of thrombomodulin (TM), CLT+TM was not affected up to 8.0 g/L oxyHb., Conclusion: These data indicate a clear inhibition of the CPU system because of hemolysis resulting in an increase of lysis in functional fibrinolysis assays. We were able to quantify the inhibitory effect and to propose cutoff values for every parameter., (© 2019 International Society on Thrombosis and Haemostasis.)
- Published
- 2019
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34. DPP8/DPP9 inhibition elicits canonical Nlrp1b inflammasome hallmarks in murine macrophages.
- Author
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de Vasconcelos NM, Vliegen G, Gonçalves A, De Hert E, Martín-Pérez R, Van Opdenbosch N, Jallapally A, Geiss-Friedlander R, Lambeir AM, Augustyns K, Van Der Veken P, De Meester I, and Lamkanfi M
- Subjects
- Alleles, Animals, Antigens, Bacterial, Apoptosis drug effects, Bacterial Toxins, Boronic Acids administration & dosage, Boronic Acids pharmacology, CARD Signaling Adaptor Proteins genetics, Caspase 1 metabolism, Cell Line, Dipeptides administration & dosage, Dipeptides pharmacology, Interleukin-18 metabolism, Interleukin-1beta metabolism, Mice, Mice, Inbred C57BL, Pyroptosis drug effects, Apoptosis Regulatory Proteins metabolism, Dipeptidyl-Peptidases and Tripeptidyl-Peptidases antagonists & inhibitors, Inflammasomes metabolism, Macrophages metabolism
- Abstract
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-1β 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-1β 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., (© 2019 de Vasconcelos et al.)
- Published
- 2019
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35. Spatiotemporal expression and inhibition of prolyl oligopeptidase contradict its involvement in key pathologic mechanisms of kainic acid-induced temporal lobe epilepsy in rats.
- Author
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Ali I, Van Eetveldt A, Van Elzen R, Kalathil Raju T, Van Der Veken P, Lambeir AM, and Dedeurwaerdere S
- Abstract
Objective: Prolyl oligopeptidase (PREP) has been implicated in neuroinflammatory processes and neuroplasticity and has been suggested as a target for the treatment of neurodegenerative disease. The aim of this investigation was to explore the involvement of PREP in the neuropathologic mechanisms relevant to temporal lobe epilepsy (TLE) using a PREP inhibitor in a well-established rat model., Methods: PREP activity and expression was studied in Sprague-Dawley rats 2 and 12 weeks following kainic acid-induced status epilepticus (KASE). Continuous video-electroencephalography monitoring was performed for 2 weeks in the 12-week cohort to identify a relationship of PREP expression/activity with epileptic seizures. In addition, the animals included in the 2-week time point were treated with a specific inhibitor of PREP, KYP-2047, or saline continuously, starting immediately after SE. PREP activity and its expression were analyzed in rat brain by using enzyme kinetics and western blot. In addition, markers for microglial activation, astrogliosis, cell loss, and cell proliferation were evaluated., Results: Enzymatic activity of PREP was unchanged following induction of SE after 2 and 12 weeks in rats. PREP activity in epileptic rats did not relate to the number of seizures/day at the 12-week time point. Moreover, continuous inhibition of PREP for 2 weeks after KASE did not alter the SE-mediated neuroinflammatory response, cell loss, or cell proliferation in the hippocampal subgranule zone measured at the 2-week time point., Significance: PREP inhibition does not affect key pathologic mechanisms, including activation of glial cells, cell loss, and neural progenitor cell proliferation, in this KASE model of TLE. The results do not support a direct role of PREP in seizure burden during the chronic epilepsy period in this model., Competing Interests: None of the authors have potential conflicts of interest to disclose. We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.
- Published
- 2018
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36. Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and β-Synuclein on the Fibrillation Process of α-Synuclein.
- Author
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Van de Vondel E, Baatsen P, Van Elzen R, Lambeir AM, Keiderling TA, Herrebout WA, and Johannessen C
- Subjects
- Amyloid ultrastructure, Benzothiazoles chemistry, Circular Dichroism, Humans, Microscopy, Atomic Force, Microscopy, Electron, Transmission, Protein Structure, Quaternary, Amyloid chemistry, alpha-Synuclein chemistry, beta-Synuclein chemistry
- Abstract
The effects of crowding, using the crowding agent Ficoll 70, and the presence of β-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while β-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.
