Physiological processes in glycobiology and immunology

Prof. dr. Herman Overkleeft

Irreversible, or mechanism-based, inhibitors are attractive tools in chemical biology. Attachment of a reporter entity, which can be a biotin, a fluorophore, a bioorthogonal tag, a radiolabel or a combination of these, allows for the identification, visualization, identification and spatiotemporal study of the modified enzyme(s) using various techniques and in complex biological systems ranging from cell extracts, living tissue to animal models. This is the field of activity-based protein profiling (ABPP). Within the group we develop activity-based probes (ABPs) for proteases, glycosidases and a range of other hydrolases and apply these in immunological and glycobiological studies related to health and disease. Together with these ABPs we develop focused libraries of (competitive) inhibitors. Together, this toolset allows us to perform a number of conceptual experiments. In comparative ABPP ABPs are used to discover new enzyme activities and to compare enzyme expression levels in different tissue. In competitive ABPP the focused inhibitor libraries are included to study efficacy and selectivity of these in complex biological samples. In fluorescence polarization ABPP we develop assays amenable for high-throughput screening to discover new chemical entities for inhibiting one of our target enzymes.

Our glycobiology research focuses on glycosphingolipid metabolism and abberations thereof in the context of lysosomal storage disorders. As an example, In Gaucher disease the enzyme, glucocerebrosidase (or lysosomal beta-glucosidase, GBA) is due to one of a series of genetic mutations less active. As a result, its substrate glucosylceramide accumulates, which is causative of the disease. In collaboration with the group of Hans Aerts research is focused on the development of inhibitors of the enzymes involved in glucosylceramide metabolism. These are glucosylceramide synthase (GCS), GBA and also the non-lysosomal hydrolase, GBA2. Each of these enzymes are putative drug targets, be it for Gaucher (GCS inhibitors are clinical practice) or diseases ranging from Parkinsonism (GBA, GBA2) and type 2 diabetes (GCS). Research is focused on the development of focused libraries of selective and cross-reactive competitive inhibitors against these enzymes. We also develop activity-based probes aimed at the glucosylceramide metabolizing enzymes and have identified a selective GBA probe, a pan-reactive retaining beta-glucosidase probe (GBA, GBA2, next to another human enzyme, GBA3) and recently also a suitable probe to detect the enzyme, ceramidase.

Our immunology research focuses on protein processing and antigen presentation. To distinct antigen presentation pathways exist by which antigenic peptides are presented to the immune system. In the Major histocompatibility complex (MHC) class I (MHCI) pathway, cytosolic and nuclear proteins are processed by the ubiquitin-proteasome system (UPS). Peptides produced by the UPS and further trimmed by downstream aminopeptidases and presented by MHCI molecules may contain virally encoded peptides, in which case immune surveillance should lead to a response aimed at eradicating the virally infected cells. The MHCII pathway consists of antigenic peptides emerging from endolysosomally processed proteins, which are internalized for this purpose by endocytosis. In this way, professional antigen presenting cells (dendritic cells, macrophages) report on infections by microorganisms. Our research, executed in collaboration with the LUMC (Ossendorp), the NKI (Neefjes, Ovaa) and in the context of the Institute of Chemical Immunology, aims at the development of inhibitors and activity-based probes directed at the individual steps that make up antigen presentation.

Projects
  • Synthesis of labeled complex glycosphingolipids (Patrick Wisse)
  • Design and synthesis of glycosidase activity based probes (Sybrin Schroder and Jianbing Jiang)
  • Design and synthesis of Glycosyl transferase inhibitors (Thomas Beenakker)
  • Subunit-selective proteasome inhibitors (Gerjan de Bruin and Bo-Tao Xin)
  • Iminosugars as glycosidase inhibitors (Bing Liu)
  1. Jiang, J., C.-L. Kuo, L. Wu, C. Franke, W.W. Kallemeijn, B.I. Florea, E. van Meel, G.A. van der Marel, et al., "Detection of Active Mammalian GH31 α-Glucosidases in Health and Disease Using In-Class, Broad-Spectrum Activity-Based Probes", ACS Central Science, vol. 2, issue 5, pp. 351 - 358, 05/2016. DOI: 10.1021/acscentsci.6b00057
  2. de Bruin, G., B.-T. Xin, B.I. Florea, H.S. Overkleeft, "Proteasome Subunit Selective Activity-Based Probes Report on Proteasome Core Particle Composition in a Native Polyacrylamide Gel Electrophoresis Fluorescence-Resonance Energy Transfer Assay", Journal of the American Chemical Society, vol. 138, issue 31, pp. 9874 - 9880, 08/2016. DOI: 10.1021/jacs.6b04207
  3. de Bruin, G., B.T. Xin, M. Kraus, M. van der Stelt, G.A. van der Marel, A.F. Kisselev, C. Driessen, B.I. Florea, et al., "A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities", Angewandte Chemie International Edition, vol. 55, issue 13, pp. 4199 - 4203, 03/2016, 2015. DOI: 10.1002/anie.201509092
  4. Willems, L.I., N. Li, B.I. Florea, M. Ruben, G.A. van der Marel, H.S. Overkleeft, "Triple Bioorthogonal Ligation Strategy for Simultaneous Labeling of Multiple Enzymatic Activities", Angewandte Chemie-International Edition, vol. 51, no. 18, pp. 4431-4434, 2012. DOI: 10.1002/anie.201200923
  5. Witte, M.D., W.W. Kallemeijn, J. Aten, K.Y. Li, A. Strijland, W.E. Donker-Koopman, A.M.C.H. van den Nieuwendijk, B. Bleijlevens, et al., "Ultrasensitive in situ visualization of active glucocerebrosidase molecules", Nature Chemical Biology, vol. 6, no. 12, pp. 907-913, Dec, 2010. DOI: 10.1038/Nchembio.466

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