Antoine Désilets

Probing the substrate specificities of matriptase, matriptase-2, hepsin and DESC1 with internally quenched fluorescent peptides

Type II transmembrane serine proteases are an emerging class of proteolytic enzymes involved in tissue homeostasis and a number of human disorders such as cancer. To better define the biochemical functions of a subset of these proteases, we compared the enzymatic properties of matriptase, matriptase‐2, hepsin and DESC1 using a series of internally quenched fluorogenic peptide substrates containing o‐aminobenzoyl and 3‐nitro‐tyrosine. We based the sequence of the peptides on the P4 to P4′ activation sequence of matriptase (RQAR‐VVGG). Positions P4, P3, P2 and P1′ were substituted with nonpolar (Ala, Leu), aromatic (Tyr), acid (Glu) and basic (Arg) amino acids, whereas P1 was fixed to Arg. Of the four type II transmembrane serine proteases studied, matriptase‐2 was the most promiscuous, and matriptase was the most discriminating, with a distinct specificity for Arg residues at P4, P3 and P2. DESC1 had a preference similar to that of matriptase, but with a propensity for small nonpolar amino acids (Ala) at P1′. Hepsin shared similarities with matriptase and DESC1, but was markedly more permissive at P2. Matriptase‐2 manifested broader specificities, as well as substrate inhibition, for selective internally quenched fluorescent substrates. Lastly, we found that antithrombin III has robust inhibitory properties toward matriptase, matriptase‐2, hepsin and DESC1, whereas plasminogen activator inhibitor‐1 and α2‐antiplasmin inhibited matriptase‐2, hepsin and DESC1, and to a much lesser extent, matriptase. In summary, our studies revealed that these enzymes have distinct substrate preferences.

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

OBJECTIVE Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA). METHODS Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low-density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis/N-terminal sequencing, while its ability to activate proteinase-activated receptor 2 (PAR-2) was determined using a synovial perfusion assay in mice. RESULTS Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase-dependent. Matriptase also induced MMP-1, MMP-3, and MMP-13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR-2, and we demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition. CONCLUSION Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR-2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium

ABSTRACT Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.

Matriptase protects against experimental colitis and promotes intestinal barrier recovery

Background: Matriptase is a membrane‐anchored serine protease encoded by suppression of tumorigenicity‐14 (ST14) that is required for epithelial barrier homeostasis. However, its functional role in inflammatory bowel disease (IBD) is unexplored. Methods: Matriptase expression in control, Crohns disease, and ulcerative colitis tissue specimens was studied by quantitative polymerase chain reaction (qPCR) and immunostaining. Matriptase function was investigated by subjecting St14 hypomorphic and control littermates to dextran sodium sulfate (DSS)‐induced colitis and by siRNA silencing in cultured monolayers. Mice were analyzed for clinical, histological, molecular, and cellular effects. Results: Matriptase protein and ST14 mRNA levels are significantly downregulated in inflamed colonic tissues from Crohns disease and ulcerative colitis patients. Matriptase‐deficient St14 hypomorphic mice administered DSS for 7 days followed by water without DSS for 3 days develop a severe colitis, with only 30% of the St14 hypomorphic mice surviving to day 14, compared with 100% of control littermates. Persistent colitis in surviving St14 hypomorphic mice was associated with sustained cytokine production, an inability to recover barrier integrity, and enhanced claudin‐2 expression. Cytokines implicated in barrier disruption during IBD suppress matriptase expression in T84 epithelial monolayers and restoration of matriptase improves barrier integrity in the cytokine‐perturbed monolayers. Conclusions: These data demonstrate a critical role for matriptase in restoring barrier function to injured intestinal mucosa during colitis, which is suppressed by excessive activation of the immune system. Strategies to enhance matriptase‐mediated barrier recovery could be important for intervening in the cycle of inflammation associated with IBD. (Inflamm Bowel Dis 2011;)

Mutation G827R in Matriptase Causing Autosomal Recessive Ichthyosis with Hypotrichosis Yields an Inactive Protease

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly827 by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.

Design and Synthesis of Potent, Selective Inhibitors of Matriptase

Matriptase is a member of the type II transmembrane serine protease family. Several studies have reported deregulated matriptase expression in several types of epithelial cancers, suggesting that matriptase constitutes a potential target for cancer therapy. We report herein a new series of slow, tight-binding inhibitors of matriptase, which mimic the P1-P4 substrate recognition sequence of the enzyme. Preliminary structure-activity relationships indicate that this benzothiazole-containing RQAR-peptidomimetic is a very potent inhibitor and possesses a good selectivity for matriptase versus other serine proteases. A molecular model was generated to elucidate the key contacts between inhibitor 1 and matriptase.

Inhibition of human matriptase by eglin c variants

Based on the enzyme specificity of matriptase, a type II transmembrane serine protease (TTSP) overexpressed in epithelial tumors, we screened a cDNA library expressing variants of the protease inhibitor eglin c in order to identify potent matriptase inhibitors. The most potent of these, R1K4′‐eglin, which had the wild‐type Pro45 (P1 position) and Tyr49 (P4′ position) residues replaced with Arg and Lys, respectively, led to the production of a selective, high affinity (K i of 4 nM) and proteolytically stable inhibitor of matriptase. Screening for eglin c variants could yield specific, potent and stable inhibitors to matriptase and to other members of the TTSP family.

Prostasin is required for matriptase activation in intestinal epithelial cells to regulate closure of the paracellular pathway

Background: Matriptase promotes intestinal epithelial barrier formation. Results: Matriptase and prostasin constitute a common proteolytic pathway where matriptase is indirectly activated by prostasin and functions on the cell surface to regulate barrier formation. Conclusion: A prostasin → matriptase protease cascade regulates barrier formation in simple intestinal epithelia. Significance: Cell surface serine proteases have complex cascade relationships that are context- and tissue-specific. The type II transmembrane serine protease matriptase is a key regulator of epithelial barriers in skin and intestine. In skin, matriptase acts upstream of the glycosylphosphatidylinositol-anchored serine protease, prostasin, to activate the prostasin zymogen and initiate a proteolytic cascade that is required for stratum corneum barrier functionality. Here, we have investigated the relationship between prostasin and matriptase in intestinal epithelial barrier function. We find that similar to skin, matriptase and prostasin are components of a common intestinal epithelial barrier-forming pathway. Depletion of prostasin by siRNA silencing in Caco-2 intestinal epithelium inhibits barrier development similar to loss of matriptase, and the addition of recombinant prostasin to the basal side of polarized Caco-2 epithelium stimulates barrier forming changes similar to the addition of recombinant matriptase. However, in contrast to the proteolytic cascade in skin, prostasin functions upstream of matriptase to activate the endogenous matriptase zymogen. Prostasin is unable to proteolytically activate the matriptase zymogen directly but induces matriptase activation indirectly. Prostasin requires expression of endogenous matriptase to stimulate barrier formation since matriptase depletion by siRNA silencing abrogates prostasin barrier-forming activity. Active recombinant matriptase, however, does not require the expression of endogenous prostasin for barrier-forming activity. Together, these data show that matriptase and not prostasin is the primary effector protease of tight junction assembly in simple columnar epithelia and further highlight a spatial and tissue-specific aspect of cell surface proteolytic cascades.

Analysis of Subpocket Selectivity and Identification of Potent Selective Inhibitors for Matriptase and Matriptase-2

We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homologous proteases matriptase and matriptase-2 across subpockets using docking simulations. We observed that the farther away a subpocket is located from the catalytic site, the more pronounced its role in selectivity. As a result of our exhaustive virtual screening, we biochemically validated novel potent and selective inhibitors of both enzymes.

Essential role of endocytosis of the type II transmembrane serine protease TMPRSS6 in regulating its functionality

The type II transmembrane serine protease TMPRSS6 (also known as matriptase-2) controls iron homeostasis through its negative regulation of expression of hepcidin, a key hormone involved in iron metabolism. Upstream of the hepcidin-regulated signaling pathway, TMPRSS6 cleaves its target substrate hemojuvelin (HJV) at the plasma membrane, but the dynamics of the cell-surface expression of the protease have not been addressed. Here, we report that TMPRSS6 undergoes constitutive internalization in transfected HEK293 cells and in two human hepatic cell lines, HepG2 and primary hepatocytes, both of which express TMPRSS6 endogenously. Cell surface-labeled TMPRSS6 was internalized and was detected in clathrin- and AP-2-positive vesicles via a dynamin-dependent pathway. The endocytosed TMPRSS6 next transited in early endosomes and then to lysosomes. Internalization of TMPRSS6 is dependent on specific residues within its N-terminal cytoplasmic domain, as site-directed mutagenesis of these residues abrogated internalization and maintained the enzyme at the cell surface. Cells coexpressing these mutants and HJV produced significantly decreased levels of hepcidin compared with wild-type TMPRSS6 due to the sustained cleavage of HJV at the cell surface by TMPRSS6 mutants. Our results underscore for the first time the importance of TMPRSS6 trafficking at the plasma membrane in the regulation of hepcidin expression, an event that is essential for iron homeostasis.