Single Molecule Force Spectroscopy of Human Angiostatin.

Angiogenesis a fundamental process in tissue development and it’s directly correlated with tumor growth and aggressivity. 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 a recent work (2) we have demonstrated by means of single molecule force spectroscopy that angiostatin mechanical properties can be modulated by the controlled reduction of its internal disulfide bonds. 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. We investigated the dependence of the unfolding forces on the natural logarithm of the pulling speed, we performed kinetic Monte Carlo simulations of the stretching experiments and (in collaboration with R. Casadio and E. Capriotti, University of Bologna) we carried out steered molecular dynamics simulations of the forced unfolding of angiostatin domains (3). All of this allowed us to map the free energy profile along the unfolding pathway and to identify a structural unfolding intermediate that could play a mechanochemical role in the activity of angiostatin. 1. Folkman, J. (2002) Semin Oncol 29, 15-18. 2. Bustanji, Y., and Samorì, B. (2002) Angew. Chem. Int. Ed. 41, 1546-1548. 3. 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.