Expression and genetic loss of function analysis of the HAT/DESC cluster proteases TMPRSS11A and HAT.

Genome mining at the turn of the millennium uncovered a new family of type II transmembrane serine proteases (TTSPs) that comprises 17 members in humans and 19 in mice. TTSPs phylogenetically belong to one of four subfamilies: matriptase, hepsin/TMPRSS, corin and HAT/DESC. Whereas a wealth of information now has been gathered as to the physiological functions of members of the hepsin/TMPRSS, matriptase, and corin subfamilies of TTSPs, comparatively little is known about the functions of the HAT/DESC subfamily of proteases.

Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice.

Hypomorphic mutations in the human SPINT2 gene cause a broad spectrum of abnormalities in organogenesis, including organ and digit duplications, atresia, fistulas, hypertelorism, cleft palate and hamartoma. SPINT2 encodes the transmembrane serine protease inhibitor HAI2 (placental bikunin), and the severe developmental effects of decreased HAI2 activity can be hypothesized to be a consequence of excess pericellular proteolytic activity. Indeed, we show here that HAI2 is a potent regulator of protease-guided cellular responses, including motogenic activity and transepithelial resistance of epithelial monolayers.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin.

Imaging specific cell surface protease activity in living cells using reengineered bacterial cytotoxins.

The scarcity of methods to visualize the activity of individual cell surface proteases in situ has hampered basic research and drug development efforts. In this chapter, we describe a simple, sensitive, and noninvasive assay that uses nontoxic reengineered bacterial cytotoxins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay takes advantage of the absolute requirement for site-specific endoproteolytic cleavage of cell surface-bound anthrax toxin protective antigen for its capacity to translocate an anthrax toxin lethal factor-beta-lactamase fusion protein to the cytoplasm.

Potent inhibition and global co-localization implicate the transmembrane Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-2 in the regulation of epithelial matriptase activity.

Hepatocyte growth factor activator inhibitors (HAI)-1 and -2 are recently identified and closely related Kunitz-type transmembrane serine protease inhibitors. Whereas HAI-1 is well established as an inhibitor of the serine proteases matriptase and hepatocyte growth factor activator, the physiological targets of HAI-2 are unknown. Here we show that HAI-2 displays potent inhibitory activity toward matriptase, forms SDS-stable complexes with the serine protease, and blocks matriptase-dependent activation of its candidate physiological substrates proprostasin and cell surface-bound pro-urokinase plasminogen activator.

Co-localization of the channel activating protease prostasin/(CAP1/PRSS8) with its candidate activator, matriptase.

Prostasin (CAP1/PRSS8) is a glycosylphosphatidylinositol-anchored membrane serine protease believed to be critical for the regulation of epithelial sodium channel (ENaC) activity. Prostasin is synthesized as an inactive zymogen that requires a site-specific endoproteolytic cleavage to be converted to an active protease. We have recently reported that the tumor-associated type II transmembrane serine protease, matriptase is necessary and sufficient for prostasin activation in the epidermis.

Neuropeptide Y induces migration, proliferation, and tube formation of endothelial cells bimodally via Y1, Y2, and Y5 receptors.

Previously we discovered that NPY induces ischemic angiogenesis by activating Y2 and Y5 receptors. The receptors that mediate specific steps of the complex process of angiogenesis are unknown. Here, we studied in vitro NPY receptors subtypes involved in migration, proliferation, and differentiation of human endothelial cells.

Imaging specific cell-surface proteolytic activity in single living cells.

We describe a simple, sensitive and noninvasive assay that uses nontoxic, reengineered anthrax toxin-beta-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell-surface proteolytic activity in single living cells. The assay could be used to specifically image endogenous cell-surface furin, urokinase plasminogen activator and metalloprotease activity. We have adapted the assay for fluorescence microscopy, flow cytometry and fluorescent plate reader formats, and it is amenable for automation and high-throughput analysis.

Matriptase-3 is a novel phylogenetically preserved membrane-anchored serine protease with broad serpin reactivity.

We report in the present study the bioinformatic identification, molecular cloning and biological characterization of matriptase-3, a novel membrane-anchored serine protease that is phylogenetically preserved in fish, birds, rodents, canines and primates. The gene encoding matriptase-3 is located on syntenic regions of human chromosome 3q13.2, mouse chromosome 16B5, rat chromosome 11q21 and chicken chromosome 1.

Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes.

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.

Role of sphingosine-1-phosphate phosphatase 1 in epidermal growth factor-induced chemotaxis.

Sphingosine-1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors that regulate a wide variety of cellular functions, including cytoskeletal rearrangements and cell motility. Because of the pivotal role of S1P, its levels are low and tightly regulated in a spatial-temporal manner through its synthesis catalyzed by sphingosine kinases and degradation by an S1P lyase and specific S1P phosphatases (SPP). Surprisingly, down-regulation of SPP-1 enhanced migration toward epidermal growth factor (EGF); conversely, overexpression of SPP-1, which is localized in the endoplasmic reticulum, attenuated migration toward EGF.

Sphingosine kinase type 1 promotes estrogen-dependent tumorigenesis of breast cancer MCF-7 cells.

The sphingolipid metabolite, sphingosine-1-phosphate (S1P), formed by phosphorylation of sphingosine, has been implicated in cell growth, suppression of apoptosis, and angiogenesis. In this study, we have examined the contribution of intracellular S1P to tumorigenesis of breast adenocarcinoma MCF-7 cells. Enforced expression of sphingosine kinase type 1 (SPHK1) increased S1P levels and blocked MCF-7 cell death induced by anti-cancer drugs, sphingosine, and TNF-alpha.

Sphingosine 1-phosphate, present in serum-derived lipoproteins, activates matriptase.

We describe here a novel biological function of sphingosine 1-phosphate (S1P): the activation of a serine protease, matriptase. Matriptase is a type II integral membrane serine protease, expressed on the surface of a variety of epithelial cells; it may play an important role in tissue remodeling. We have previously reported that the activation of matriptase is regulated by serum.