- Published
- 2018
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37. Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) inhibition stimulates the fibrinolytic rate in different in vitro models.
- Author
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Leenaerts D, Loyau S, Mertens JC, Boisseau W, Michel JB, Lambeir AM, Jandrot-Perrus M, and Hendriks D
- Subjects
- Carboxypeptidase B2 blood, Humans, Kinetics, Blood Coagulation Tests methods, Butyrates pharmacology, Carboxypeptidase B2 antagonists & inhibitors, Fibrinolysis drug effects, Fibrinolytic Agents pharmacology, Protease Inhibitors pharmacology, Pyridines pharmacology
- Abstract
Essentials AZD9684 is a potent inhibitor of carboxypeptidase U (CPU, TAFIa, CPB2). The effect of AZD9684 on fibrinolysis was investigated in four in vitro systems. The CPU system also attenuates fibrinolysis in more advanced hemostatic systems. The size of the observed effect on fibrinolysis is dependent on the exact experimental conditions., Summary: Background Carboxypeptidase U (CPU, carboxypeptidase B2, activated thrombin-activatable fibrinolysis inhibitor) is a basic carboxypeptidase that attenuates fibrinolysis. This characteristic has raised interest in the scientific community and pharmaceutical industry for the development of inhibitors as profibrinolytic agents. Objectives Little is known about the contribution of CPU to clot resistance in more advanced hemostatic models, which include blood cells and shear stress. The aim of this study was to evaluate the effects of the CPU system in in vitro systems for fibrinolysis with different grades of complexity. Methods The contribution of the CPU system was evaluated in the following systems: (i) plasma clot lysis; (ii) rotational thromboelastometry (ROTEM) in whole blood; (iii) front lysis with confocal microscopy in platelet-free and platelet-rich plasma; and (iv) a microfluidic system with whole blood under arterial shear stress. Experiments were carried out in the presence or absence of AZD9684, a specific CPU inhibitor. Results During plasma clot lysis, addition of AZD9684 resulted in 33% faster lysis. In ROTEM, the lysis onset time was decreased by 38%. For both clot lysis and ROTEM, an AZD9684 dose-dependent response was observed. CPU inhibition in front lysis experiments resulted in 47% and 50% faster lysis for platelet-free plasma and platelet-rich plasma, respectively. Finally, a tendency for faster lysis was observed only in the microfluidic system when AZD9684 was added. Conclusions Overall, these experiments provide novel evidence that the CPU system can also modulate fibrinolysis in more advanced hemostatic systems. The extent of the effects appears to be dependent upon the exact experimental conditions., (© 2018 International Society on Thrombosis and Haemostasis.)
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- 2018
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38. Newly developed serine protease inhibitors decrease visceral hypersensitivity in a post-inflammatory rat model for irritable bowel syndrome.
- Author
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Ceuleers H, Hanning N, Heirbaut J, Van Remoortel S, Joossens J, Van Der Veken P, Francque SM, De Bruyn M, Lambeir AM, De Man JG, Timmermans JP, Augustyns K, De Meester I, and De Winter BY
- Subjects
- Animals, Disease Models, Animal, Irritable Bowel Syndrome physiopathology, Male, Rats, Rats, Sprague-Dawley, Irritable Bowel Syndrome drug therapy, Serine Proteinase Inhibitors therapeutic use, Visceral Pain physiopathology
- Abstract
Background and Purpose: Serine proteases have been re suggested as important mediators of visceral pain. We investigated their effect by using newly developed serine protease inhibitors with a well-characterized inhibitory profile in a rat model of post-inflammatory irritable bowel syndrome (IBS)., Experimental Approach: Colitis was induced in rats receiving intrarectal trinitrobenzenesulphonic acid; controls received 0.9% NaCl. Colonoscopies were performed on day 3, to confirm colitis, and later until mucosal healing. Visceral hypersensitivity was quantified by visceromotor responses (VMRs) to colorectal distension, 30 min after i.p. injection of the serine protease inhibitors nafamostat, UAMC-00050 or UAMC-01162. Serine proteases, protease-activated receptors (PARs) and TRP channels were quantified by qPCR and immunohistochemistry. Proteolytic activity was characterized using fluorogenic substrates., Key Results: VMR was significantly elevated in post-colitis rats. Nafamostat normalized VMRs at the lowest dose tested. UAMC-00050 and UAMC-01162 significantly decreased VMR dose-dependently. Expression of mRNA for tryptase-αβ-1and PAR4, and tryptase immunoreactivity was significantly increased in the colon of post-colitis animals. Trypsin-like activity was also significantly increased in the colon but not in the faeces. PAR2 and TRPA1 immunoreactivity co-localized with CGRP-positive nerve fibres in control and post-colitis animals., Conclusions and Implications: Increased expression of serine proteases and activity together with increased expression of downstream molecules at the colonic and DRG level and in CGRP-positive sensory nerve fibres imply a role for serine proteases in post-inflammatory visceral hypersensitivity. Our results support further investigation of serine protease inhibitors as an interesting treatment strategy for IBS-related visceral pain., (© 2018 The British Pharmacological Society.)
