Research team

Medical Biochemistry

Expertise

- Prolyl oligopeptidase in the central nervous system and prolyl oligopeptidase inhibitors: new therapeutic strategies in neurodegenerative disease and Carboxypeptidase M: substrates and interaction partners in health and disease (kidney, adipocytes). - Eiwit engineering en structuurgebaseerde ontwikkeling van geneesmiddelen: aspecten van enzymologie, eiwitchemie en moleculaire biologie. - Know-how in enzymology: research on catalytic mechanisms, kinetics and characterisation of enzyme inhibitors. Enzymes currently under investigation: prolyl oligopeptidase, carboxypeptidase M, dipeptidyl peptidase 4 (porphyromonas gingivalis). Other enzymes: proline specific peptidases, serine proteases of the coagulation and fibrinolysis systems, glucose isomerase, amylases and glucoamylases, glycolytic enzymes (such as triosephosphate isomerase, glyceraldehydephosphate dehydrogenase, glycerol kinase, hexokinase), thiolases, glutathionyl-spermidine synthase, peroxidases, prostaglandin H synthase (COX-1).-

Carboxypeptidase U - a new drug target for the improvement of treatment in acute ischemic stroke. 01/10/2018 - 30/09/2020

Abstract

Thrombolysis with tissue plasminogen activator remains the only approved pharmacological treatment for acute ischemic stroke, AIS. Besides the narrow therapeutic time window, its use is limited by its efficacy: in up to 50% of the treated patients, timely recanalization is not achieved. Moreover, administration involves serious side effects such as intracranial hemorrhage and neurotoxicity. Consequently, the search for new agents for improvement of AIS treatment is urgently needed. Research has demonstrated that the enzyme carboxypeptidase U (CPU, TAFIa) is an important player in thrombus lysis. After activation from its precursor proCPU, the released CPU is able to potently attenuate fibrinolysis. Consequently, inhibition of CPU activity is a novel approach to enhance fibrinolysis. We want to explore the involvement of this enzyme in AIS in more detail. The usefulness of CPU as a diagnostic marker to discriminate ischemic from hemorrhagic stroke and the relationship of CPU with clinical outcome and thrombolytic treatment efficacy will be investigated. We plan to optimize the Thrombodynamics assay in order to assess the effect of CPU-inhibition on clot lysis during thrombolysis. Furthermore, in a preclinical setting, we will evaluate the effect of CPU inhibition in an experimental stroke model in rats. This research will provide essential information on the role of the CPU system and the usefulness of CPU inhibitors as potentially efficient and safer treatment of AIS.

Researcher(s)

Research team(s)

The Biomolecular Interaction Platform (BIP) at UAntwerp. 01/05/2018 - 30/04/2021

Abstract

Physical and functional interactions between biomolecules play pivotal roles in all aspects of human health and disease. Gaining a greater understanding of these biomolecular interactions will further expand our understanding of diseases such as cancer, metabolic diseases and neurodegeneration. At UAntwerp, 7 research groups have joined forces to obtain the absolutely necessary equipment to measure these interactions with a Biomolecular Interactions Platform (BIP). This will allow to detect interactions and precisely determine binding affinities between any kind of molecule, from ions and small molecules to high-molecular weight and multi-protein complexes. The BIP will also allow to identify collateral off- targets, crucial in the drug discovery field. Access to a BIP will strongly support ongoing research projects and bring research within the BIP-consortium to a higher level. Since biomolecular interactions are highly influenced by the methodology, it is recommended to measure the interaction by several, independent techniques and continue with the most appropriate one. For this reason, the consortium aims at installing a BIP, consisting of several complementary instruments that each measure biomolecular interactions based on different physical principles. They wish to expand the existing Isothermal Titration Calorimetry with two complementary state-of-the- art techniques: MicroScale Thermophoresis and Grating- Coupled waveguide Interferometry.

