Search

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found that Cryptococcus neoformans independently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing, Cryptococcus initiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity against C. neoformans. Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to kill C. neoformans.

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors.

During tumor progression, epithelial ovarian cancer (EOC) cells undergo epithelial-to-mesenchymal transition (EMT), which influences metastatic success. Mutation-dependent activation of Wnt/β-catenin signaling has been implicated in gain of mesenchymal phenotype and loss of differentiation in several solid tumors; however, similar mutations are rare in most EOC histotypes. Nevertheless, evidence for activated Wnt/β-catenin signaling in EOC has been reported, and immunohistochemical analysis of human EOC tumors demonstrates nuclear staining in all histotypes. This study addresses the hypothesis that the bioactive lipid lysophosphatidic acid (LPA), prevalent in the EOC microenvironment, functions to regulate EMT in EOC. Our results demonstrate that LPA induces loss of junctional β-catenin, stimulates clustering of β1 integrins, and enhances the conformationally active population of surface β1 integrins. Furthermore, LPA treatment initiates nuclear translocation of β-catenin and transcriptional activation of Wnt/β-catenin target genes resulting in gain of mesenchymal marker expression. Together these data suggest that LPA initiates EMT in ovarian tumors through β1-integrin-dependent activation of Wnt/β-catenin signaling, providing a novel mechanism for mutation-independent activation of this pathway in EOC progression.

Vaccinia virus encodes a number of proteins that inhibit and manipulate innate immune signaling pathways that also have a role in virulence. These include A52, a protein shown to inhibit IL-1- and Toll-like receptor-stimulated NFκB activation, via interaction with interleukin-1 receptor-associated kinase 2 (IRAK2). Interestingly, A52 was also found to activate p38 MAPK and thus enhance Toll-like receptor-dependent IL-10 induction, which was TRAF6-dependent, but the manner in which A52 manipulates TRAF6 to stimulate p38 activation was unclear. Here, we show that A52 has a non-canonical TRAF6-binding motif that is essential for TRAF6 binding and p38 activation but dispensable for NFκB inhibition and IRAK2 interaction. Wild-type A52, but not a mutant defective in p38 activation and TRAF6 binding (F154A), caused TRAF6 oligomerization and subsequent TRAF6-TAK1 association. The crystal structure of A52 shows that it adopts a Bcl2-like fold and exists as a dimer in solution. Residue Met-65 was identified as being located in the A52 dimer interface, and consistent with that, A52-M65E was impaired in its ability to dimerize. A52-M65E although capable of interacting with TRAF6, was unable to cause either TRAF6 self-association, induce the TRAF6-TAK1 association, or activate p38 MAPK. The results suggest that an A52 dimer causes TRAF6 self-association, leading to TAK1 recruitment and p38 activation. This reveals a molecular mechanism whereby poxviruses manipulate TRAF6 to activate MAPKs (which can be proviral) without stimulating antiviral NFκB activation.

Toll-like receptors (TLRs) have an anti-viral role in that they detect viruses, leading to cytokine and IFN induction, and as such are targeted by viruses for immune evasion. TLR4, although best known for its role in recognizing bacterial LPS, is also strongly implicated in the immune response to viruses. We previously showed that the poxviral protein A46 inhibits TLR4 signaling and interacts with Toll-IL-1 receptor (TIR) domain-containing proteins of the receptor complex. However the exact molecular mechanism whereby A46 disrupts TLR4 signaling remains to be established, and may yield insight into how the TLR4 complex functions, since viruses often optimally target key residues and motifs on host proteins for maximal efficiency. Here we show that A46 targets the BB loop motif of TIR proteins and thereby disrupts receptor:adaptor (TLR4:Mal and TLR4:TRAM), but not receptor:receptor (TLR4:TLR4) nor adaptor:adaptor (Mal:MyD88, TRAM:TRIF, and Mal:Mal) TIR interactions. The requirement for an intact BB loop for TIR adaptor interactions correlated with the protein:protein interfaces antagonized by A46. We previously discovered a peptide fragment derived from A46 termed VIPER (Viral Inhibitory Peptide of TLR4), which specifically inhibits TLR4 responses. Here we demonstrate that the region of A46 from which VIPER is derived represents the TLR4-specific inhibitory motif of the intact protein, and is essential for A46:TRAM interactions. This study provides the molecular basis for pathogen subversion of TLR4 signaling and clarifies the importance of TIR motif BB loops, which have been selected for viral antagonism, in the formation of the TLR4 complex. Background: TLR4 signaling is inhibited by poxviral protein A46, but the mechanism is unknown. Results: We identify the protein interaction surfaces within the TLR4 complex that A46 antagonizes, and characterize the interaction between A46 and TRAM. Conclusion: A46 prevents receptor:adaptor interactions, and has a TRAM-specific interaction motif. Significance: This work reveals the molecular basis for poxviral antagonism of TLR4.