- Published
- 2018
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39. Prolyl carboxypeptidase activity in the circulation and its correlation with body weight and adipose tissue in lean and obese subjects.
- Author
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Kehoe K, Noels H, Theelen W, De Hert E, Xu S, Verrijken A, Arnould T, Fransen E, Hermans N, Lambeir AM, Venge P, Van Gaal L, and De Meester I
- Subjects
- Adult, Anthropometry, Aorta, Bariatric Surgery, Blood Cells enzymology, Diet, Reducing, Endothelial Cells enzymology, Female, Humans, Macrophages enzymology, Male, Middle Aged, Myocytes, Smooth Muscle enzymology, Obesity diet therapy, Obesity surgery, Plasma enzymology, Platelet Activation, Platelet-Rich Plasma enzymology, Weight Loss, Adipose Tissue chemistry, Body Weight, Carboxypeptidases blood, Obesity enzymology, Thinness enzymology
- Abstract
Background: Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight., Methods: PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0-29.9; 30.0-39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group., Results: White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found., Conclusion: Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2018
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40. Procarboxypeptidase U (proCPU, TAFI, proCPB2) in cerebrospinal fluid during ischemic stroke is associated with stroke progression, outcome and blood-brain barrier dysfunction.
- Author
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Mertens JC, Leenaerts D, Brouns R, Engelborghs S, Ieven M, De Deyn PP, Lambeir AM, and Hendriks D
- Subjects
- Aged, Aged, 80 and over, Biomarkers cerebrospinal fluid, Blood-Brain Barrier physiopathology, Brain Ischemia diagnosis, Brain Ischemia physiopathology, Capillary Permeability, Case-Control Studies, Disease Progression, Female, Humans, Male, Middle Aged, Prognosis, Stroke diagnosis, Stroke physiopathology, Time Factors, Up-Regulation, Blood-Brain Barrier metabolism, Brain Ischemia cerebrospinal fluid, Carboxypeptidase B2 cerebrospinal fluid, Enzyme Precursors cerebrospinal fluid, Stroke cerebrospinal fluid
- Abstract
Essentials Little is known of procarboxypeptidase U (proCPU) in cerebrospinal fluid (CSF) of stroke patients. ProCPU levels were studied in CSF of controls and non-thrombolyzed acute ischemic stroke patients. ProCPU is elevated in CSF of stroke patients compared with controls. ProCPU in CSF correlates with stroke progression, outcome, and blood-brain barrier dysfunction., Summary: Background Procarboxypeptidase U (proCPU, TAFI, proCPB2), the zymogen of CPU, which is a potent antifibrinolytic enzyme and a modulator of inflammation, has previously been investigated in plasma of stroke patients, but so far, no information on the proCPU levels in cerebrospinal fluid (CSF) during acute ischemic stroke (AIS) is available. Objectives This case-control observational study investigates proCPU in CSF of AIS patients compared with controls with an intact blood-brain barrier (BBB) and evaluates the relationship of CSF/plasma proCPU ratios with stroke parameters. Methods A sensitive HPLC-based enzymatic assay was used to determine proCPU levels in CSF of non-thrombolyzed patients in the hyperacute phase (< 24 h after onset) of AIS (n = 72). Individuals (n = 32) without stroke, an intact BBB and no apparent abnormalities in biochemical and microbiological tests, served as controls. Relations between the CSF/plasma proCPU ratio and (i) stroke severity, (ii) stroke progression/recurrence, (iii) stroke outcome and (iv) BBB dysfunction (CSF/serum albumin ratio) were assessed. Results Mean (SEM) proCPU levels were elevated in the CSF of stroke patients compared with controls (4.36 (0.23) U L
-1 vs. 3.50 (0.23) U L-1 ). Higher median [IQR] CSF/plasma proCPU ratios were found in patients with stroke progression ((6.0 [4.2-6.9]) × 10-3 ) and poor outcome ((6.4 [3.9-7.0]) × 10-3 ) after 3 months (modified Rankin Scale; mRS > 3) compared with patients without progression ((3.9 [2.7-5.4]) × 10-3 ) or better outcome ((4.0 [2.8-5.0]) × 10-3 ). In stroke patients with a disrupted BBB, proCPU ratios were higher compared with stroke patients with an intact BBB ((6.4 [5.8-9.0]) × 10-3 vs. (3.7 [2.8-5.0]) × 10-3 ). Conclusions ProCPU is increased in CSF during hyperacute ischemic stroke and is associated with stroke progression and outcome after 3 months, most likely due to BBB dysfunction in the hyperacute phase of ischemic stroke., (© 2017 International Society on Thrombosis and Haemostasis.)- Published
- 2018
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41. Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.