Researcher(s)

Research team(s)

Inhibitors of prolyl oligopeptidase (PREP) as novel candidates for tackling synucleinopathies: insight in structural, thermodynamic and kinetic parameters that determine inhibitor potential to block PREP-promoted alpha-Synuclein aggregation 01/10/2017 - 30/09/2020

Abstract

Inadequate perfusion, oxygen limitation and cell metabolic changes, are key factors contributing to the formation of an acidic microenvironment in tumors.1 Two pivotal adaptations of tumor cells, related to maintaining intracellular pH and homeostasis in an acidic environment, have recently received significant attention: (1) the presence of chronic autophagy and (2) the overexpression of carbonic anhydrases (CAs), mainly CA IX and CA XII. Aiming to counter these essential tumor survival processes, the proposal deals with discovering and thoroughly evaluating novel autophagy inhibitors and and dual [autophagy-CA] inhibitors. The following three Work Packages will be elaborated during the course of the project: 1) Biopharmaceutical optimization of the host's Atg4B-inhibitor set. 2) Design and synthesis of dual [Atg4B-CA] inhibitors. 3) Biological characterization of Atg4B- and hybrid [Atg4B-CA] inhibitors in acidic cancer cell cultures and in an animal model of colorectal cancer.

Researcher(s)

Research team(s)

TRP channel sensitization as target for treatment of hypersensitivity (TRP-sensation). 01/07/2017 - 30/06/2021

Abstract

IBS affects around 18% of the general population. It is one of the most common disorders seen by physicians. However, the IBS market is commercially weak due to the limited understanding of its pathophysiology and the availability of limited treatment options. In fact, IBS is largely seen as a syndrome rather than a disease. By increasing the understanding of the underlying mechanisms of IBS coupled to validation of therapeutic and diagnostic targets, we have the ambition to turn IBS "from a syndrome into a disease". To achieve this, we want to establish an academic knowledge platform and an industrial network in Flanders that is able to tackle the major challenges in the IBS field, to identify and validate novel therapeutic and diagnostic targets and to develop them into novel therapeutic and diagnostic solutions. When available, this network will put Flanders at the forefront of innovation in the emerging IBS field.

Researcher(s)

Research team(s)

Carboxypeptidase U - a new drug target for the improvement of treatment in acute ischemic stroke. 01/10/2016 - 30/09/2018

Abstract

Thrombolysis with tissue plasminogen activator remains the only approved pharmacological treatment for acute ischemic stroke, AIS. Besides the narrow therapeutic time window, its use is limited by its efficacy: in up to 50% of the treated patients, timely recanalization is not achieved. Moreover, administration involves serious side effects such as intracranial hemorrhage and neurotoxicity. Consequently, the search for new agents for improvement of AIS treatment is urgently needed. Research has demonstrated that the enzyme carboxypeptidase U (CPU, TAFIa) is an important player in thrombus lysis. After activation from its precursor proCPU, the released CPU is able to potently attenuate fibrinolysis. Consequently, inhibition of CPU activity is a novel approach to enhance fibrinolysis. We want to explore the involvement of this enzyme in AIS in more detail. The usefulness of CPU as a diagnostic marker to discriminate ischemic from hemorrhagic stroke and the relationship of CPU with clinical outcome and thrombolytic treatment efficacy will be investigated. We plan to optimize the Thrombodynamics assay in order to assess the effect of CPU-inhibition on clot lysis during thrombolysis. Furthermore, in a preclinical setting, we will evaluate the effect of CPU inhibition in an experimental stroke model in rats. This research will provide essential information on the role of the CPU system and the usefulness of CPU inhibitors as potentially efficient and safer treatment of AIS.

Researcher(s)

Research team(s)

Prolyl carboxypeptidase in peripheral body weight control and glucose homeostasis: is cleavage of apelin-13 playing a role? 01/10/2016 - 31/12/2016

Abstract

The complex mechanism involved in the pathogenesis of these metabolic disorders remains poorly understood. Recent findings indicate that the enzyme prolyl carboxypeptidase (PRCP) plays a role in body weight control and glucose homeostasis by inactivating melanocyt-stimulating hormone MSH, a neuropeptide which causes a loss of appetite. We aim to investigate whether 'peripheral' (outside the central nervous system) actions of PRCP also contribute to this function. Our preliminary experiments have identified apelin, which also regulates feeding behaviour and glucose metabolism, as a novel PRCP substrate. Firstly, we will characterize this cleavage in vitro and investigate whether truncation by PRCP alters the function of apelin and other peptide substrates. Secondly, the role of PRCP on in vivo cleavage of peripheral apelin will be investigated.

