Matriptase,HAT, and TMPRSS2 Activate the Hemagglutinin of H9N2 Influenza A Viruses

Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.     

Hepsin activates prostasin and cleaves the extracellular domain of the epidermal growth factor receptor

The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates prostasin by cleaving in the amino-terminal pro-peptide region of prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human prostasin. Using an Arg-to-Ala mutant (R44A) human prostasin, we showed in this report that the cleavage of prostasin by matriptase is at Arg44. This prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the prostasin-activating protease in cells co-transfected with EGFR, we showed that active prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase–prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, prostasin, and EGFR.     

Enteroviral protease 2A directly cleaves dystrophin and is inhibited by a dystrophin-based substrate analogue

Enteroviruses such as Coxsackievirus B3 can cause dilated cardiomyopathy through unknown pathological mechanism(s). Dystrophin is a large extrasarcomeric cytoskeletal protein whose genetic deficiency causes hereditary dilated cardiomyopathy. In addition, we have recently shown that dystrophin is proteolytically cleaved by the Coxsackievirus protease 2A leading to functional impairment and morphological disruption. However, the mechanism of dystrophin cleavage and the exact cleavage site remained to be identified. Antibody epitope mapping of endogenous dystrophin indicated protease 2A-mediated cleavage at the site in the hinge 3 region predicted by a neural network algorithm (human, amino acid 2434; mouse, amino acid 2427). Using site-directed mutagenesis, peptide sequencing, and fluorescence resonance energy transfer assays with recombinant dystrophin, we demonstrate that this putative site in mouse and human dystrophin is a direct substrate for the Coxsackieviral protease 2A both in vitro and in vivo. The substrate analogue protease inhibitor z-LSTT-fmk was designed based on the dystrophin sequence that interacts with the protease 2A and was found to have an IC(50) of 550 nM in vitro. Dystrophin is the first cellular substrate of the enteroviral protease 2A that was identified using by a bioinformatic approach and for which the cleavage site was molecularly mapped within living cells.     

The activation of matriptase requires its noncatalytic domains,serine protease domain,and its cognate inhibitor

The activation of matriptase requires proteolytic cleavage at a canonical activation motif that converts the enzyme from a one-chain zymogen to an active, two-chain protease. In this study, matriptase bearing a mutation in its catalytic triad was unable to undergo this activational cleavage, suggesting that the activating cleavage occurs via a transactivation mechanism where interaction between matriptase zymogen molecules leads to activation of the protease. Using additional point and deletion mutants, we showed that activation of matriptase requires proteolytic processing at Gly-149 in the SEA domain of the protease, glycosylation of the first CUB domain and the serine protease domain, and intact low density lipoprotein receptor class A domains. Its cognate inhibitor, hepatocyte growth factor activator inhibitor-1, may also participate in the activation of matriptase, based on the observation that matriptase activation did not occur when the protease was co-expressed with hepatocyte growth factor activator inhibitor-1 mutated in its low density lipoprotein receptor class A domain. These results suggest that besides matriptase catalytic activity, matriptase activation requires post-translational modification of the protease, intact noncatalytic domains, and its cognate inhibitor.     

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin–Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)₆] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)₆ exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.     

Strong expression association between matriptase and its substrate prostasin in breast cancer

Breast cancer tumorigenesis is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and signaling receptors that are critically involved in neoplastic progression. Multiple studies implicate the membrane anchored serine protease matriptase (also known as MT-SP1 and epithin) in breast cancer. The pro-form of the GPI-anchored serine protease prostasin has recently been identified as a physiological substrate of matriptase and the two proteases are co-expressed in multiple healthy tissues. In this study, the inter-relationship between the two membrane-anchored serine proteases in breast cancer was investigated using breast cancer cell lines and breast cancer patient samples to delineate the association between matriptase and prostasin. We used Western blotting to determine the expression of matriptase and prostasin proteins in a panel of breast cancer cell lines and immunohistochemistry to assess the expression in serial sections from breast cancer tissue arrays. We demonstrate that the expression of matriptase and prostasin is closely correlated in breast cancer cell lines as well as in breast cancer tissue samples. Furthermore, matriptase and prostasin display a near identical spatial expression pattern in the epithelial compartment of breast cancer tissue. These data suggest that the matriptase-prostasin cascade might play a critical role in breast cancer.     

Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)

Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) modulates cellular adhesion and growth in an IGF/insulin-dependent or independent manner. It also shows tumor-suppressive activity in vivo. We recently found that a single-chain IGFB-rP1 is proteolytically cleaved to a two-chain form by a trypsin-like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP-rP1-processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP-rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN-45), highly produced the cleaved IGFBP-rP1. Serine proteinase inhibitors effectively blocked the IGFBP-rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP-rP1, but their membrane fraction had an IGFBP-rP1-cleaving activity. The membrane fraction contained an 80-kDa gelatinolytic enzyme, which was identified as the membrane-type serine proteinase matriptase (MT-SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor-free form efficiently cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is processed to the two-chain form by matriptase on the cell surface.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Matriptase: potent proteolysis on the cell surface

Matriptase is a type II transmembrane serine protease expressed in most human epithelia, where it is coexpressed with its cognate transmembrane inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. Activation of the matriptase zymogen requires sequential N-terminal cleavage, activation site autocleavage, and transient association with HAI-1. Matriptase has an essential physiological role in profilaggrin processing, corneocyte maturation, and lipid matrix formation associated with terminal differentiation of the oral epithelium and the epidermis, and is also critical for hair follicle growth. Matriptase and HAI expression are frequently dysregulated in human cancer, and matriptase expression that is unopposed by HAI-1 potently promotes carcinogenesis and metastatic dissemination in animal models.     

Increased matriptase zymogen activation in inflammatory skin disorders

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.     

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. To further explore the potential functional relationship between HAI-2 and matriptase, we generated a transgenic mouse strain with a promoterless beta-galactosidase marker gene inserted into the endogenous locus encoding HAI-2 protein and performed a global high resolution mapping of the expression of HAI-2, matriptase, and HAI-1 proteins in all adult tissues. This analysis showed striking co-localization of HAI-2 with matriptase and HAI-1 in epithelial cells of all major organ systems, thus strongly supporting a role of HAI-2 as a physiological regulator of matriptase activity, possibly acting in a redundant or partially redundant manner with HAI-1. Unlike HAI-1 and matriptase, however, HAI-2 expression was also detected in non-epithelial cells of brain and lymph nodes, suggesting that HAI-2 may also be involved in inhibition of serine proteases other than matriptase.     

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

Hepatocyte growth factor activator inhibitor type I (HAI-1) is a membrane-bound, serine protease inhibitor with two protease-inhibitory domains (Kunitz domain I and II). HAI-1 is known as a physiological inhibitor of a membrane-bound serine protease, matriptase. Paradoxically, however, HAI-1 has been found to be required for the extracellular appearance of the protease in an expression system using a monkey kidney COS-1 cell line. In the present study, we show using COS-1 cells that co-expression of recombinant variants of HAI-1 with the inhibition activity toward matriptase, including a variant consisting only of Kunitz domain I (the domain responsible for inhibition of matriptase), allowed for the appearance of this protease in the conditioned medium, whereas that of the variants without the activity did not. These findings suggest that the inhibition activity toward matriptase is critical for the extracellular appearance of protease in COS-1 cells.     

Molecular cloning of cDNA for matriptase, a matrix-degrading serine protease with trypsin-like activity.

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.     

Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acid-sensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the two-chain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.     

Differential Subcellular Localization Renders HAI-2 a Matriptase Inhibitor in Breast Cancer Cells but Not in Mammary Epithelial Cells

The type 2 transmembrane serine protease matriptase is under tight control primarily by the actions of the integral membrane Kunitz-type serine protease inhibitor HAI-1. Growing evidence indicates that HAI-2 might also be involved in matriptase inhibition in some contexts. Here we showed that matriptase inhibition by HAI-2 depends on the subcellular localizations of HAI-2, and is observed in breast cancer cells but not in mammary epithelial cells. HAI-2 is co-expressed with matriptase in 21 out of 26 human epithelial and carcinoma cells examined. HAI-2 is also a potent matriptase inhibitor in solution, but in spite of this, HAI-2 inhibition of matriptase is not observed in all contexts where HAI-2 is expressed, unlike what is seen for HAI-1. Induction of matriptase zymogen activation in mammary epithelial cells results in the formation of matriptase-HAI-1 complexes, but matriptase-HAI-2 complexes are not observed. In breast cancer cells, however, in addition to the appearance of matriptase-HAI-1 complex, three different matriptase-HAI-2 complexes, are formed following the induction of matriptase activation. Immunofluorescent staining reveals that activated matriptase is focused at the cell-cell junctions upon the induction of matriptase zymogen activation in both mammary epithelial cells and breast cancer cells. HAI-2, in contrast, remains localized in vesicle/granule-like structures during matriptase zymogen activation in human mammary epithelial cells. In breast cancer cells, however, a proportion of the HAI-2 reaches the cell surface where it can gain access to and inhibit active matriptase. Collectively, these data suggest that matriptase inhibition by HAI-2 requires the translocation of HAI-2 to the cell surface, a process which is observed in some breast cancer cells but not in mammary epithelial cells.     

HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-1 regulates this protease. First, we observed that after S1P treatment HAI-1 was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-1 to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-1 small interfering RNA or anti-HAI-1 MAb M19, spontaneous activation of matriptase occurred in the absence of S1P-induced translocation; S1P-induced matriptase activation was also enhanced. These results support a role for HAI-1 in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-1 in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-1; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-1, but not HAI-1 mutants altered in its Kunitz domain 1, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-1. These results are also consistent with a role for HAI-1 to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-1 to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition. protease-activated receptor-2; hepatocyte growth factor; urokinase; sphingosine 1-phosphate; Kunitz domain     

Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells

We describe a versatile system for monitoring the activity of the NS3-4A serine protease of the hepatitis C virus (HCV) in mammalian cells. The system relies on coexpression of the protease and of an artificial substrate containing a reporter domain and an intracellular targeting sequence separated by a NS3-4A-specific cleavage site. We constructed two different substrates suitable for different applications. The first substrate secretory alkaline phosphatase-1 (SEAP-1) harbors the NS3-4A cleavage site inserted between the SEAP and a membrane anchor featuring an endoplasmic reticulum retention sequence. The arrangement of this substrate is such that SEAP is secreted in the extracellular medium depending on the NS3 protease activity. We show that SEAP-1 can be used to evaluate the activity of NS3-4A inhibitors in living cells. In the second substrate (CD8-1), SEAP is replaced by the extracellular domain of the lymphocyte surface antigen CD8 alpha. The arrangement of this substrate is such that the CD8 alpha domain is transported to the cell surface upon NS3-4Ap cleavage and remains associated with the plasma membrane as an integral membrane protein. We show that CD8-1 can be used for selecting cells capable of supporting HCV replication.     

Dynamic Regulation of Platelet-derived Growth Factor D (PDGF-D) Activity and Extracellular Spatial Distribution by Matriptase-mediated Proteolysis

The oncogenic roles of PDGF-D and its proteolytic activator, matriptase, have been strongly implicated in human prostate cancer. Latent full-length PDGF-D (FL-D) consists of a CUB domain, a growth factor domain (GFD), and the hinge region in between. Matriptase processes the FL-D dimer into a GFD dimer (GFD-D) in a stepwise manner, involving generation of a hemidimer (HD), an intermediate product containing one FL-D subunit and one GFD subunit. Although the HD is a pro-growth factor that can be processed into the GFD-D by matriptase, the HD can also act as a dominant-negative ligand that prevents PDGF-B-mediated β-PDGF receptor activation in fibroblasts. The active GFD-D can be further cleaved into a smaller and yet inactive form if matriptase-mediated proteolysis persists. Through mutagenesis and functional analyses, we found that the R³⁴⁰R³⁴¹GR³⁴³A (P4–P1/P1′) motif within the GFD is the matriptase cleavage site through which matriptase can deactivate PDGF-D. Comparative sequence analysis based on the published crystal structure of PDGF-B predicted that the matriptase cleavage site R³⁴⁰R³⁴¹GR³⁴³A is within loop III of the GFD, a critical structural element for its binding with the β-PDGF receptor. Interestingly, we also found that matriptase processing regulates the deposition of PDGF-D dimer species into the extracellular matrix (ECM) with increased binding from the FL-D dimer, to the HD, and to the GFD-D. Furthermore, we provide evidence that R³⁴⁰R³⁴¹GR³⁴³A within the GFD is critical for PDGF-D deposition and binding to the ECM. In this study, we report a structural element crucial for the biological function and ECM deposition of PDGF-D and provide molecular insight into the dynamic functional interplay between the serine protease matriptase and PDGF-D.     

Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice

Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes.