Prion diseases are a group of neurodegenerative diseases, which can progress rapidly. Previous data have demonstrated that prion protein (PrP) stimulates activation of plasminogen (Plg) by tissue plasminogen activator (tPA). In this study, using spectroscopic method, we aimed to determine whether PrP's role in activating Plg is influenced by metal binding. We also investigated the region in PrP involved in binding to tPA and Plg, and whether PrP in fibrillar form behaves the same way as PrP unbound to any metal ion i.e., apo-PrP. We investigated the effect of recombinant mouse PrP (residues 23-231) refolded with nickel, manganese, copper, and a variant devoid of any metal ions, on tPA-catalyzed Plg activation. Using mutant PrP (H95A, H110A), we also investigated whether histidine residues outside the octarepeat region in PrP, which is known to bind tPA and Plg, are also involved in their binding. We demonstrated that apo-PrP is most effective at stimulating Plg. PrP refolded with nickle or manganese behave similar to apo-PrP, and PrP refolded with copper is least effective. The mutant form of PrP did not stimulate Plg activation to the same degree as apo-PrP indicating that the histidine residues outside the octarepeat region are also involved in binding to tPA and Plg. Similarly, the fibrillar form of PrP was ineffective at stimulating Plg activation. Our data suggest that upon loss of copper specifically, a structural rearrangement of PrP occurs that exposes binding sites to Plg and tPA, enhancing the stimulation of Plg activation.

Plasminogen activation, catalyzed by the serine proteases urokinase-type plasminogen activator (u-PA) and tissue plasminogen activator (t-PA), is thought to play an important role in a variety of physiological and pathological processes. The potency of the plasminogen activation system indicates that specific mechanisms must exist to control the initiation, progression, localization, and termination of the system. The principal role of u-PAR is to provide a facile mechanism for the confinement of u-PA to the cell surface, and this is accomplished by the high-affinity interaction between the growth factor domain of u-PA and the amino-terminal domain of u-PAR. The explanation for the discrepancy in the effects of u-PAR in purified and cell-associated systems lies in the binding of plasminogen to the cell surface. The cellular potentiation of plasminogen activation has been shown to involve effects on the activation of both plasminogen and pro-u-PA, which together account for the overall effect.

TMEFF2 is a type I transmembrane protein with two follistatin (FS) and one EGF-like domain over-expressed in prostate cancer; however its biological role in prostate cancer development and progression remains unclear, which may, at least in part, be explained by its proteolytic processing. The extracellular part of TMEFF2 (TMEFF2-ECD) is cleaved by ADAM17 and the membrane-retained fragment is further processed by the gamma-secretase complex. TMEFF2 shedding is increased with cell crowding, a condition associated with the tumour microenvironment, which was mediated by oxidative stress signalling, requiring jun-kinase (JNK) activation. Moreover, we have identified that TMEFF2 is also a novel substrate for other proteases implicated in prostate cancer, including two ADAMs (ADAM9 and ADAM12) and the type II transmembrane serine proteinases (TTSPs) matriptase-1 and hepsin. Whereas cleavage by ADAM9 and ADAM12 generates previously identified TMEFF2-ECD, proteolytic processing by matriptase-1 and hepsin produced TMEFF2 fragments, composed of TMEFF2-ECD or FS and/or EGF-like domains as well as novel membrane retained fragments. Differential TMEFF2 processing from a single transmembrane protein may be a general mechanism to modulate transmembrane protein levels and domains, dependent on the repertoire of ADAMs or TTSPs expressed by the target cell.

4 School of Biological Sciences, University of East Anglia, Norwich, Norfolk, NR4 7TJ, UK 5 Anglia Ruskin IT Research Institute (ARITI), Anglia Ruskin University, Cambridge, CB1 1PT, UK 6 Department of Applied Sciences, Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, UK. Corresponding author: rosemary.bass@northumbria.ac.uk, telephone +44 (0)191 2274899, no fax number.

