Single Molecule Force Spectroscopy of Human Angiostatin

Angiogenesis is a complex multistep process by which new blood vessels are formed from pre-existent ones. It’s a fundamental process in tissue development and it’s directly correlated with tumor growth and aggressivity. Tumors appear to activate an angiogenesis switch from vascular quiescence by changing the balance between angiogenesis inhibitors and promoters. The multimodular protein angiostatin physiologically inhibits angiogenesis by suppressing endothelial cell growth and migration (1). Being angiostatin already in clinical trials, it is essential to understand the underlying molecular mechanism of inhibition. The scanning force microscope in the force spectroscopy mode allows to mechanically unfold multimodular proteins by working at the single molecule level. In this kind of experiments, a single protein bridge is settled between the microscope tip and a substrate; the force required to strech the protein is monitored as a function of its extension, showing a typical saw tooth profile in which each peak corresponds to the unfolding of a single domain (2). In a recent work (3) we have demonstrated by means of single molecule force spectroscopy that angiostatin mechanical properties can be modulated by the controlled reduction of its internal disulphide bonds. In the same work we have also shown how this technique allows to identify the different thiol/disulphide intermediates at the single domain level. Here we demonstrate by force spectroscopy techniques that angiostatin disulphide bonds reduction can be performed by human thioredoxin, an extracellular reductase overexpressed in the proximity of tumor tissues. This result indicate that angiostatin mechanical properties might be modulated in vivo and that angiostatin might play a mechanochemical role in angiogenesis. On this basis a mechanochemical model of the angiostatin inhibitory effect on angiogenesis is proposed. We investigated the dependence of the unfolding forces on the natural logarithm of the pulling speed and found the expected linear dependence (4). We performed kinetic Monte Carlo simulations of the stretching experiments (5) and (in collaboration with Rita Casadio and Emidio Capriotti, University of Bologna) we carried out steered molecular dynamics simulations of the forced unfolding of angiostatin domains (6). The comparison between the simulations results and the experimental data allowed us to get quantitative information about the free energy profile along the unfolding pathway and to identify a structural unfolding intermediate that could play a mechanochemical role in the angiostatin inhibitory effect on angiogenesis. 1. Folkman, J. (2002) Semin Oncol 29, 15-18. 2. Samori, B. (2000) Chemistry 6, 4249-4255. Best, R. B., and Clarke, J. (2002) Chem Commun (Camb), 183-192. 3. Bustanji, Y., and Samorì, B. (2002) Angew. Chem. Int. Ed. 41, 1546-1548. 4. Bustanji, Y., Arciola, C. R., Conti, M., Mandello, E., Montanaro, L., and Samori, B. (2003) Proc Natl Acad Sci U S A 100, 13292-13297. 5. Carrion-Vazquez, M., Oberhauser, A. F., Fowler, S. B., Marszalek, P. E., Broedel, S. E., Clarke, J., and Fernandez, J. M. (1999) Proc Natl Acad Sci U S A 96, 3694-3699. 6. Lu, H., Isralewitz, B., Krammer, A., Vogel, V., and Schulten, K. (1998) Biophys J 75, 662-671.