UC San Diego

The urokinase-type plasminogen activator (uPA) is a serine protease involved in fibrinolysis, tissue remodeling, angiogenesis, and cancer metastasis. This dissertation investigated the factors that affect the activity of this enzyme beyond the three amino acids that constitute the catalytic triad. In Chapter 2, a new method to purify single-chain uPA from inclusion bodies was developed. This method increased the time of storage of single-chain uPA to 2 months. In Chapter 3, the effect of uPA receptor (uPAR) binding on the protease activity of uPA was studied. I showed that monomeric uPAR does not affect the catalytic activity of uPA in solution. In Chapter 4, the ability of single-chain uPA to autoactivate was demonstrated. Two-chain uPA can cleave single-chain uPA after K135 in the disordered part of the light chain and after K158. Only this last cleavage causes activation of uPA. In addition, hydrogen deuterium exchange mass spectrometry (HDX-MS) was used to characterize the dynamic differences between the uPA zymogen and its active form. A mechanism was proposed in which the dimerization of the uPAR could promote autoactivation of single-chain uPA on cell surfaces. In Chapter 5, the effect of the amino terminal fragment (ATF) on the protease domain of uPA was evaluated. Full-length uPA was faster at catalyzing the hydrolysis of plasminogen than the form of uPA that was missing the ATF. Surprisingly, the behavior was opposite for a small chromogenic substrate. In addition, full-length uPA also had less affinity for a small substrate inhibitor than the protease domain alone form. HDX-MS revealed that when ATF is present, the deuterium uptake of the 8-strand that is located below the S1 specificity pocket is reduced. In Chapter 6, the effect of the disordered part of the light chain on the protease domain of murine uPA was assessed. The light chain increases the activity of the protease domain 5-fold and key interactions between the light-chain and the protease domain were confirmed by mutagenesis. HDX-MS showed that the light chain orders the 220s loop where the S1 specificity pocket is located. Even more, the decrease of deuterium uptake in the 140s loop was inversely proportional to the activity of murine uPA. A model was proposed to account for how allostery is transferred through the protein.