Researcher(s)

Research team(s)

The role of carboxypeptidase U in atherosclerosis and its thrombotic complications. 01/10/2015 - 30/09/2017

Abstract

In the first stage of the project we will extend the knowledge about assays for the measurement of CPU, i.e. the clot lysis assay and an ultrasensitive activity-based method. Next we will use these techniques to investigate the role of CPU in patients with acute myocardial infarction. A third objective is to explore the localization of CPU in atherosclerotic plaques and thrombi. Finally, we will evaluate the role of CPU in a novel plaque rupture model. Investigating the role of CPU in atherosclerosis and its clinical manifestations may open new approaches for plaque stabilising therapies and treatment of acute ischemic syndromes.

Researcher(s)

Research team(s)

Medicinal Chemistry-Drug Discovery (ADDN). 01/01/2015 - 31/12/2020

Abstract

This project represents a research contract awarded by the University of Antwerp. The supervisor provides the Antwerp University research mentioned in the title of the project under the conditions stipulated by the university.

Researcher(s)

Research team(s)

INFLA-MED - Fundamental research in the pathophysiological processes of inflammatory diseases. 01/01/2015 - 31/12/2019

Abstract

The Infla-Med consortium performs fundamental research on the pathophysiological processes of inflammatory diseases (cardiovascular, gastrointestinal, renal and infectious disease) by using a multidisciplinary approach (pathophysiology, pharmacology, biochemistry and medicinal chemistry). The consortium is embedded within the research priorities 'Drug Research' and 'Infectious Diseases' of the University of Antwerp. Recently, the University of Antwerp assigned the Infla-Med consortium as Research Consortium of Excellence.

Researcher(s)

Research team(s)

Prolyl oligopeptidase: partners and pathways in neuronal function and neurodegeneration. 01/01/2015 - 31/12/2018

Abstract

In this project we aim (1) to characterize at the molecular level the role of PREP in hallmark processes of neurodegenerative disease: neuronal death, synapse loss and neuroinflammation; and (2) to investigate whether PREP-inhibitors can be used to modulate these processes.

Researcher(s)

Research team(s)

Expression and role of dipeptidyl peptidases and related peptidases in acute lung injury. 01/01/2015 - 31/12/2018

Abstract

Acute lung injury remains the third major cause of mortality worldwide, and it is assumed that excessive inflammatory responses could be involved. The precise role of dipeptidyl peptidases (DPPs; a family of enzymes that cleave off dipeptides from the amino terminus of peptides) in the pathophysiology of acute lung injury is poorly understood. Taken broadly, the DPP family consists of DPPIV, fibroblast activation protein alpha (FAP), prolyl oligopeptidase (PREP), DPP8 and DPP9. DPPIV inhibitors are used in the treatment of diabetes type 2, but evidence for other roles of DPPIV is also emerging. Despite a presumed role of individual peptidases in lung disease, knowledge on DPPs in acute lung injury remains limited. Previously, we have shown that DPPIV inhibitors protect against lung ischemia-reperfusion induced injury. Apart from that, we discovered that DPP9 has a role in macrophage activation, which is an important component of acute lung injury. The current project aims to explore the hypothesis that DPPIV, DPP9 and related peptidases play a role in the pathophysiology of acute lung injury. We will study the expression of DPPs in both an infectious and a non-infectious mouse model of acute lung injury. Subsequently, we will determine the effect of DPPIV inhibition on the outcome, and will assess whether DPPs have a role in lung macrophages. We will compare the animal findings with measurements in human tissue to study the translational potential of our results.

Researcher(s)

Research team(s)

Prolylcarboxypeptidase in body weight control: a role for peripheral peptide cleavage? 01/10/2014 - 30/09/2016

Abstract

In a first part of the project we will investigate the protein expression and enzymatic activity of PRCP in the circulation. Thereafter, we will perform in vitro experiments to study the cleavage of apelin and enterostatin by PRCP and compare cleavage efficiency with other known PRCP substrates. A third objective is to investigate apelin and enterostatin cleavage by PRCP in vivo and to find out whether PRCP influences their effect on body weight and metabolism. Understanding how peptide hormones are degraded by PRCP may open new approaches for therapeutic intervention.

