Nicolai Sidenius
e-mail: nicolai.sidenius AT ifom.eu
affiliation: IFOM-FIRC Institute of Molecular Oncology
research area(s): Cell Biology
Course:
Molecular Medicine: Molecular Oncology and Computational Biology
University/Istitution: Università di Milano, UNIMI-SEMM
University/Istitution: Università di Milano, UNIMI-SEMM
Cell matrix signaling
Current lines of research in the lab are:
1) Indentification of the molecular mechanisms of signal transduction induced by cell adhesion to the extracellular matrix.
Our long-standing interest in the molecular basis for uPAR’s ability to induce signal transduction has over the last few years led to a focused effort aimed at understanding, at the molecular level, how membrane receptors translate extracellular signals (i.e. ligand binding) into defined intracellular biochemical signaling events. Of specific interest is the mechanism by which membrane receptors lacking transmembrane and cytoplasmic domains signal. This type of receptors, including uPAR, cannot transduce signals by propagation of a conformational change over the plasma membrane and must therefore rely on different mechanisms. The prevailing paradigm is that these adhesion receptors utilize direct lateral interactions with other signaling competent receptors (such as integrins or receptor tyrosine kinases) to transmit the signal. We have, however, recently documented that direct lateral interaction with other membrane receptors play no functional role in the ability of uPAR to signal and that the signaling mechanism downstream of uPAR is likely to be shared with most, if not all, membrane receptors with high-affinity ligands in the extracellular matrix. Our findings strongly question the importance of the prevailing paradigm of “adapter mediated” uPAR signaling, but the real mechanism still has to be established. In the lab we have some promising “leads” on this mechanism and these are currently being exploited using state-of-the-art “genetics-in-cell-culture” and optical imaging techniques.
2) The importance of the uPAR/Vn-interaction in vivo.
Our demonstration of the pivotal importance of the direct molecular interaction with Vn for the function of uPAR in vitro clearly identifies this interaction as a promising target for the pharmaceutical interference with cancer invasion and metastasis (and in other diseases where uPA/uPAR is known or suspected to be of functional relevance). To address the importance of this interaction in vivo we are currently conducting a systematic comparison of the phenotypes of uPAR and Vn null mice in a variety of processes where these genes have been published to be of functional relevance including inflammation, tumor invasion and metastasis, as well as in HSC/HPC mobilization (in collaboration with Dr. Marc Tjwa, Leuven, Belgium). If functional and relevant phenocopying will be observed between these two lines of null mice the final evidence for importance of the molecular interaction between the two proteins will be provided by the generation of a uPAR knock-in mouse in which we have specifically and selectively disrupted the Vn-binding activity of the receptor.
3)Determination of the structural basis for the regulation of the molecular interaction between uPAR and Vn.
Several crystal structures of the bi-molecular complex between uPAR and uPA, as well as a structure of the ternary complex between uPAR, uPA and Vn have been published, providing a certain degree of insight into the structural basis for the molecular interactions between these three proteins. Despite this structural insight the published structures, besides roughly defining the molecular interfaces, fall short of explaining the biological activity of uPAR as a cell adhesion receptor for vitronectin and the importance of uPA in this process. Firstly, efficient uPAR binding to Vn requires dimerization while uPAR is monomeric in the crystal structure(s). Secondly, the structures only partially explain the structural requirements to uPAR and Vn as determined by complete alanine scans of binding regions of these proteins. Thirdly, the stoichiometry of the component observed in the crystal structures, as well as the experimentally determined affinities, are entirely insufficient to explain the uPA-dependence of soluble uPAR binding and cell adhesion to matrix Vn. To fill this knowledge gab a series of structural and biochemical approaches are being exploited in the lab to determine the stoichiometry and topology of the high-affinity uPAR/uPA/Vn-interaction as well as to understand its regulation on the surface of living cells by single molecule imaging/nano-spectroscopy techniques conducted in the collaboration with the Caiolfa group at the San Raffaele Scientific Institute in Milan, Italy.
Current lines of research in the lab are:
1) Indentification of the molecular mechanisms of signal transduction induced by cell adhesion to the extracellular matrix.
Our long-standing interest in the molecular basis for uPAR’s ability to induce signal transduction has over the last few years led to a focused effort aimed at understanding, at the molecular level, how membrane receptors translate extracellular signals (i.e. ligand binding) into defined intracellular biochemical signaling events. Of specific interest is the mechanism by which membrane receptors lacking transmembrane and cytoplasmic domains signal. This type of receptors, including uPAR, cannot transduce signals by propagation of a conformational change over the plasma membrane and must therefore rely on different mechanisms. The prevailing paradigm is that these adhesion receptors utilize direct lateral interactions with other signaling competent receptors (such as integrins or receptor tyrosine kinases) to transmit the signal. We have, however, recently documented that direct lateral interaction with other membrane receptors play no functional role in the ability of uPAR to signal and that the signaling mechanism downstream of uPAR is likely to be shared with most, if not all, membrane receptors with high-affinity ligands in the extracellular matrix. Our findings strongly question the importance of the prevailing paradigm of “adapter mediated” uPAR signaling, but the real mechanism still has to be established. In the lab we have some promising “leads” on this mechanism and these are currently being exploited using state-of-the-art “genetics-in-cell-culture” and optical imaging techniques.
2) The importance of the uPAR/Vn-interaction in vivo.
Our demonstration of the pivotal importance of the direct molecular interaction with Vn for the function of uPAR in vitro clearly identifies this interaction as a promising target for the pharmaceutical interference with cancer invasion and metastasis (and in other diseases where uPA/uPAR is known or suspected to be of functional relevance). To address the importance of this interaction in vivo we are currently conducting a systematic comparison of the phenotypes of uPAR and Vn null mice in a variety of processes where these genes have been published to be of functional relevance including inflammation, tumor invasion and metastasis, as well as in HSC/HPC mobilization (in collaboration with Dr. Marc Tjwa, Leuven, Belgium). If functional and relevant phenocopying will be observed between these two lines of null mice the final evidence for importance of the molecular interaction between the two proteins will be provided by the generation of a uPAR knock-in mouse in which we have specifically and selectively disrupted the Vn-binding activity of the receptor.
3)Determination of the structural basis for the regulation of the molecular interaction between uPAR and Vn.
Several crystal structures of the bi-molecular complex between uPAR and uPA, as well as a structure of the ternary complex between uPAR, uPA and Vn have been published, providing a certain degree of insight into the structural basis for the molecular interactions between these three proteins. Despite this structural insight the published structures, besides roughly defining the molecular interfaces, fall short of explaining the biological activity of uPAR as a cell adhesion receptor for vitronectin and the importance of uPA in this process. Firstly, efficient uPAR binding to Vn requires dimerization while uPAR is monomeric in the crystal structure(s). Secondly, the structures only partially explain the structural requirements to uPAR and Vn as determined by complete alanine scans of binding regions of these proteins. Thirdly, the stoichiometry of the component observed in the crystal structures, as well as the experimentally determined affinities, are entirely insufficient to explain the uPA-dependence of soluble uPAR binding and cell adhesion to matrix Vn. To fill this knowledge gab a series of structural and biochemical approaches are being exploited in the lab to determine the stoichiometry and topology of the high-affinity uPAR/uPA/Vn-interaction as well as to understand its regulation on the surface of living cells by single molecule imaging/nano-spectroscopy techniques conducted in the collaboration with the Caiolfa group at the San Raffaele Scientific Institute in Milan, Italy.
1. Hellriegel C, Caiolfa VR, Corti V, Sidenius N, Zamai M.
Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. FASEB J. 2011 May 20. [Epub ahead of print]
2. Blasi F, Sidenius N.
The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling.
FEBS Lett. 2010 May 3;584(9):1923-30. Epub 2009 Dec 27. Review.
3. Blasi F, Sidenius N.
Efferocytosis: another function of uPAR.
Blood. 2009 Jul 23;114(4):752-3.
4. Cunningham O, Campion S, Perry VH, Murray C, Sidenius N, Docagne F, Cunningham C.
Microglia and the urokinase plasminogen activator receptor/uPA system in innate brain inflammation.
Glia. 2009 Dec;57(16):1802-14.
5. Tjwa M, Sidenius N, Moura R, Jansen S, Theunissen K, Andolfo A, De Mol M, Dewerchin M, Moons L, Blasi F, Verfaillie C, Carmeliet P.
Membrane-anchored uPAR regulates the proliferation, marrow pool size, engraftment, and mobilization of mouse hematopoietic stem/progenitor cells.
J Clin Invest. 2009 Apr;119(4):1008-18. doi: 10.1172/JCI36010. Epub 2009 Mar 9.
6. Nebuloni M, Cinque P, Sidenius N, Ferri A, Lauri E, Omodeo-Zorini E, Zerbi P, Vago L.
Expression of the urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) in brain tissues of human immunodeficiency virus patients with opportunistic cerebral diseases.
J Neurovirol. 2009 Jan;15(1):99-107.
7. Malengo G, Andolfo A, Sidenius N, Gratton E, Zamai M, Caiolfa VR.
Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor.
J Biomed Opt. 2008 May-Jun;13(3):031215.
8. Madsen, C.D. and Sidenius, N.
The interaction between urokinase receptor and vitronectin in cell adhesion and signaling.