It has recently been shown that hepatocyte growth factor activator inhibitor‐2 (HAI‐2) is able to suppress carcinogenesis induced by overexpression of matriptase, as well as cause regression of individual established tumors in a mouse model system. However, the role of HAI‐2 is poorly understood. In this study, we describe 3 mutations in the binding loop of the HAI‐2 Kunitz domain 1 (K42N, C47F and R48L) that cause a delay in the SEA domain cleavage of matriptase, leading to accumulation of non‐SEA domain cleaved matriptase in the endoplasmic reticulum (ER). We suggest that, like other known SEA domains, the matriptase SEA domain auto‐cleaves and reflects that correct oligomerization, maturation, and/or folding has been obtained. Our results suggest that the HAI‐2 Kunitz domain 1 mutants influence the flux of matriptase to the plasma membrane by affecting the oligomerization, maturation and/or folding of matriptase, and as a result the SEA domain cleavage of matriptase. Two of the HAI‐2 Kunitz domain 1 mutants investigated (C47F, R48L and C47F/R48L) also displayed a reduced ability to proteolytically silence matriptase. Hence, HAI‐2 separately stabilizes matriptase, regulates the secretory transport, possibly via maturation/oligomerization and inhibits the proteolytic activity of matriptase in the ER, and possible throughout the secretory pathway.

Recent developments in quantitative image analysis allow us to interrogate confocal microscopy images to answer biological questions. Clumped and layered cell nuclei and cytoplasm in confocal images challenges the ability to identify subcellular compartments. To date, there is no perfect image analysis method to identify cytoskeletal changes in confocal images. Here, we present a multidisciplinary study where an image analysis model was developed to allow quantitative measurements of changes in the cytoskeleton of cells with different maspin exposure. Maspin, a noninhibitory serpin influences cell migration, adhesion, invasion, proliferation, and apoptosis in ways that are consistent with its identification as a tumor metastasis suppressor. Using different cell types, we tested the hypothesis that reduction in cell migration by maspin would be reflected in the architecture of the actin cytoskeleton. A hybrid marker-controlled watershed segmentation technique was used to segment the nuclei, cytoplasm, and ruffling regions before measuring cytoskeletal changes. This was informed by immunohistochemical staining of cells transfected stably or transiently with maspin proteins, or with added bioactive peptides or protein. Image analysis results showed that the effects of maspin were mirrored by effects on cell architecture, in a way that could be described quantitatively.

Matriptase is a type-II transmembrane serine protease involved in epithelial homeostasis in both health and disease, and is implicated in the development and progression of a variety of cancers. Matriptase mediates its biological effects both via as yet undefined substrates and pathways, and also by proteolytic cleavage of a variety of well-defined protein substrates, several of which it shares with the closely-related protease hepsin. Development of targeted therapeutic strategies will require discrimination between these proteases. Here we have investigated cyclic microproteins of the squash Momordica cochinchinensis trypsin-inhibitor family (generated by total chemical synthesis) and found MCoTI-II to be a high-affinity (Ki 9 nM) and highly selective (> 1,000-fold) inhibitor of matriptase. MCoTI-II efficiently inhibited the proteolytic activation of pro-hepatocyte growth factor (HGF) by matriptase but not by hepsin, in both purified and cell-based systems, and inhibited HGF-dependent cell scattering. MCoTI-II also selectively inhibited the invasion of matriptase-expressing prostate cancer cells. Using a model of epithelial cell tight junction assembly, we also found that MCoTI-II could effectively inhibit the re-establishment of tight junctions and epithelial barrier function in MDCK-I cells after disruption, consistent with the role of matriptase in regulating epithelial integrity. Surprisingly, MCoTI-II was unable to inhibit matriptase-dependent proteolytic activation of prostasin, a GPI-anchored serine protease also implicated in epithelial homeostasis. These observations suggest that the unusually high selectivity afforded by MCoTI-II and its biological effectiveness might represent a useful starting point for the development of therapeutic inhibitors, and further highlight the role of matriptase in epithelial maintenance.