Researcher(s)

Research team(s)

An in vitro and ex vivo Study on the Expression and Role of Dipeptidyl Peptidase 9 in the Lung. 01/10/2014 - 30/09/2015

Abstract

This project fits in the study of the role and expression of DPP9 in lung pathophysiology with an emphasis on lung fibrosis. However, DPP9 is part of a larger family of DPPs with structural similarities and overlapping substrate specificities. Initially, we want to establish a DPP atlas of the lung to provide context to the study of DPP9 specifically. Our second and main objective is to form a picture of DPP9's role and expression in the lung, starting from our current knowledge of DPP9 in human macrophages, towards a broader take on DPP9 and lung fibrosis.

Researcher(s)

Research team(s)

Equipment for high-speed refrigerated, preparative ultracentrifugation, automated gradient formation and fraction collection and analysis. 19/05/2014 - 31/12/2018

Abstract

This project represents a formal research agreement between UA and on the other hand the Hercules Foundation. UA provides the Hercules Foundation research results mentioned in the title of the project under the conditions as stipulated in this contract.

Researcher(s)

Research team(s)

Creation of a preclinical platform at the UA for testing novel therapeutic approaches against ocular surface diseases. 01/01/2014 - 31/12/2015

Abstract

Ocular Surface diseases (OSD) such as dry eye syndrome (DES) show an estimated prevalence between 15 and 29%. The only FDA approved and on subscription dry-eye treatment is cyclosporine 0.05% (Restasis®), but this formulation is not available in the EU. Novel therapies for OSD are therefore needed. The expertise within ADDN fosters a unique opportunity to set up a preclinical platform on OSD leading to an increased collaboration with industrial partners.

Researcher(s)

Research team(s)

The role of carboxypeptidase U in atherosclerosis and its thrombotic complications. 01/10/2013 - 30/09/2015

Abstract

In the first stage of the project we will extend the knowledge about assays for the measurement of CPU, i.e. the clot lysis assay and an ultrasensitive activity-based method. Next we will use these techniques to investigate the role of CPU in patients with acute myocardial infarction. A third objective is to explore the localization of CPU in atherosclerotic plaques and thrombi. Finally, we will evaluate the role of CPU in a novel plaque rupture model. Investigating the role of CPU in atherosclerosis and its clinical manifestations may open new approaches for plaque stabilising therapies and treatment of acute ischemic syndromes.

Researcher(s)

Research team(s)

4D Protein Structure. 01/01/2013 - 31/12/2016

Abstract

In our research we will investigate two recent, complementary biophysical approaches, 'native' ion mobility/mass spectrometry (IM-MS) and Vibrational Optical Activity (VOA), to investigate structure and dynamics of proteins of biomedical interest. Specifically we will benchmark these techniques against well-studied protein systems, and apply them to probe changes in the conformational and assembly space of globins and oligopeptidases.

Researcher(s)

Research team(s)

    Developoing new tools for (un)structural biology by ion mobility-mass spectrometry and related methods.. 01/10/2012 - 31/01/2016

    Abstract

    In our research we will use state-of-the-art 'native' mass spectrometric techniques to investi-gate both the dynamic as well as structural characteristics of membrane (associated) proteins. Furthermore will we probe the changes in conformational space of proteins when they interact with ligand or other proteins using ion mobility-mass spectrometry, with a special focus on intrinsically disordered proteins.

    Researcher(s)

    Research team(s)

      Prolylcarboxypeptidase in body weight control: a role for peripheral peptide cleavage? 01/10/2012 - 30/09/2014

      Abstract

      In a first part of the project we will investigate the protein expression and enzymatic activity of PRCP in the circulation. Thereafter, we will perform in vitro experiments to study the cleavage of apelin and enterostatin by PRCP and compare cleavage efficiency with other known PRCP substrates. A third objective is to investigate apelin and enterostatin cleavage by PRCP in vivo and to find out whether PRCP influences their effect on body weight and metabolism. Understanding how peptide hormones are degraded by PRCP may open new approaches for therapeutic intervention.

      Researcher(s)

      Research team(s)

      Carboxypeptidase M, substrates and ligands. 01/10/2010 - 31/03/2012

      Abstract

      The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

      Researcher(s)

      Research team(s)

      Prolyl oligopeptidase and prolyl oligopeptidase inhibitors in models of neurodegeneration. 01/10/2009 - 31/03/2010

      Abstract

      The objective of this project is to investigate the interactions between prolyl oligopeptidase and relevant biomolecules, in vitro and in the cell. We envisage three lines of research: (1) the interaction with alpha-synuclein and the connection with Parkinson's disease, (2) the interaction with tubulin, intracellular transport and secretion, (3) co-localisation with other cytosolic peptidases and the connection with the aggresome.

      Researcher(s)

      Research team(s)

      Dipeptidyl peptidases beyond glucose homeostasis: from biochemistry to physiological importance. 01/01/2009 - 31/12/2012

      Abstract

      This project aims to better understand the effects of chronic dipeptidyl peptidase (DPP) inhibition on pre-defined aspects of cardiovascular, renal and bone (patho)physiology. Inhibitors with defined selectivity profiles will be developed as tools. Expression and inhibition of DPP4 and related peptidases will be studied on the molecular level, in cultured cells and in rat models of ischemia/reperfusion injury of heart and kidney.

      Researcher(s)

      Research team(s)

      Comparative biochemical and functional study of the different dipeptidyl peptidases. 01/01/2009 - 31/12/2012

      Abstract

      this research project studies proline selective peptidases. It has the following aims : 1. Characterization of inhibitors and selection of potent and specific inhibitors of DPP4 related enzymes DPP8, DPP9 and FAP. 2. Study of the expression of the DPPP mambers in endothelia of different origin and this under normoxia as well as hypoxia. 3. In vitro study of the effect of selective ans not selective inhibition on endothelial cell activation. 4. The in vitro study of the effect of selective and non-selective DPP inhibition on collagen metabolism of fibroblasts.

      Researcher(s)

      Research team(s)

      Bacterial virulence as new target for protease inhibitors. 01/01/2009 - 31/12/2012

      Abstract

      The goal of this project is to gain insight in the role and applicability of protease (DPP4) inhibitors in bacterial infections. The proof-of-concept will be obtained in Porphyromonas gingivalis models with the following objectives and work packages: 1. Development of in vitro and in vivo virulence models for P. gingivalis. 2. Evaluation of enzyme inhibitors using purified recombinant P. gingivalis DPP4. 3. Evaluation of DPP/protease inhibitors in bacterial in vitro and in vivo models. 4. SAR and optimisation of the lead compounds. 5. Biochemical characterisation of lead compound-target enzyme interactions.

      Researcher(s)

      Research team(s)

      High performance LC-tandem mass spectrometry as necessary equipment for strategic research elucidation of chemical biomolecules and quantitative determination of medicines, metabolites and biomolecules in biological matrices. 19/12/2008 - 18/12/2013

      Abstract

      This project represents a research contract awarded by the University of Antwerp. The supervisor provides the Antwerp University research mentioned in the title of the project under the conditions stipulated by the university.

      Researcher(s)

      Research team(s)

      Oligopeptidase inhibitors in brain function and dysfunction: towards new therapeutic strategies for neuroprotection (NEUROPRO). 01/10/2008 - 30/09/2012

      Abstract

      The three main objectives of the consortium are: 1) to unravel the mode of action of PREP and PREP-like enzymes in health and disease; 2) to develop new drugs: 3) to discover new therapeutic targets. In order to achieve this, studies will be performed to identify the physiological substrates PREP and PREP-like enzymes and to characterize the pathways in which they are involved.

      Researcher(s)

      Research team(s)

      Carboxypeptidase M, substrates and ligands. 01/10/2008 - 30/09/2010

      Abstract

      The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

      Researcher(s)

      Research team(s)

      Investigation of prolyl oligopeptidase as a therapeutic target for neuropathological diseases: inhibitors, substrates and ligands. 01/01/2008 - 31/12/2011

      Abstract

      The objective of this project is to investigate the interactions between P0 and relevant biomolecules, in vitro and in the cell, We envisage four lines of research: (1) the interaction with alpha-synuclein and the connection with Parkinsons disease, (2) the interaction with tubulin, intracellular transport and secretion, (3) co-localisation with other cytosolic peptidases and the connection with the aggresome, (4) comparative peptidomics of mouse brain after administration of a P0 inhibitor. The four research lines are interconnected.

