Local protease signaling contributes to neural tube closure in the mouse embryo.

We report an unexpected role for protease signaling in neural tube closure and the formation of the central nervous system. Mouse embryos lacking protease-activated receptors 1 and 2 showed defective hindbrain and posterior neuropore closure and developed exencephaly and spina bifida, important human congenital anomalies. Par1 and Par2 were expressed in surface ectoderm, and Par2 was expressed selectively along the line of closure. Ablation of G(i/z) and Rac1 function in these Par2-expressing cells disrupted neural tube closure, further implicating G protein-coupled receptors and identifying a likely effector pathway. Cluster analysis of protease and Par2 expression patterns revealed a group of membrane-tethered proteases often coexpressed with Par2. Among these, matriptase activated Par2 with picomolar potency, and hepsin and prostasin activated matriptase. Together, our results suggest a role for protease-activated receptor signaling in neural tube closure and identify a local protease network that may trigger Par2 signaling and monitor and regulate epithelial integrity in this context.

Tumor detection by imaging proteolytic activity.

The cell surface protease membrane-type serine protease-1 (MT-SP1), also known as matriptase, is often upregulated in epithelial cancers. We hypothesized that dysregulation of MT-SP1 with regard to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1), a situation that increases proteolytic activity, might be exploited for imaging purposes to differentiate malignant from normal tissue. In this study, we show that MT-SP1 is active on cancer cells and that its activity may be targeted in vivo for tumor detection. A proteolytic activity assay with several MT-SP1-positive human cancer cell lines showed that MT-SP1 antibodies that inhibit recombinant enzyme activity in vitro also bind and inhibit the full-length enzyme expressed on cells. In contrast, in the same assay, MT-SP1-negative cancer cell lines were inactive. Fluorescence microscopy confirmed the cell surface localization of labeled antibodies bound to MT-SP1-positive cells. To evaluate in vivo targeting capability, 0.7 to 2 nmoles of fluorescently labeled antibodies were administered to mice bearing tumors that were positive or negative for MT-SP1. Antibodies localized to MT-SP1-positive tumors (n = 3), permitting visualization of MT-SP1 activity, whereas MT-SP1-negative tumors (n = 2) were not visualized. Our findings define MT-SP1 activity as a useful biomarker to visualize epithelial cancers using a noninvasive antibody-based method.

Structure of an Fab-protease complex reveals a highly specific non-canonical mechanism of inhibition.

The vast majority of protein protease inhibitors bind their targets in a substrate-like manner. This is a robust and efficient mechanism of inhibition but, due to the highly conserved architecture of protease active sites, these inhibitors often exhibit promiscuity. Inhibitors that show strict specificity for one protease usually achieve this selectivity by combining substrate-like binding in the active site with exosite binding on the protease surface. The development of new, specific inhibitors can be aided greatly by binding to non-conserved regions of proteases if potency can be maintained. Due to their ability to bind specifically to nearly any antigen, antibodies provide an excellent scaffold for creating inhibitors targeted to a single member of a family of highly homologous enzymes. The 2.2 A resolution crystal structure of an Fab antibody inhibitor in complex with the serine protease membrane-type serine protease 1 (MT-SP1/matriptase) reveals the molecular basis of its picomolar potency and specificity. The inhibitor has a distinct mechanism of inhibition; it gains potency and specificity through interactions with the protease surface loops, and inhibits by binding in the active site in a catalytically non-competent manner. In contrast to most naturally occurring protease inhibitors, which have diverse structures but converge to a similar inhibitory archetype, antibody inhibitors provide an opportunity to develop divergent mechanisms of inhibition from a single scaffold.

MT-SP1 proteolysis and regulation of cell-microenvironment interactions.

MT-SP1 is a type II transmembrane serine protease implicated in a range of human cancers including those of the breast, cervix, ovaries, prostate, colon and gastrointestinal tract. Mouse models have shown it to be critical for proper epidermal development and postnatal survival. However, the role of this enzyme in normal and malignant biology has not yet been fully elucidated. Several groups have identified putative substrates of MT-SP1 in an effort to understand the possible biological processes in which this protease may be involved. Methods for substrate identification include comparing known protein cleavage sequences with MT-SP1 specificity data, in vitro cleavage assays, examining genetic microarrays for enzyme/substrate coexpression, immunohistochemistry for colocalization, and a variety of phenotypic observations using cell culture and mouse models. Given the inherent limitations of each individual method, substrate plausibility is best substantiated using a combination of experimental approaches. Here we review MT-SP1 substrates identified to date and the possible physiological implications of substrate cleavage in cell-microenvironment interactions. This data indicates that MT-SP1 is capable of playing roles in growth factor activation, receptor activation and inactivation, protease activation, and ectodomain shedding. We also present for the first time vascular endothelial growth factor receptor 2 (VEGFR-2) as a putative substrate for MT-SP1.

The mechanism of inhibition of antibody-based inhibitors of membrane-type serine protease 1 (MT-SP1).

The mechanisms of inhibition of two novel scFv antibody inhibitors of the serine protease MT-SP1/matriptase reveal the basis of their potency and specificity. Kinetic experiments characterize the inhibitors as extremely potent inhibitors with K(I) values in the low picomolar range that compete with substrate binding in the S1 site. Alanine scanning of the loops surrounding the protease active site provides a rationale for inhibitor specificity. Each antibody binds to a number of residues flanking the active site, forming a unique three-dimensional binding epitope. Interestingly, one inhibitor binds in the active site cleft in a substrate-like manner, can be processed by MT-SP1 at low pH, and is a standard mechanism inhibitor of the protease. The mechanisms of inhibition provide a rationale for the effectiveness of these inhibitors, and suggest that the development of specific antibody-based inhibitors against individual members of closely related enzyme families is feasible, and an effective way to develop tools to tease apart complex biological processes.