- Author
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Rea D, Van Elzen R, De Winter H, Van Goethem S, Landuyt B, Luyten W, Schoofs L, Van Der Veken P, Augustyns K, De Meester I, Fülöp V, and Lambeir AM
- Subjects
- Crystallography, X-Ray, Dipeptidyl Peptidase 4 chemistry, Dipeptidyl-Peptidase IV Inhibitors chemical synthesis, Dipeptidyl-Peptidase IV Inhibitors chemistry, Dose-Response Relationship, Drug, Humans, Models, Molecular, Molecular Structure, Structure-Activity Relationship, Dipeptidyl Peptidase 4 metabolism, Dipeptidyl-Peptidase IV Inhibitors pharmacology, Porphyromonas gingivalis enzymology
- Abstract
The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)
- Published
- 2017
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42. Plasma carboxypeptidase U (CPU, CPB2, TAFIa) generation during in vitro clot lysis and its interplay between coagulation and fibrinolysis.
- Author
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Leenaerts D, Aernouts J, Van Der Veken P, Sim Y, Lambeir AM, and Hendriks D
- Subjects
- Adult, Aged, Aged, 80 and over, Algorithms, Biomarkers blood, Blood Coagulation Tests, Carboxypeptidase B2 genetics, Enzyme Activation, Female, Fibrinolysin metabolism, Genotype, Healthy Volunteers, Humans, Male, Middle Aged, Phenotype, Polymorphism, Genetic, Thrombin metabolism, Thrombomodulin blood, Time Factors, Young Adult, Blood Coagulation genetics, Carboxypeptidase B2 blood, Fibrinolysis genetics
- Abstract
Carboxypeptidase U (CPU, CPB2, TAFIa) is a basic carboxypeptidase that is able to attenuate fibrinolysis. The inactive precursor procarboxypeptidase U is converted to its active form by thrombin, the thrombin-thrombomodulin complex or plasmin. The aim of this study was to investigate and characterise the time course of CPU generation in healthy individuals. In plasma of 29 healthy volunteers, CPU generation was monitored during in vitro clot lysis. CPU activity was measured by means of an enzymatic assay that uses the specific substrate Bz-o-cyano-Phe-Arg. An algorithm was written to plot the CPU generation curve and calculate the parameters that define it. In all individuals, CPU generation was biphasic. Marked inter-individual differences were present and a reference range was determined. The endogenous CPU generation potential is the composite effect of multiple factors. With respect to the first CPU activity peak characteristics, we found correlations with baseline proCPU concentration, proCPU Thr325Ile polymorphism, time to clot initiation and the clot lysis time. The second CPU peak related with baseline proCPU levels and with the maximum turbidity of the clot lysis profile. In conclusion, our method offers a technique to determine the endogenous CPU generation potential of an individual. The parameters obtained by the method quantitatively describe the different mechanisms that influence CPU generation during the complex interplay between coagulation and fibrinolysis, which are in line with the threshold hypothesis.
- Published
- 2017
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43. Dynamics and ligand-induced conformational changes in human prolyl oligopeptidase analyzed by hydrogen/deuterium exchange mass spectrometry.