Helminth pathogens prepare a Th2 type immunological environment in their hosts to ensure their longevity. They achieve this by secreting molecules that not only actively drive type 2 responses but also suppress type 1 responses. Here, we show that the major cysteine proteases secreted from the helminth pathogens Fasciola hepatica (FheCL1) and Schistosoma mansoni (SmCB1) protect mice from the lethal effects of lipopolysaccharide by preventing the release of inflammatory mediators, nitric oxide, interleukin-6, tumor necrosis factor alpha, and interleukin-12, from macrophages. The proteases specifically block the MyD88-independent TRIF-dependent signaling pathway of Toll-like receptor (TLR)4 and TLR3. Microscopical and flow cytometric studies, however, show that alteration of macrophage function by cysteine protease is not mediated by cleavage of components of the TLR4 complex on the cell surface but occurs by degradation of TLR3 within the endosome. This is the first study to describe a parasite molecule that degrades this receptor and pinpoints a novel mechanism by which helminth parasites modulate the innate immune responses of their hosts to suppress the development of Th1 responses.

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported forhuman cathepsin K. The 1.4-Å three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and Kprovided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Amino acids generated from the catabolism of hemoglobin by intra-erythrocytic malaria parasites are not only essential for protein synthesis but also function in maintaining an osmotically stable environment, and creating a gradient by which amino acids that are rare or not present in hemoglobin are drawn into the parasite from host serum. We have proposed that a Plasmodium falciparum M17 leucyl aminopeptidase (PfLAP) generates and regulates the internal pool of free amino acids and therefore represents a target for novel antimalarial drugs. This enzyme has been expressed in insect cells as a functional 320-kDa homo-hexamer that is optimally active at neutral or alkaline pH, is dependent on metal ions for activity, and exhibits a substrate preference for N-terminally exposed hydrophobic amino acids, particularly leucine. PfLAP is produced by all stages in the intra-erythrocytic developmental cycle of malaria but was most highly expressed by trophozoites, a stage at which hemoglobin degradation and parasite protein synthesis are elevated. The enzyme was located by immunohistochemical methods and by transfecting malaria cells with a PfLAP-green fluorescent protein construct, to the cytosolic compartment of the cell at all developmental stages, including segregated merozoites. Amino acid dipeptide analogs, such as bestatin and its derivatives, are potent inhibitors of the protease and also block the growth of P. falciparum malaria parasites in culture. This study provides a biochemical basis for the antimalarial activity of aminopeptidase inhibitors. Availability of functionally active recombinant PfLAP, coupled with a simple enzymatic readout, will aid medicinal chemistry and/or high throughput approaches for the future design/discovery of new antimalarial drugs.

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin alpha3beta1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of alpha3beta1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of alpha3beta1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/alpha3beta1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (-1967) site in the uPA promoter blocked the uPAR/integrin-mediated transcriptional activation. Because uPAR is redistributed to clustered alpha3beta1 integrins, the requirement for uPAR/alpha3beta1 interaction in uPA regulation was assessed. Clustering of alpha3beta1 in the presence of a peptide (alpha325) that disrupts uPAR/alpha3beta1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin alpha3beta1 clustering. These results were confirmed using a genetic strategy in which alpha3 null epithelial cells reconstituted with wild type alpha3 integrin, but not a mutant alpha3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/alpha3beta1 binding using peptide alpha325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering ofalpha3beta1 integrin promotes uPAR/alpha3beta1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wild-type parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 μm) compared with the parent parasites (EC50 25 μm). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.

Acquisition of matrix metalloproteinase-2 (MMP-2) activity is temporally associated with increased migration and invasiveness of cancer cells. ProMMP-2 activation requires multimolecular complex assembly involving proMMP-2, membrane type 1-MMP (MT1-MMP, MMP-14), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Because transforming growth factor-beta1 (TGF-beta1) promotes tumor invasion in advanced squamous cell carcinomas, the role of TGF-beta1 in the regulation of MMP activity in a cellular model of invasive oral squamous cell carcinoma was examined. Treatment of oral squamous cell carcinoma cells with TGF-beta1 promoted MMP-dependent cell scattering and collagen invasion, increased expression of MMP-2 and MT1-MMP, and enhanced MMP-2 activation. TGF-beta1 induced concomitant activation of ERK1/2 and p38 MAPK, and kinase inhibition studies revealed a negative regulatory role for ERK1/2 in modulating acquisition of MMP-2 activity. Thus, a reciprocal effect on proMMP-2 activation was observed whereupon blocking ERK1/2 phosphorylation promoted proMMP-2 activation and MT1-MMP activity, whereas inhibiting p38 MAPK activity decreased proteolytic potential. The cellular mechanism for the control of MT1-MMP catalytic activity involved concurrent reciprocal modulation of TIMP-2 expression by ERK1/2 and p38 MAPKs, such that inhibition of ERK1/2 phosphorylation decreased TIMP-2 production, and down-regulation of p38 MAPK activity enhanced TIMP-2 synthesis. Further, p38 MAPK inhibition promoted ERK1/2 phosphorylation, providing additional evidence for cross-talk between MAPK pathways. These observations demonstrate the complex reciprocal effects of ERK1/2 and p38 MAPK in the regulation of MMP activity, which could complicate the use of MAPK-specific inhibitors as therapeutic agents to down-regulate the biologic effects of TGF-beta1 on pericellular collagen degradation and tumor invasion.

Matrix metalloproteinases (MMPs) play an important role in the invasive behavior of a number of cancers including oral squamous cell cancer (OSCC), and increased expression of MMP-9 is correlated with invasive and metastatic OSCC. Because calcium is an important regulator of keratinocyte function, the effect of modulating extracellular calcium on MMP-9 expression in OSCC cell lines was evaluated. Increasing extracellular calcium induced a dose-dependent increase in MMP-9 expression in immortalized normal and premalignant oral keratinocytes, but not in two highly invasive OSCC cell lines. Differential activation of MAPK signaling was also induced by calcium. p38 MAPK activity was down-regulated, whereas ERK1/2 activity was enhanced. Pharmacologic inhibition of p38 MAPK activity or expression of a catalytically inactive mutant of the upstream kinase MAPK kinase 3 (MKK3) increased the calcium induced MMP-9 gene expression, demonstrating that p38 MAPK activity negatively regulated this process. Interestingly blocking p38 MAPK activity enhanced ERK1/2 phosphorylation, suggesting reciprocal regulation between the ERK1/2 and p38 MAPK pathways. Together these data support a model wherein calcium-induced MMP-9 expression is differentially regulated by the ERK1/2 and p38 MAPK pathways in oral keratinocytes, and the data suggest that a loss of this regulatory mechanism accompanies malignant transformation of the oral epithelium.

The epidermal growth factor receptor (EGFR) has been proposed as a key modulator of cadherin-containing intercellular junctions, particularly in tumors that overexpress this tyrosine kinase. Here the EGFR tyrosine kinase inhibitor PKI166 and EGFR blocking antibody C225, both of which are used clinically to treat head and neck cancers, were used to determine the effects of EGFR inhibition on intercellular junction assembly and adhesion in oral squamous cell carcinoma cells. EGFR inhibition resulted in a transition from a fibroblastic morphology to a more epithelial phenotype in cells grown in low calcium; under these conditions cadherin-mediated cell-cell adhesion is normally reduced, and desmosomes are absent. The accumulated levels of desmoglein 2 (Dsg2) and desmocollin 2 increased 1.7-2.0-fold, and both desmosomal cadherin and plaque components were recruited to cell-cell borders. This redistribution was paralleled by an increase in Dsg2 and desmoplakin in the Triton-insoluble cell fraction, suggesting that EGFR blockade promotes desmosome assembly. Importantly, E-cadherin expression and solubility were unchanged. Furthermore, PKI166 blocked tyrosine phosphorylation of Dsg2 and plakoglobin following epidermal growth factor stimulation, whereas no change in phosphorylation was detected for E-cadherin and beta-catenin. The increase in Dsg2 protein was in part due to the inhibition of matrix metalloproteinase-dependent proteolysis of this desmosomal cadherin. These morphological and biochemical changes were accompanied by an increase in intercellular adhesion based on functional assays at all calcium concentrations tested. Our results suggest that EGFR inhibition promotes desmosome assembly in oral squamous cell carcinoma cells, resulting in increased cell-cell adhesion.

The role of proteases in the tumor cell invasion process is multifaceted. Members of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and degradation of extracellular matrix (ECM) components. Differentiating between the up-regulation of MMP production and the presence of activated MMPs can be difficult but may well dictate which MMPs are critical to invasion. Because the hydrolysis of collagens is one of the committed steps in ECM turnover, we have investigated selective MMP action on collagenous substrates as a means to evaluate active MMPs. Two triple-helical peptide (THP) models of the MMP-9 cleavage site in type V collagen, alpha1(V)436-450 THP and alpha1(V)436-447 fTHP, were hydrolyzed by MMP-2 and MMP-9 at the Gly-Val bond, analogous to the bond cleaved by MMP-9 in the corresponding native collagen. Kinetic analyses showed k(cat)/K(m) values of 14,002 and 5,449 s(-1)m(-1) for MMP-2 and -9 hydrolysis of alpha1(V)436-447 fTHP, respectively. These values, along with individual k(cat) and K(m) values, are comparable with collagen hydrolysis by MMP-2 and -9. Neither THP was hydrolyzed by MMP-1, -3, -13, or -14. alpha1(V)436-447 fTHP and a general fluorogenic THP were used to screen for triple-helical peptidase activity in alpha(2)beta(1) integrin-stimulated melanoma cells. Binding of the alpha(2)beta(1) integrin resulted in the production of substantial triple-helical peptidase activity, the majority (>95%) of which was non-MMP-2/-9. THPs were found to provide highly selective substrates for members of the MMP family and can be used to evaluate active MMP production in cellular systems.