      Researcher(s)

      Research team(s)

      Carboxypeptidase M, substrates and ligands. 01/10/2007 - 30/09/2008

      Abstract

      The aim of the project is to identify interaction partners of human carboxypeptidase M (CPM). The research plan has two components. On the one hand we study the interaction with bioactive peptides that are substrates for the carboxypeptidase enzymatic activity. On the other hand we investigate a potential role of this membrane bound protein in cell-cell interactions, extracellular matrix interactions and cell migration.

      Researcher(s)

      Research team(s)

      Substrate specificity of basic carboxypeptidases. 01/10/2006 - 31/12/2008

      Abstract

      The aims are to study systematically the substrate specificity of the basic carboxypeptidases CPU, CPN, CPM, proCPU and the pancreatic enzyme CPB. This will be done by means of a library of short peptides where 1 or 2 of the amino acids preceding the C-terminal arginine are varied. In addition the degradation of postulated natural substrates will be investigated. The knowledge of the substrate speicificty will be applied for the development of specifici enzymatic methods for CPU, CPN, and CPM in plasma or serum and for CPM on cell surfaces and membrane preparations.

      Researcher(s)

      Research team(s)

      Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2006 - 30/09/2008

      Abstract

      Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

      Researcher(s)

      Research team(s)

      Biochemical study of recently discovered human dipeptidyl peptidases. 01/01/2005 - 31/12/2008

      Abstract

      This research plan aims to answer the following specific questions concerning FAP, DPPII, DP8 and DP9. The experimental objectives can be grouped around 3 themes: chromogenic and fluorogenic substrates, peptide substrates and inhibitors. The experimental results will be interpreted with the aid of available structural information (crystal structure of DPIV is published recently; DP8 and 9 show primary sequence homology, DPPII does not) in order to obtain a better insight in substrate binding and catalysis by the members of this family of serine type peptidases.

      Researcher(s)

      Research team(s)

      Substrate specificity and structure-function relationships in the prolyl oligopeptidase family of serine proteases. 01/10/2004 - 30/09/2006

      Abstract

      Prolyloligopeptidase (PO) and dipeptidyl peptidase IV (DPP-IV) are proline specific peptidases. Their biological function is connected with the metabolism of biologically active peptides. It is generally assumed that they exert their catalytic function through the classical serine protease mechanism. The specificity and the selectivity are mainly determined by the primary binding site and a few sub-binding sites for adjacent amino acids. The substrate-specificity of DPP-IV for natural peptides could not be predicted based on these assumptions and the data obtained by using small dipeptide-derived substrates. It was not possible to unambiguously identify the rate limiting step in the mechanism from solvent isotope effects. Under certain conditions a conformational change seems to be rate limiting for the hydrolysis of chromogenic substrates by PO. The interpretation of structure-activity-relationships of inhibitors also remains very difficult. The object of this project is to get a better insight in the details of the catalytic mechanism of PO and DPP-IV. Different approaches are being considered: kinetic experiments, site-directed mutagenesis in the active site and the substrate binding sites in PO, kwantitative structure-activity relationships of inhibitors and ligands.

      Researcher(s)

      Research team(s)

      Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2004 - 30/09/2006

      Abstract

      Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

      Researcher(s)

      Research team(s)

      Molecular characterisation of carboxypeptidase M, an extracellular membrane-bound peptidase involved in the tissue-specific response toward biologically active peptides. 01/10/2003 - 30/09/2004