BACKGROUND:Abnormal liver blood tests are common in Epstein-Barr virus (EBV) infection, but symptomatic hepatitis is rare. The demographics, clinical features and outcome of EBV hepatitis are incompletely understood, particularly in the elderly people.AIM:To identify the demographics, presenting features and natural history of EBV hepatitis.METHODS:Retrospective review of 1995 consecutive patients attending the jaundice hotline clinic over a 13-year period. Data collected included demographic information, presenting features, clinical and laboratory parameters, radiology imaging and clinical outcome.RESULTS:Seventeen of 1995 (0.85%) had EBV hepatitis. The median age was 40 years (range 18-68 years). Ten of 17 (59%) patients were aged >30 years, and seven of 17 (41%) patients were aged ≥60 years. Fifteen of 17 (88%) patients presented with clinical/biochemical evidence of jaundice. Seventeen of 17 (100%) patients had a serum lymphocytosis at presentation. 2/17 (12%) patients with EBV hepatitis presented with the classical features of infectious mononucleosis (fever, sore throat and lymphadenopathy). Splenomegaly was present in 15/17 (88%) of patients. Symptoms lasted for a median 8 weeks (range 1-12 weeks). Three of 17 (18%) patients required a brief hospital admission.CONCLUSIONS:In patients presenting with jaundice/hepatitis, EBV hepatitis is an uncommon diagnosis and causes a self-limiting hepatitis. The diagnosis is suggested by the presence of a lymphocytosis and/or splenomegaly. The majority of patients do not have infectious mononucleosis. Compared with infectious mononucleosis, EBV hepatitis affects an older age group, with nearly half of patients being aged more than 60 years. The diagnosis should be considered in all patients with unexplained hepatitis irrespective of their age.

BACKGROUND Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin. METHODS H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry. RESULTS Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions. CONCLUSIONS The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases.

Background:  Integrins initiate signalling in response to the extracellular matrix (ECM), which is important in wound healing and cancer. Previous studies have shown that over-expression of the αvβ6 integrin in oral squamous cell carcinoma (OSCC) cells results in enhanced motility and expression of matrix-degrading proteases, and the aim of this study was to investigate whether this is also the case for the α9β1 integrin. Methods:  H357 OSCCcells were transfected with the α9 integrin subunit and proliferation, adhesion and migration assays were performed on these along with null vector control and wild-type cells. The effect of ligand engagement on matrix metalloproteinase expression and the plasminogen activator system was measured using ELISA and chromogenic assays. Expression of α9 integrin was examined in oral squamous cell carcinoma tissue by immunohistochemistry. Results:  Functionally active α9 integrin mediated specific upregulation of adhesion and migration towards the TNfn3RAA fragment of tenascin-C but reduced proliferation. Migration towards collagen I was also enhanced in transfected cells. Matrix metalloproteinase-2 and metalloproteinase-9 expression was increased upon TNfn3RAA ligand engagement. Cell surface plasmin generation was also enhanced in α9-expressing cells and was the result of enhanced expression of urokinase receptor. In normal oral mucosa, α9 integrin expression was restricted to the suprabasal and prickle cell layers, and expression was heterogeneous in tumours but present in islands infiltrating connective tissue particularly in moderately and well-differentiated lesions. Conclusions:  The α9β1 integrin may play a key role in modulation of tumour behaviour including enhanced cell migration and expression of matrix-degrading proteases.

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

HGF (hepatocyte growth factor) is a pleiotropic cytokine homologous to the serine protease zymogen plasminogen that requires canonical proteolytic cleavage to gain functional activity. The activating proteases are key components of its regulation, but controversy surrounds their identity. Using quantitative analysis we found no evidence for activation by uPA (urokinase plasminogen activator), despite reports that this is a principal activator of pro-HGF. This was unaffected by a wide range of experimental conditions, including the use of various molecular forms of both HGF and uPA, and the presence of uPAR (uPA receptor) or heparin. In contrast the catalytic domains of the TTSPs (type-II transmembrane serine proteases) matriptase and hepsin were highly efficient activators (50% activation at 0.1 and 3.4 nM respectively), at least four orders of magnitude more efficient than uPA. PS-SCL (positional-scanning synthetic combinatorial peptide libraries) were used to identify consensus sequences for the TTSPs, which in the case of hepsin corresponded to the pro-HGF activation sequence, demonstrating a high specificity for this reaction. Both TTSPs were also found to be efficient activators at the cell surface. Activation of pro-HGF by PC3 prostate carcinoma cells was abolished by both protease inhibition and matriptase-targeting siRNA (small interfering RNA), and scattering of MDCK (Madin-Darby canine kidney) cells in the presence of pro-HGF was abolished by inhibition of matriptase. Hepsin-transfected HEK (human embryonic kidney)-293 cells also activated pro-HGF. These observations demonstrate that, in contrast with the uPA/uPAR system, the TTSPs matriptase and hepsin are direct pericellular activators of pro-HGF, and that together these proteins may form a pathway contributing to their involvement in pathological situations, including cancer.