- Author
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Tsirigotaki A, Elzen RV, Veken PV, Lambeir AM, and Economou A
- Subjects
- Amino Acid Sequence, Catalytic Domain, Cloning, Molecular, Crystallography, X-Ray, Deuterium Exchange Measurement methods, Dipeptides metabolism, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Humans, Kinetics, Ligands, Mass Spectrometry methods, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Models, Molecular, Proline chemistry, Proline metabolism, Protease Inhibitors metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Substrate Specificity, Dipeptides chemistry, Mitochondrial Proteins chemistry, Proline analogs & derivatives, Protease Inhibitors chemistry, Serine Endopeptidases chemistry
- Abstract
Prolyl oligopeptidase (PREP) is conserved in many organisms across life. It is involved in numerous processes including brain function and neuropathology, that require more than its strict proteolytic role. It consists of a seven-bladed β-propeller juxtaposed to a catalytic α/β-hydrolase domain. The conformational dynamics of PREP involved in domain motions and the gating mechanism that allows substrate accessibility remain elusive. Here we used Hydrogen Deuterium eXchange Mass Spectrometry (HDX-MS) to derive the first near-residue resolution analysis of global PREP dynamics in the presence or absence of inhibitor bound in the active site. Clear roles are revealed for parts that would be critical for the activation mechanism. In the free state, the inter-domain interface is loose, providing access to the catalytic site. Inhibitor binding "locks" the two domains together exploiting prominent interactions between the loop of the first β-propeller blade and its proximal helix from the α/β-hydrolase domain. Loop A, thought to drive gating, is partially stabilized but remains flexible and dynamic. These findings provide a conformational guide for further dissection of the gating mechanism of PREP, that would impact drug development. Moreover, they offer a structural framework against which to study proteolysis-independent interactions with disordered proteins like α-synuclein involved in neurodegenerative disease.
- Published
- 2017
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44. Regulation of intestinal permeability: The role of proteases.
- Author
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Van Spaendonk H, Ceuleers H, Witters L, Patteet E, Joossens J, Augustyns K, Lambeir AM, De Meester I, De Man JG, and De Winter BY
- Subjects
- Animals, Diabetes Mellitus, Type 1 physiopathology, Electrolytes, Epithelial Cells metabolism, Humans, Inflammatory Bowel Diseases physiopathology, Matrix Metalloproteinase Inhibitors chemistry, Mice, Permeability, Protease Inhibitors chemistry, Serine Proteinase Inhibitors chemistry, Tight Junctions, Treatment Outcome, Inflammation physiopathology, Intestines pathology, Peptide Hydrolases metabolism
- Abstract
The gastrointestinal barrier is - with approximately 400 m
2 - the human body's largest surface separating the external environment from the internal milieu. This barrier serves a dual function: permitting the absorption of nutrients, water and electrolytes on the one hand, while limiting host contact with noxious luminal antigens on the other hand. To maintain this selective barrier, junction protein complexes seal the intercellular space between adjacent epithelial cells and regulate the paracellular transport. Increased intestinal permeability is associated with and suggested as a player in the pathophysiology of various gastrointestinal and extra-intestinal diseases such as inflammatory bowel disease, celiac disease and type 1 diabetes. The gastrointestinal tract is exposed to high levels of endogenous and exogenous proteases, both in the lumen and in the mucosa. There is increasing evidence to suggest that a dysregulation of the protease/antiprotease balance in the gut contributes to epithelial damage and increased permeability. Excessive proteolysis leads to direct cleavage of intercellular junction proteins, or to opening of the junction proteins via activation of protease activated receptors. In addition, proteases regulate the activity and availability of cytokines and growth factors, which are also known modulators of intestinal permeability. This review aims at outlining the mechanisms by which proteases alter the intestinal permeability. More knowledge on the role of proteases in mucosal homeostasis and gastrointestinal barrier function will definitely contribute to the identification of new therapeutic targets for permeability-related diseases., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest.- Published
- 2017
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45. The expression of proline-specific enzymes in the human lung.