Eur J Cell Biol 2008; 87: 617-29
9. Caiolfa, V. R., Zamai, M., Malengo, G., Andolfo, A., Madsen, C. D., Gaudesi, D., Sutin, J., Digman, M., Gratton, E., Blasi, F., and Sidenius, N.
Cell surface protein assemblies determine the location, diffusion and monomer-dimer dynamics of the GPI-anchored receptor uPAR.
J Cell Biol 2007; 179: 1067-82.
10. Madsen, C. D., Sarra Ferraris, G. M., Andolfo, A., Cunningham, O. M., and Sidenius, N.
uPAR-dependent cell migration: Vitronectin provides the key.
J Cell Biol 2007; 177: 927-939.
11. Elia, C., Cassol, E., Sidenius, N., Blasi, B., Castagna, A., Poli, G. and Alfano, M. Inhibition of HIV Replication by Plasminogen Activator Is Dependent Upon
Vitronectin-Mediated Cell Adhesion.
J Leukoc Biol 2007; 82: 1212-1220.
12 Selleri, C., Montuori, N., Ricci, P., Visconte, V., Carriero, M. V., Sidenius, N., Serio, B., Blasi, F., Rotoli, B., Rossi, G., and Ragno, P.
Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization.
Blood 2005; 105: 2198-2205.
Number and brightness image analysis reveals ATF-induced dimerization kinetics of uPAR in the cell membrane. FASEB J. 2011 May 20. [Epub ahead of print]
2. Blasi F, Sidenius N.
The urokinase receptor: focused cell surface proteolysis, cell adhesion and signaling.
FEBS Lett. 2010 May 3;584(9):1923-30. Epub 2009 Dec 27. Review.
3. Blasi F, Sidenius N.
Efferocytosis: another function of uPAR.
Blood. 2009 Jul 23;114(4):752-3.
4. Cunningham O, Campion S, Perry VH, Murray C, Sidenius N, Docagne F, Cunningham C.
Microglia and the urokinase plasminogen activator receptor/uPA system in innate brain inflammation.
Glia. 2009 Dec;57(16):1802-14.
5. Tjwa M, Sidenius N, Moura R, Jansen S, Theunissen K, Andolfo A, De Mol M, Dewerchin M, Moons L, Blasi F, Verfaillie C, Carmeliet P.
Membrane-anchored uPAR regulates the proliferation, marrow pool size, engraftment, and mobilization of mouse hematopoietic stem/progenitor cells.
J Clin Invest. 2009 Apr;119(4):1008-18. doi: 10.1172/JCI36010. Epub 2009 Mar 9.
6. Nebuloni M, Cinque P, Sidenius N, Ferri A, Lauri E, Omodeo-Zorini E, Zerbi P, Vago L.
Expression of the urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) in brain tissues of human immunodeficiency virus patients with opportunistic cerebral diseases.
J Neurovirol. 2009 Jan;15(1):99-107.
7. Malengo G, Andolfo A, Sidenius N, Gratton E, Zamai M, Caiolfa VR.
Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor.
J Biomed Opt. 2008 May-Jun;13(3):031215.
8. Madsen, C.D. and Sidenius, N.
The interaction between urokinase receptor and vitronectin in cell adhesion and signaling.
Eur J Cell Biol 2008; 87: 617-29
9. Caiolfa, V. R., Zamai, M., Malengo, G., Andolfo, A., Madsen, C. D., Gaudesi, D., Sutin, J., Digman, M., Gratton, E., Blasi, F., and Sidenius, N.
Cell surface protein assemblies determine the location, diffusion and monomer-dimer dynamics of the GPI-anchored receptor uPAR.
J Cell Biol 2007; 179: 1067-82.
10. Madsen, C. D., Sarra Ferraris, G. M., Andolfo, A., Cunningham, O. M., and Sidenius, N.
uPAR-dependent cell migration: Vitronectin provides the key.
J Cell Biol 2007; 177: 927-939.
11. Elia, C., Cassol, E., Sidenius, N., Blasi, B., Castagna, A., Poli, G. and Alfano, M. Inhibition of HIV Replication by Plasminogen Activator Is Dependent Upon
Vitronectin-Mediated Cell Adhesion.
J Leukoc Biol 2007; 82: 1212-1220.
12 Selleri, C., Montuori, N., Ricci, P., Visconte, V., Carriero, M. V., Sidenius, N., Serio, B., Blasi, F., Rotoli, B., Rossi, G., and Ragno, P.
Involvement of the urokinase-type plasminogen activator receptor in hematopoietic stem cell mobilization.
Blood 2005; 105: 2198-2205.
Project Title: