Molekulare Zellbiologie der Eukaryonten, mit besonderer Betonung folgender Themenkreise: intrazelluläre Transportprozesse, posttranslationale Modifikationen von Proteinen, Tumorinvasion und Metastasierung, Lysosomen und deren pathophysiologische Bedeutung.
Leiter: Ao.Univ.Prof. DI Dr. Lukas Mach
In unserer Forschungstätigkeit beschäftigen wir uns in erster Linie mit folgenden Fragestellungen:
- pathophysiologische Funktionen von lysosomalen Proteinen und deren Rezeptoren
- intrazellulärer Transport zu Endosomen und Lysosomen
- N-Glykosylierung von Proteinen und die an diesem Prozess beteiligten Enzyme
- proteolytische Reifung von Proteinvorstufen
- zelluläre Rollen von Proteinasen und deren Inhibitoren
The current research projects under study in our group are addressing the following topics:
A) The role of proteinases and their receptors in tumour invasion and metastasis
B) Pathophysiological functions of novel lysosomal proteins
C) Posttranslational modifications of proteins in plants
- A) Proteinases and their receptors in tumour invasion and metastasis
The spread of cancerous cells from a primary tumour to distant sites represents a major obstacle in current cancer therapies. During formation of such metastases, tumour cells invade the surrounding tissue, enter the circulation and migrate to other organs within the affected body. Substances which block this invasive and migratory capacity of malignant tumour cells could be used to treat cancer patients. One important step in the development of metastasis is the degradation of connective-tissue components, a process which involves various proteinases and their cellular receptors.
We are currently focussing on the characterisation of the anti-metastatic potential of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R). M6P/IGF2R is a multifunctional protein which binds a number of tissue-degrading proteinases and other factors that impinge on the migration and invasiveness of cancer cells. The gene encoding this receptor is frequently lost or mutated in a variety of malignant cancers. We have recently demonstrated that reconstitution of functional M6P/IGF2R expression in receptor-deficient cancer cells reduces their tumourigenicity and invasiveness. These findings illustrate that the M6P/IGF2R status influences the metastatic propensity of malignant cancer cells. Future studies will aim at the identification of the M6P/IGF2R ligands responsible for the anti-invasive activity of the receptor, which could prove the starting point for the development of novel cancer treatment strategies.
- B) Pathophysiological functions of novel lysosomal proteins
In eukaryotic cells, dedicated subcellular structures referred to as lysosomes play a pivotal role in the breakdown of cellular macromolecules. Deficiencies in individual lysosomal proteins result in the accumulation of undigested macromolecules which ultimately interferes with the functions of these compartments. In humans, about 45 lysosomal storage diseases have been directly linked to the deficiency of certain lysosomal proteins. However, the molecular etiology of some lysosomal storage diseases is still unknown. Therefore, considerable efforts have been recently undertaken to establish the complete proteome of lysosomes. This has led to the discovery of novel lysosomal proteins with as yet unknown functions.
Cellular Repressor of E1A-stimulated Genes (CREG) is a novel lysosomal protein expressed in most mammalian tissues and cell types. Evidence has been provided that CREG might be involved in the control of cell growth and differentiation. Interestingly, the genomes of several model organisms (e.g. Arabidopsis thaliana, Drosophila melanogaster) encode putative orthologues of mammalian CREG, indicating an evolutionary conserved function of the protein. However, the biochemical basis for its cellular activities has not been unravelled so far. We have recently started to investigate the pathophysiological relevance of this protein by studying the consequences of CREG ablation in mammalian cells and the model plant A. thaliana.
- C) Posttranslational modifications of proteins in plants
N-glycosylation is considered to be one of the most important posttranslational protein modifications. In both animal and plant cells, a highly ordered biosynthetic pathway accounts for the maturation of N-linked oligosaccharides attached to newly synthesised proteins. The initial steps of the N-glycosylation pathway are evolutionarily highly conserved. However, the structures of mature N-glycans differ between plants and mammals due to major differences in the final processing events. We have recently characterized the enzymatic properties of two plant-specific glycosyltransferases as well as of several other enzymes involved in N-glycan maturation in planta, thus setting the stage for transgenic approaches to improve the N-glycosylation capacity of plant-based expression platforms used for the production of monoclonal antibodies and other protein therapeutics.
The production of recombinant monoclonal antibodies in plants is frequently hampered by unwanted proteolysis. This is also the case in Nicotiana benthamiana, a tobacco-related plant species widely used for such purposes. In close collaboration with other DAGZ groups, we have now started to characterize the proteolytic activities accounting for the fragmentation of antibodies in N. benthamiana. Future efforts will be directed at the prevention of antibody proteolysis in this plant species by removing or inhibiting the responsible enzymes using both transgenic and gene silencing approaches.
Probst, O.C., Puxbaum, V., Svoboda, B., Leksa, V., Stockinger, H., Mikula, M., Mikulits, W., Mach, L. (2009) The mannose 6-phosphate/insulin-like growth factor II receptor restricts the tumourigenicity and invasiveness of squamous cell carcinoma cells. Int. J. Cancer 124, 2559-2567
Schähs, P., Weidinger, P., Probst, O.C., Svoboda, B., Stadlmann, J., Beug, H., Waerner, T., Mach, L. (2008) Cellular Repressor of E1A-stimulated Genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis. Exp. Cell Res. 314, 3036-3047
Strasser, R., Bondili, J.S., Schoberer, J., Svoboda, B., Liebminger, E., Glössl, J., Altmann, F., Steinkellner, H., Mach, L. (2007) Enzymatic properties and subcellular localization of Arabidopsis β-N-acetyl-hexosaminidases. Plant Physiol. 145, 5-16
Strasser, R., Bondili, J.S., Vavra, U., Schoberer, J., Svoboda, B., Glössl, J., Leonard, R., Stadlmann, J., Altmann, F., Steinkellner, H., Mach, L. (2007) A unique β1,3-galactosyltransferase is indispensable for the biosynthesis of Lewis a containing N-glycans in Arabidopsis thaliana. Plant Cell 19, 2278-2292
Probst, O.C., Ton, P., Svoboda, B., Gannon, A., Schuhmann, W., Wieser, J., Pohlmann, R., Mach, L. (2006) The 46-kDa mannose 6-phosphate receptor does not depend on endosomal acidification for delivery of hydrolases to lysosomes. J. Cell Sci. 119, 4935-4943.
Antonio Baici, Institut für Biochemie, Universität Zürich, Schweiz
Hartmut Beug, Institut für Molekulare Pathologie, Wien, Österreich
Wolfgang Mikulits, Institut für Tumorbiologie und Krebsforschung, Medizinische Universität Wien, Österreich
Regina Pohlmann, Institut für Physiologische Chemie und Pathobiochemie, Universität Münster, Deutschland
Hannes Stockinger, Institut für Molekulare Immunologie, Medizinische Universität Wien, Österreich