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Vliegen G, Raju TK, Adriaensen D, Lambeir AM, and De Meester I
- Abstract
The pathophysiology of lung diseases is very complex and proteolytic enzymes may play a role or could be used as biomarkers. In this review, the literature was searched to make an overview of what is known on the expression of the proline-specific peptidases dipeptidyl peptidase (DPP) 4, 8, 9, prolyl oligopeptidase (PREP) and fibroblast activation protein α (FAP) in the healthy and diseased lung. Search terms included asthma, chronic obstructive pulmonary disease (COPD), lung cancer, fibrosis, ischemia reperfusion injury and pneumonia. Knowledge on the loss or gain of protein expression and activity during disease might tie these enzymes to certain cell types, substrates or interaction partners that are involved in the pathophysiology of the disease, ultimately leading to the elucidation of their functional roles and a potential therapeutic target. Most data could be found on DPP4, while the other enzymes are less explored. Published data however often appear to be conflicting, the applied methods divers and the specificity of the assays used questionable. In conclusion, information on the expression of the proline-specific peptidases in the healthy and diseased lung is lacking, begging for further well-designed research., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.
- Published
- 2017
- Full Text
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46. Ligand-induced conformational changes in prolyl oligopeptidase: a kinetic approach.
- Author
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Van Elzen R, Schoenmakers E, Brandt I, Van Der Veken P, and Lambeir AM
- Subjects
- Animals, Enzyme Stability, Humans, Hydrogen-Ion Concentration, Kinetics, Mitochondrial Proteins metabolism, Protein Domains, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Serine Endopeptidases metabolism, Swine, Mitochondrial Proteins chemistry, Serine Endopeptidases chemistry
- Abstract
Most kinetic studies of prolyl oligopeptidase (PREP) were performed with the porcine enzyme using modified peptide substrates. Yet recent biophysical studies used the human homolog. Therefore, the aim of this study was to compare the kinetic behavior of human and porcine PREP, as well as to find a suitable method to study enzyme kinetics with an unmodified biological substrate. It was found that human PREP behaves identically to the porcine homolog, displaying a double bell-shaped pH profile and a pH-dependent solvent kinetic isotope effect of the kcat/Km, features that set it apart from the related exopeptidase dipeptidyl peptidase IV (DPP IV). However, the empirical temperature coefficient Q10, describing the temperature dependency of the kinetic parameters and the non-linear Arrhenius plot of kcat/Km are common characteristics between PREP and DPP IV. The results also demonstrate the feasibility of microcalorimetry for measuring turn-over of proline containing peptides., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
- Published
- 2017
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47. Visceral hypersensitivity in inflammatory bowel diseases and irritable bowel syndrome: The role of proteases.
- Author
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Ceuleers H, Van Spaendonk H, Hanning N, Heirbaut J, Lambeir AM, Joossens J, Augustyns K, De Man JG, De Meester I, and De Winter BY
- Subjects
- Abdominal Pain drug therapy, Abdominal Pain enzymology, Abdominal Pain physiopathology, Animals, Humans, Hyperalgesia drug therapy, Hyperalgesia enzymology, Hyperalgesia physiopathology, Inflammatory Bowel Diseases drug therapy, Inflammatory Bowel Diseases enzymology, Inflammatory Bowel Diseases physiopathology, Intestinal Absorption, Intestines innervation, Irritable Bowel Syndrome drug therapy, Irritable Bowel Syndrome enzymology, Irritable Bowel Syndrome physiopathology, Permeability, Protease Inhibitors therapeutic use, Receptors, Proteinase-Activated metabolism, Signal Transduction, Abdominal Pain etiology, Hyperalgesia etiology, Inflammatory Bowel Diseases complications, Intestines enzymology, Irritable Bowel Syndrome complications, Peptide Hydrolases metabolism
- Abstract
Proteases, enzymes catalyzing the hydrolysis of peptide bonds, are present at high concentrations in the gastrointestinal tract. Besides their well-known role in the digestive process, they also function as signaling molecules through the activation of protease-activated receptors (PARs). Based on their chemical mechanism for catalysis, proteases can be classified into several classes: serine, cysteine, aspartic, metallo- and threonine proteases represent the mammalian protease families. In particular, the class of serine proteases will play a significant role in this review. In the last decades, proteases have been suggested to play a key role in the pathogenesis of visceral hypersensitivity, which is a major factor contributing to abdominal pain in patients with inflammatory bowel diseases and/or irritable bowel syndrome. So far, only a few preclinical animal studies have investigated the effect of protease inhibitors specifically on visceral sensitivity while their effect on inflammation is described in more detail. In our accompanying review we describe their effect on gastrointestinal permeability. On account of their promising results in the field of visceral hypersensitivity, further research is warranted. The aim of this review is to give an overview on the concept of visceral hypersensitivity as well as on the physiological and pathophysiological functions of proteases herein., Competing Interests: Conflict-of-interest statement: No potential conflicts of interest.