Maspin is a serpin that has multiple effects on cell behavior, including inhibition of migration. How maspin mediates these diverse effects remains unclear, as it is devoid of protease inhibitory activity. We have previously shown that maspin rapidly inhibits the migration of vascular smooth muscle cells(VSMC), suggesting the involvement of direct interactions with cell surface proteins. Here, using immunofluorescence microscopy, we demonstrate that maspin binds specifically to the surface of VSMC in the dedifferentiated, but not the differentiated, phenotype. Ligand blotting of VSMC lysates revealed the presence of several maspin-binding proteins, with a protein of 150 kDa differentially expressed between the two VSMC phenotypes. Western blotting suggested that this protein was the beta 1 integrin subunit, and subsequently both alpha 3 beta 1 and alpha 5 beta 1, but not alpha v beta 3, were shown to associate with maspin by coimmunoprecipitation. Specific binding of these integrins was also observed using maspin-affinity chromatography, using HT1080 cell lysates. Direct binding of maspin to alpha 5 beta 1 was confirmed using a recombinant alpha 5 beta 1-Fc fusion protein. Using conformation-dependent anti-beta 1 antibodies, maspin binding to VSMC was found to lead to a decrease in the activation status of the integrin. The functional involvement of alpha 5 beta 1 in mediating the effect of maspin was established by the inhibition of migration of CHO cells overexpressing human alpha 5 integrin, but not those lacking alpha 5 expression. Our observations suggest that maspin engages in specific interactions with a limited number of integrins on VSMC, leading to their inactivation, and that these interactions are responsible for the effects of maspin in the pericellular environment.

Interactions between the uPA receptor (uPAR) and various integrins, including alpha(5)beta(1), are known to modulate integrin-dependent cell adhesion, and we have shown that the integrin-associated tetraspanin protein CD82 down-regulates uPAR-dependent plasminogen activation by affecting alpha(5)beta(1) cellular localisation. Here we have investigated whether overexpression Of alpha(5)beta(1) directly affects uPAR-dependent pericellular proteolysis. CHO cells overexpressing alpha(5)beta(1) were found to activate plasminogen at a rate up to 18-fold faster than B2CHO cells which are alpha(5)-deficient. This effect was dependent on the activation state of alpha(5)beta(1), as it was maximal in the presence of Mn2+. To determine the role of uPAR-alpha(5)beta(1), interactions in this effect, we determined the adhesion of these cells to immobilised soluble uPAR (suPAR). Neither cell-type was found to adhere to suPAR, but both cell types were found to adhere to an anti-uPAR monoclonal antibody in a uPAR- and integrin-dependent manner. This adhesion was 10-fold greater in the absence of alpha(5)beta(1), possibly implicating the involvement of non-alpha(5)-integrins. Soluble forms of the various components were used to investigate the molecular basis of these effects, but no direct interactions could be demonstrated between alpha(5)beta(1) and either uPAR, uPA or uPA-uPAR complex. This suggests that assembly of these components on the plasma membrane is required to influence uPAR function, increasing uPAR-dependent pericellular proteolysis and decreasing uPAR-dependent cell adhesion. These interactions may be modified by other integrins, suggesting a complex interplay between uPAR and integrins on the cell surface with the potential to regulate invasive cell migration.

The incidence of hepatitis A is falling. In contrast, autochthonous hepatitis E is an emerging infection in developed countries. The objective of this study was to compare both laboratory-confirmed cases of hepatitis A and autochthonous hepatitis E over a 2-year period in Cornwall and Devon and anti-hepatitis A virus (HAV) IgG and anti-hepatitis E virus (HEV) IgG seroprevalence in blood donors. The databases of microbiology laboratories in Cornwall and Devon were searched for the number of diagnostic HEV and HAV assays performed during 2005-2006 and the number of confirmed cases of acute hepatitis A and hepatitis E detected. Patients were followed up until recovery or death. Sera from 500 blood donors from the regional centre were tested for HEV and HAV IgG. In total, 28 cases of autochthonous hepatitis E were identified from 838 assays, and 20 cases of hepatitis A were identified from 4503 assays. Compared to hepatitis A cases, patients with hepatitis E were older (mean age 61 vs. 45 years, P = 0.003), less likely to present in winter (P = 0.028) and had more complications (five vs. one). The IgG seroprevalence rates in blood donors were 45% for HAV and 16% for HEV. There was no relationship between HAV and HEV IgG seropositivity. Autochthonous hepatitis E may be more common than hepatitis A, affects older patients, is less likely to occur in winter and may be associated with more complications. Patients with acute hepatitis, whatever their age or travel history, should be tested for HEV.