Nonproteolytic induction of catalytic activity into the single-chain form of urokinase-type plasminogen activator by dipeptides.

Serine proteases are initially synthesized as single-chain proenzymes with activities that are many orders of magnitude lower than those of the mature enzyme. Proteolytic cleavage of an exposed loop liberates a new amino terminus that inserts into a hydrophobic pocket and forms a stabilizing salt bridge with a ubiquitously conserved aspartate residue, resulting in a conformational change organizing the mature oxyanion hole. In a decisive 1976 work, Huber and Bode [Bode, W., and Huber, R. (1976) FEBS Lett. 68, 231-236] demonstrated that peptides sequentially similar to the new amino terminus in combination with a catalytic site inhibitor could specifically induce a trypsin-like conformation in trypsinogen. We now demonstrate that an Ile-Ile or Ile-Val dipeptide can induce limited enzyme activity in the single-chain zymogen form of urokinase-type plasminogen activator (uPA) or its K158A variant, which cannot be activated proteolytically. Furthermore, the slow formation of a covalent serpin-protease complex between single-chain uPA and PAI-1 is significantly accelerated in the presence of specific dipeptide sequences. The technique of using a dipeptide mimic as a surrogate for the liberated amino terminus further provides a novel means by which to covalently label the immature active site of single-chain uPA with a fluorescent probe, permitting fluorescence approaches for direct observations of conformational changes within the protease domain during zymogen activation. These data demonstrate the structural plasticity of the protease domain, reinforce the notion of "molecular sexuality", and provide a novel way of studying conformational changes of zymogens during proteolytic activation.

A novel mode of intervention with serine protease activity: targeting zymogen activation.

Serine proteases are secreted from cells as single-chain zymogens, typically having activities orders of magnitude lower than those of the mature two-chain enzymes. Activation occurs by a conformational change initiated by cleavage of a specific peptide bond. We have derived a monoclonal antibody (mAb-112) which binds with subnanomolar affinity to pro-uPA, the zymogen form of urokinase-type plasminogen activator (uPA). We mapped the epitope of the antibody to the autolysis loop, one of the structural elements known to change conformation during zymogen activation. A mechanistic evaluation with biophysical methods elucidated a novel bifunctional inhibitory mechanism whereby mAb-112 not only delays the proteolytic conversion of single-chain pro-uPA into the two-chain form but also subsequently averts the conformational transition to a mature protease by sequestering the two-chain form in a zymogen-like, noncatalytic state. Functional studies employing two variants of human HT-1080 cells, exhibiting high and low levels of dissemination in a chorioallantoic membrane assay, demonstrate that mAb-112 is an effective inhibitor of tumor cell intravasation. These findings show that pharmacological interference with zymogen activation is a plausible and robust means to regulate uPA activity and the downstream effects of plasminogen activation in the spread of cancer and other processes of pathological tissue remodeling. A strategy that targets regions related to pro-enzyme activation likely provide a unique inhibitor-protease interaction surface and is, thus, expected to enhance the chances of engineering high inhibitor specificity. Our results provide new information about the structural flexibility underlying the equilibrium between active and inactive forms of serine proteases.