- Published
- 2016
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48. Inhibitor screening and enzymatic activity determination for autophagy target Atg4B using a gel electrophoresis-based assay.
- Author
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Cleenewerck M, Grootaert MOJ, Gladysz R, Adriaenssens Y, Roelandt R, Joossens J, Lambeir AM, De Meyer GRY, Declercq W, Augustyns K, Martinet W, and Van der Veken P
- Subjects
- Cysteine Proteinase Inhibitors metabolism, Drug Evaluation, Preclinical, Electrophoresis, Humans, Temperature, Autophagy drug effects, Autophagy-Related Proteins antagonists & inhibitors, Autophagy-Related Proteins metabolism, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors pharmacology, Enzyme Assays
- Abstract
Atg4B is a cysteine hydrolase that plays a key role in autophagy. Although it has been proposed as an attractive drug target, inhibitor discovery has proven highly challenging. The absence of a standardized, easily implementable enzyme activity/inhibition assay for Atg4B most likely contributes to this situation. Therefore, three different assay types for Atg4B activity/inhibition quantification were first compared: (1) an approach using fluorogenic Atg4B-substrates, (2) an in-gel densitometric quantification assay and (3) a thermal shift protocol. The gel-based approach showed the most promising results and was validated for screening of potential Atg4B inhibitors. A set of 8 literature inhibitors was included. Remarkably, in our hands only 2 literature references were found to have measurable Atg4B affinity. Furthermore, a fragment library (n = 182) was tested for Atg4B inhibition. One library member showed inhibition at high micromolar concentration and was found fit for further, fragment-based inhibitor design., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)
- Published
- 2016
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49. Prolyl carboxypeptidase purified from human placenta: its characterization and identification as an apelin-cleaving enzyme.
- Author
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Kehoe K, Van Elzen R, Verkerk R, Sim Y, Van der Veken P, Lambeir AM, and De Meester I
- Subjects
- Amino Acid Sequence, Animals, Apelin, Carboxypeptidases genetics, Carboxypeptidases isolation & purification, Carboxypeptidases metabolism, Colipases chemistry, Colipases genetics, Colipases metabolism, Energy Metabolism genetics, Enzyme Precursors chemistry, Enzyme Precursors genetics, Enzyme Precursors metabolism, Female, Gene Expression, Ghrelin chemistry, Ghrelin genetics, Ghrelin metabolism, Human Umbilical Vein Endothelial Cells, Humans, Intercellular Signaling Peptides and Proteins genetics, Intercellular Signaling Peptides and Proteins metabolism, Kinetics, Placenta enzymology, Pregnancy, Proteolysis, Substrate Specificity, alpha-MSH genetics, alpha-MSH metabolism, Carboxypeptidases chemistry, Intercellular Signaling Peptides and Proteins chemistry, Placenta chemistry, alpha-MSH chemistry
- Published
- 2016
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50. Prolyl endopeptidase is involved in the degradation of neural cell adhesion molecules in vitro.
- Author
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Jaako K, Waniek A, Parik K, Klimaviciusa L, Aonurm-Helm A, Noortoots A, Anier K, Van Elzen R, Gérard M, Lambeir AM, Roßner S, Morawski M, and Zharkovsky A
- Subjects
- Animals, Antibodies, Neutralizing metabolism, Blotting, Western, Cell Differentiation drug effects, Cell Line, Tumor, Cells, Cultured, Culture Media, ErbB Receptors metabolism, Gene Knockdown Techniques, Immunohistochemistry, Matrix Metalloproteinase 9 metabolism, Neural Cell Adhesion Molecule L1 metabolism, Neuroblastoma metabolism, Neurons drug effects, Neurons metabolism, Phosphorylation drug effects, Prolyl Oligopeptidases, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Recombinant Proteins pharmacology, Sialic Acids metabolism, Sialyltransferases metabolism, Neural Cell Adhesion Molecules metabolism, Proteolysis drug effects, Serine Endopeptidases metabolism
- Abstract
Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120 kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM., (© 2016. Published by The Company of Biologists Ltd.)
- Published
- 2016
- Full Text
- View/download PDF
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