      Abstract

      Carboxypeptidase M (CPM) is a cell-surface bound carboxypeptidase which removes C-terminal basic aminoacids from bioactive peptides. CPM has been reported to be distributed in many organs and fluids. CPM is also present on immunological and inflammatory cells. However, a physiological role of CPM in these tissues has not yet been clearly defined. Commonly, it is accepted that CPM can be a player in the processing or inactivation of peptide hormones, growth factors, inflammatory important peptides, et cetera. As there is very little knowledge of the molecular properties of CPM, we aim to systematically characterise the catalytic mechanisme and substrate specificity of CPM. These investigations must allow to differentiate between the various carboxypeptidases, specifically concerning the substrate specificity. Carboxypeptidase U (CPU) inhibitors are currently under clinical investigation as adjuvans in the fibrinolytic therapy. The importance of studying the selectivity of the CPU inhibitors with regard to other carboxypeptidases is self-evident. In this context, CPM is particulary important because it is constitutionally expressed in many tissues. In the first phase of our project, CPM will be cloned, expressed and purified. A comparative study of the recombinant and natural enzyme will be performed. We wish to study the different elements of the catalytic proces using enzymological methods. The substrate specificity of CPM will be studied using site-directed mutagenesis. In the second phase, we aim to identify naturally occuring substrates of CPM in tissue extracts (proteomics analysis). Extracted polypeptides will be fractionated and analysed for the presence of substrate (LC-MS). The enzyme kinetics of these substrates will be studied in vitro. A last aim is to set up a database of CPM values in normal and clinical samples. The results of our study can be used in the development of new CPU inhibitors. New therapeutic strategies can be proposed if the involvement of CPM in inflammation is confirmed. The possibility of administering therpeutic peptides as aerosol is under research. The presence of CPM on type I alveolar cells can influence the absorption and pharmacokinetics of these drugs.

      Researcher(s)

      Research team(s)

      Substrate specificity and structure-function relationships in the prolyl oligopeptidase family of serine proteases. 01/10/2002 - 30/09/2004

      Abstract

      Prolyloligopeptidase (PO) and dipeptidyl peptidase IV (DPP-IV) are proline specific peptidases. Their biological function is connected with the metabolism of biologically active peptides. It is generally assumed that they exert their catalytic function through the classical serine protease mechanism. The specificity and the selectivity are mainly determined by the primary binding site and a few sub-binding sites for adjacent amino acids. The substrate-specificity of DPP-IV for natural peptides could not be predicted based on these assumptions and the data obtained by using small dipeptide-derived substrates. It was not possible to unambiguously identify the rate limiting step in the mechanism from solvent isotope effects. Under certain conditions a conformational change seems to be rate limiting for the hydrolysis of chromogenic substrates by PO. The interpretation of structure-activity-relationships of inhibitors also remains very difficult. The object of this project is to get a better insight in the details of the catalytic mechanism of PO and DPP-IV. Different approaches are being considered: kinetic experiments, site-directed mutagenesis in the active site and the substrate binding sites in PO, kwantitative structure-activity relationships of inhibitors and ligands.

      Researcher(s)

      Research team(s)

      Selective immunosuppresion via the CD26/DPPIV T cell activation marker. 01/01/1999 - 31/12/1999

      Abstract

      Evidence for the involvement of CD26 in T cell activation and proliferation is provided by several approaches using mAb and CD26 transfection in human leukemic T cell lines. Application of CD26 mAb recognizing the 5/9 epitope to deplete bone marrow for allegeneic bone marrow transplantation in leukemia results in low graft versus hart disease.

      Researcher(s)

      Research team(s)

        Selective immunosuppresion via the CD26/DPPIV T cell activation marker. 01/01/1997 - 31/12/2000

        Abstract

        Evidence for the involvement of CD26 in T cell activation and proliferation is provided by several approaches using mAb and CD26 transfection in human leukemic T cell lines. Application of CD26 mAb recognizing the 5/9 epitope to deplete bone marrow for allegeneic bone marrow transplantation in leukemia results in low graft versus hart disease.

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          Protein design studies with two monomeric TlM-barrel proteins: towards new active sites of chitinase and monomeric triosephosphate isomerase.(MONOTIM) 01/11/1996 - 31/10/1999

          Abstract

          In this project we aim to study the structural determinants of the enzymatic activity, substrate specificity and stability of monomeric TlM-barrel proteins, in particular of monoTlM and two related chitinases. This understanding should in the end permit the use of the TlM-barrel scaffold for the design and construction of enzymes with desired properties. By changing sequences in specified loops it will be attempted to change the specificity of monoTlM into a xylose isomerase. The chitinase studies will be aimed at making a more simple, more active and more stable chitinase with a rationally designed substrate specificity.

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