The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

Our laboratory's research focuses on regulation of the initiating steps of protease cascades. Protease cascades mediate a number of important biological processes, including hemostasis, cell migration, and apoptosis. A central conundrum in understanding regulation of protease cascades is how the system starts, as the cascade initially consists only of zymogens. Furthermore, once active, the first enzyme activates the second zymogen, and the second enzyme activates the initial zymogen. This constitutes a positive feedback loop, raising the additional question of how the system stops.

Using urokinase mediating cell surface associated plasminogen activation as a model, our data suggests a hypothesis which addresses each of the conundra. 1) The zymogen of the initial enzyme in the cascade has a small, but measurable, amount of enzymatic activity. This activity is masked by a novel reversible interaction with its cognate serpin type inhibitor. This yields a ready supply of enzyme, without inappropriate expression of activity. 2) Proteolysis is initiated by expression of a cellular receptor, which acts as an activating co-factor. The initiating zymogen partitions from the inhibitor to the receptor, acquiring full proteolytic activity without having been cleaved from the zymogen to the mature enzyme form. Importantly, the active zymogen bound to the receptor is poorly inhibited by the cognate serpin. 3) The activated second protease, besides other downstream functions, cleaves the receptor bound initiating zymogen to the mature form. The mature enzyme is now susceptible to irreversible inhibition by the serpin, bringing the process to a halt unless new activating co-factor (receptor) is expressed on the cell surface.

In our current work, we are testing the kinetic and structural determinants for each step in this hypothesis with urokinase (For instance, are the relative affinities of the interactions consistent with the hypothesis at physiologic protein concentrations? What are the implications for conformation of the active site as each form of the enzyme interacts with substrate, vs. with inhibitor?). We are also testing the hypothesis in other protease cascades to determine how widespread this framework of regulation is in mammalian biology (We have demonstrated this regulatory framework is operative for tissue type plasminogen activator, and would like to test the hypothesis in coagulation, and apoptosis related pathways).