Up-regulation of the collagenolytic membrane type-1 matrix metalloproteinase (MT1-MMP) leads to increased MMP2 (gelatinase A) activation and MT1-MMP autolysis. The autocatalytic degradation product is a cell surface 44-kDa fragment of MT1-MMP (Gly285–Val582) in which the ectodomain consists of only the linker, hemopexin C domain and the stalk segment found before the transmembrane sequence. In the collagenases, hemopexin C domain exosites bind native collagen, which is required for triple helicase activity during collagen cleavage. Here we investigated the collagen binding properties and the role of the hemopexin C domain of MT1-MMP and of the 44-kDa MT1-MMP ectodomain in collagenolysis. Recombinant proteins, MT1-LCD (Gly285–Cys508), consisting of the linker and the hemopexin C domain, and MT1-CD (Gly315–Cys508), which consists of the hemopexin C domain only, were found to bind native type I collagen but not gelatin. Functionally, MT1-LCD inhibited collagen-induced MMP2 activation in fibroblasts, suggesting that interactions between collagen and endogenous MT1-MMP directly stimulate the cellular activation of pro-MMP2. MT1-LCD, but not MT1-CD, also blocked the cleavage of native type I collagen by MT1-MMP in vitro, indicating an important role for the MT1-MMP linker region in triple helicase activity. Similarly, soluble MT1-LCD, but not MT1-CD or peptide analogs of the MT1-MMP linker, reduced the invasion of type I collagen matrices by MDA-MB-231 cells as did the expression of recombinant 44-kDa MT1-MMP on the cell surface. Together, these studies demonstrate that generation of the 44-kDa MT1-MMP autolysis product regulates collagenolytic activity and subsequent invasive potential, suggesting a novel feedback mechanism for the control of pericellular proteolysis.

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.

Type I collagen stimulation of pro-matrix metalloproteinase (pro-MMP)-2 activation by ovarian cancer cells involves beta(1) integrin receptor clustering; however, the specific cellular and biochemical events that accompany MMP processing are not well characterized. Collagenolysis is not required for stimulation of pro-MMP-2 activation, and denatured collagen does not elicit an MMP-2 activation response. Similarly, DOV13 cells bind to intact collagen utilizing both alpha(2)beta(1) and alpha(3)beta(1) integrins but interact poorly with collagenase-treated or thermally denatured collagen. Antibody-induced clustering of alpha(3)beta(1) strongly promotes activation of pro-MMP-2, whereas alpha(2)beta(1) integrin clustering has only marginal effects. Membrane-type 1 (MT1)-MMP is present on the DOV13 cell surface as both an active 55-kDa TIMP-2-binding species and a stable catalytically inactive 43-kDa form. Integrin clustering stimulates cell surface expression of MT1-MMP and co-localization of the proteinase to aggregated integrin complexes. Furthermore, cell surface proteolysis of the 55-kDa MT1-MMP species occurs in the absence of active MMP-2, suggesting MT1-MMP autolysis. Cellular invasion of type I collagen matrices requires collagenase activity, is blocked by tissue inhibitor of metalloproteinases-2 (TIMP-2) and collagenase-resistant collagen, is unaffected by TIMP-1, and is accompanied by pro-MMP-2 activation. Together, these data indicate that integrin stimulation of MT1-MMP activity is a rate-limiting step for type I collagen invasion and provide a mechanism by which this activity can be down-regulated following collagen clearance.

Cells in complex tissues contact extracellular matrix that interacts with integrin receptors to influence gene expression, proliferation, apoptosis, adhesion, and motility. During development, tissue remodeling, and tumorigenesis, matrix components are modified by enzymatic digestion with subsequent effects on integrin binding and signaling. We are interested in understanding the mechanisms by which broad spectrum proteinases such as plasmin are targeted to their extracellular matrix protein substrates. We have utilized plasmin-mediated cleavage of the epithelial basement membrane glycoprotein laminin-5 as a model to evaluate molecular events that direct plasmin activity to specific structural domains. We report that plasminogen and tissue plasminogen activator (tPA) exhibit high affinity, specific binding to the G(1) subdomain of the N terminus of the laminin-5 alpha(3) subunit, with equilibrium dissociation constants of 50 nm for plasminogen and 80 nm for tPA. No high affinity binding to the G(2), G(3), and G(4) subdomains was observed. As a result of binding to the G(1) subdomain, the catalytic efficiency of tPA-catalyzed plasminogen activation is enhanced 32-fold, leading to increased matrix-associated plasmin that is positioned favorably for cleavage within the G(4) subdomain as we have reported previously (Goldfinger, L. E., Stack, M. S., and Jones, J. C. R. (1998) J. Cell Biol. 141, 255-265). Thus, physical constraints dictated by interaction of proteinase and matrix macromolecule control not only enzymatic activity but may regulate substrate targeting of proteinases.

