den Hertog: Protein-tyrosine phosphatases in development

We are interested in the function of PTPs in development and disease. Using biochemical and cell biological approaches, we study PTP-signaling in order to obtain insight into conceptual questions about substrates, regulation, ligands and effectors. Over the years, we established that the proto-typical RPTPalpha is regulated by dimerization, oxidation and phosphorylation.

PTEN

PTEN is a tumor suppressor gene that belongs to the PTP superfamily and exhibits lipid phosphatase activity with high selectivity for the 3-position of phosphatidylinositol(3,4,5)trisphosphate. The zebrafish genome encodes two PTEN genes: ptena and ptenb. We isolated null mutants of both PTEN genes and found that adult zebrafish with a single wild type PTEN allele develop hemangiosarcomas, blood-filled clusters of blood vessels with endothelial origin. Double homozygous ptena-/-ptenb-/- mutant embryos are embryonic lethal and display a pleiotropic phenotype, including hyperproliferation of endothelial cells, resulting in hyperbranching of blood vessels. Hematopoiesis is also affected in ptena-/-ptenb-/- mutants. Hematopoietic stem and progenitor cells (HSPCs) hyperproliferate and arrest during differentiation in embryos lacking functional PTEN, resulting in enhanced numbers of blood cell progenitors, but no mature blood cells.

In parallel to the research line above, a second line of research was developed in the den Hertog lab over the years, aimed at identification of biologically active molecules. Due to an ageing world population, globalization and drug resistance, there is a constant need for new and more effective drugs. The number of drugs that are being used in the clinic is limited and the number of biologically active molecules in these drugs is only ~1,500. The goal of this project is to identify novel chemical scaffolds with biological activity that may provide the starting point for the development of new drugs. Fungi are well-known for the production of a wide variety of secondary metabolites with biological activity. Commonly used drugs, including penicillin, cyclosporin and lovastatin all originate from fungi. Continuous discovery of novel compounds from fungi proves that the fungal kingdom as a source of new compounds is far from depleted. We have generated a library of crude growth media from >10,000 fungal species in collaboration with the Westerdijk Fungal Biodiversity Institute. These growth media contain the secreted secondary metabolites from these fungi. We have assessed biological activity of these fungal filtrates using zebrafish embryogenesis as read-out. Following incubation from 6 hpf onwards, 1,535 fungal secondary metabolites induced morphological abnormalities at 2 dpf, varying from an undulating notochord to pigmentation, heart and tail defects, amongst others. Fungal strains were selected based on the phenotype they induced and in collaboration with the chemistry department at Utrecht University, we have set up a pipeline to identify bioactive compounds by activity-guided purification using HPLC. Purified active compounds were identified using LCMS, UV-Vis spectrometry, high resolution mass spectrometry and NMR. We have established the biological activity in zebrafish of more than 60 known fungal metabolites and we identified several bioactive metabolites that have not been described before.