Within the circulation, sophisticated networks of interactions between proteins and other macromolecules are important for maintaining stasis. Ongoing research in my lab focuses on the structural and functional impact that the interaction between two circulatory and tissue-associated proteins, vitronectin and plasminogen activator inhibitor-1 (PAI-1), have on each other. This interaction is involved in the regulation of many diverse processes, including coagulation, fibrinolysis, tumor metastasis, the inflammatory response, cellular migration, obesity and the metabolic syndrome, infection and the circadian rhythm. Although PAI-1 and vitronectin are commonly called “cofactors” and most often are co-localized, the consequences of their association are ill defined. Vitronectin is a multi-domain glycoprotein that participates in the above regulatory circuits via extensive interactions, providing an adhesive function for cells with the extracellular matrix, and serving in a pro-coagulant role by binding to heparin-like molecules on the vasculature and neutralizing their anticoagulant function. Vitronectin also regulates the process of matrix degradation or fibrinolysis by modulating interactions of cells with integrins and other receptors or maintaining protease inhibitor molecules in an active conformation. Vitronectin stabilizes the active conformation of the serine protease inhibitor (serpin), PAI-1, which is the chief inhibitor of proteases (tissue plasminogen activator, tPA, and urokinase plasminogen activator, uPA) that activate the fibrinolytic protease plasmin from its zymogen form. This complex formed between PAI-1 and vitronectin is more comprehensive than a simple 1:1 complex, leading to the formation of higher-order PAI-1/vitronectin oligomers. Also, metals have an unexpected and pronounced impact on the stability of PAI-1, the effect of which is vitronectin-dependent. Furthermore, some functions of PAI-1 appear independent of its protease activity, and the binding to vitronectin is required in many of these cases. From this variety of conformational effects and activities, it appears that structural and/or dynamical changes in PAI-1 and PAI-1-vitronectin complexes give rise to its broad spectrum of functions. Despite the fact that the two have been studied extensively, their mechanism of interaction remains elusive. We maintain that the conformation and dynamics are PAI-1 are regulated by vitronectin binding via an extensive binding region that encompasses dual sites recently identified through our work. We postulate that the entire interaction surface is required for the full range of conformational and functional effects. Also, we propose that PAI-1 has altered functions that are influenced by bioavailability of metals in the extracellular compartment. To test these hypotheses, a variety of experimental approaches from molecular biology to biophysics are used, including eukaryotic cell culture/transfection; eukaryotic and prokaryotic expression systems; site-directed mutagenesis; protein purification; mass spectrometry; analytical ultracentrifugation; fluorescence spectroscopy; calorimetry; surface plasmon resonance; and electron paramagnetic resonance.
Behrens, M. A., Botkjaer, K. A., Goswami, S., Oliveira, C. L., Jensen, J. K., Schar, C. R., Declerck, P. J., Peterson, C. B., Andreasen, P. A., and Pedersen, J. S. (2011) J Mol Biol 411, 417-429. “Activation of the zymogen to urokinase-type plasminogen activator is associated with increased interdomain flexibility”
Jensen, J. K., Thompson, L. C., Bucci, J. C., Nissen, P., Gettins, P. G., Peterson, C. B., Andreasen, P. A., and Morth, J. P. (2011) J Biol Chem, in press. “Crystal Structure of Plasminogen Activator Inhibitor-1 in an Active Conformation with Normal Thermodynamic Stability”
Zmijewski, J. W., Bae, H. B., Deshane, J. S., Peterson, C. B., Chaplin, D. D., and Abraham, E. (2011) Am J Physiol Lung Cell Mol Physiol 301, L247-254. “Inhibition of neutrophil apoptosis by PAI-1”
Thompson, L., C., Goswami, S., Ginsberg, D. S., Day, D. E., Verhamme, I. M. and Peterson, C. B. (2011) Protein Sci. 20, 353-365. “Metals Affect the Structure and Activity of Human Plasminogen Activator Inhibitor-1: I. Modulation of stability and protease inhibition.”
Thompson, L. C., Goswami, S., and Peterson, C. B. (2011) Protein Sci. 20, 366-378. “Metals Affect the Structure and Activity of Human Plasminogen Activator Inhibitor-1: II. Binding Affinity and Conformational Effects.”
Mou, X., Peterson, C. B., and Prosser, R. A. (2009) Eur. J. Neuroscience 30, 1451-1460. “Tissue-type Plasminogen Activator-Plasmin-BDNF Modulate of Glutamate-Induced Phase-Shifts of the Mouse Suprachiasmatic Circadian Clock in Vitro.”
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Cynthia Peterson