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.

A new glycoamidase, peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase (PNGase) At, was discovered in the eukaryote Aspergillus tubigensis. The enzyme was purified to homogeneity, and the DNA sequence was determined by cloning in Escherichia coli. Over 80% of the deduced amino acid sequence was verified independently by Edman analysis and/or electrospray ionization-mass spectrometry of protease fragments of native PNGase At. This glycoamidase contains 12 potential asparagine-linked glycosylation sites, of which at least 9 sites are occupied with typical high mannose oligosaccharides. PNGase At consists of two non-identical glycosylated subunits that are derived from a single polypeptide gene precursor. Evidence is presented suggesting that autocatalysis is involved in subunit formation. PNGase At is an important new tool for analysis of asparagine-linked glycans; it can hydrolyze a broad range of glycopeptides, including those with core-linked alpha1-->6 or alpha1-->3 fucose, under conditions not favorable with existing glycoamidases.

Hepatocyte growth factor (HGF) is a potent mitogen, motogen, and morphogen for various epithelial cell types. The pleiotropic effects of HGF are mediated by its binding to a specific high affinity receptor, c-Met. In addition, HGF binds to heparan sulfate proteoglycans on cell surfaces and within the extracellular matrix. Incubation of HGF with 0.1, 1.0, and 10 micrograms/ml of heparin, heparan sulfate, or dextran sulfate resulted in a concentration-dependent increase in mitogenic potency in a primary rat hepatocyte bioassay, whereas sodium sulfate or fucoidan did not. Although co-incubation of HGF with sulfated compounds that enhanced HGF-dependent mitogenesis did not alter the binding isotherm of HGF for the c-Met receptor in a solid phase assay, an increase in autophosphorylation of the c-Met receptor in intact A549 cells was observed upon their addition. A series of chemically sulfated malto-oligosaccharides varying in unit size and charge was tested in the bioassay in order to provide additional insights into the nature of the HGF-heparin interaction. While sulfated di-, tri-, tetra-, and pentasaccharides did not significantly potentiate HGF-dependent mitogenesis, larger oligosaccharides such as the sulfated hexa-, hepta-, or a sulfated oligosaccharide mixture containing decasaccharides resulted in an approximate 2-, 4-, and 7-fold enhancement, respectively. We observed a correlation between the sulfated oligosaccharide preparations that enhanced mitogenic potency and those that promoted HGF oligomerization in vitro, as measured by gel filtration and analytical ultracentrifugation. These findings indicate that heparin-like molecules can stabilize HGF oligomers, which may facilitate c-Met receptor dimerization and activation.

The Vps15 protein kinase and the Vps34 phosphatidylinositol 3-kinase have been shown to function as a membrane-associated complex which facilitates the delivery of proteins to the vacuole in yeast. Biochemical characterization of the autophosphorylation reaction catalyzed by Vps15p demonstrates that it is a functional serine/threonine protein kinase. In addition, we show that the Vps34 phosphatidylinositol 3-kinase undergoes an autophosphorylation event both in vivo and in vitro, indicating that it represents a novel multiple specificity kinase capable of phosphorylating both protein and lipid substrates. Vps34p is phosphorylated predominately on serine in vivo and is able to phosphorylate serine, threonine, and tyrosine residues in vitro. Mutant Vps34 proteins containing alterations in conserved amino acids in the lipid kinase domain are severely defective for both PI 3-kinase activity and autophosphorylation. Characterization of the PI 3-kinase activity of Vps34p demonstrates that it, unlike the mammalian p110 PI 3-kinase, is highly resistant to the PI 3-kinase inhibitors wortmannin and LY294002. We also find that Vps34p is a phosphatidylinositol-specific 3-kinase, as it is able to utilize phosphatidylinositol (PtdIns) but not PtdIns(4)P or PtdIns(4,5)P2 as substrates in an in vitro PI kinase reaction. The substrate specificity, wortmannin resistance, and other biochemical characteristics of its PtdIns 3-kinase activity suggest that Vps34p is quite similar to a PtdIns-specific 3-kinase activity recently characterized from mammalian cells. These data indicate the existence of a family of PI 3-kinases composed of p110-like PI 3-kinases and Vps34p-like PtdIns-specific 3-kinases. On the basis of the role for Vps34p in vacuolar protein sorting, we propose that the production of a specific phosphoinositide, PtdIns(3)P, is involved in regulating intracellular protein sorting reactions in eukaryotic cells.

Human lung mast cell tryptase is a trypsin-like serine proteinase that is stored in mast cell granules and released by activated mast cells. Here we report that mast cell tryptase is a potent activator of single-chain urinary-type plasminogen activator (scu-PA, or prourokinase), the zymogen form of urinary-type plasminogen activator (u-PA). Activation was complete within 75 min using an enzyme:substrate molar ratio of 1:50 and was accompanied by cleavage of scu-PA at Lys158-Ile159, generating active two-chain u-PA. The reaction was dependent on enzyme concentration and obeyed Michaelis-Menten kinetics. Kinetic constants calculated for scu-PA activation by mast cell tryptase are Km = 34 microM, Vmax = 3.6 pmol of u-PA/min, and kcat = 0.08 s-1. These data suggest that tryptase from tumor-associated mast cells may participate in the activation of scu-PA.

This study describes the binding of plasminogen and tissue-type plasminogen activator (t-PA) to the extracellular matrix proteins fibronectin and laminin. Plasminogen bound specifically and saturably to both fibronectin and laminin immobilized on microtiter wells, with Kd(app) values of 115 and 18 nM, respectively. Limited proteolysis by endoproteinase V8 coupled with ligand blotting analysis showed that both plasminogen and t-PA preferentially bind to a 55-kDa fibronectin fragment and a 38-kDa laminin fragment. Amino acid sequence analysis demonstrated that the 5-kDa fragment originates with the fibronectin amino terminus whereas the laminin fragment was derived from the carboxyl-terminal globular domain of the laminin A chain. Ligand blotting experiments using isolated plasminogen domains were also used to identify distinct regions of the plasminogen molecule involved in fibronectin and laminin binding. Solution phase fibronectin binding to immobilized plasminogen was mediated primarily via lysine binding site-dependent interactions with plasminogen kringles 1-4. Lysine binding site-dependent binding of soluble laminin to immobilized plasminogen kringles 1-5 as well as an additional lysine binding site-independent interaction between mini-plasminogen and the 38-kDa laminin A chain fragment were also observed. These studies demonstrate binding of plasminogen and tissue-type plasminogen activator to specific regions of the extracellular matrix glycoproteins laminin and fibronectin and provide further insight into the mechanism of regulation of plasminogen activation by components of the extracellular matrix.

We demonstrated previously that heparin-derived hexasaccharides are the smallest fragments of the polysaccharide with comparable basic fibroblast growth factor (bFGF)-modulating activity in vitro (Ishihara, M., Tyrrell, D.J., Stauber, G.B., Brown, S., Cousens, L., and Stack, R.J. (1993) J. Biol. Chem. 268, 4675-4683. In this report, a specific hexasaccharide having high affinity for recombinant human bFGF was isolated and its structure deduced by analysis of its reduced disaccharide products after treatment with nitrous acid at pH 1.5, and by 1H NMR spectroscopy. The hexasaccharide has the structure [IdoA(2-OSO3)alpha 1-4GlcNSO3(6-OSO3)alpha 1-4]2IdoA(2-OSO3)alpha 1-4 AManR(6-OSO3). The hexasaccharide effectively inhibits the binding of syndecan-transfected RO-12 UC cells to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315), prevents the binding of 125I-bFGF to confluent monolayers of adrenocortical endothelial (ACE) cells, and inhibits the bFGF-dependent proliferation of ACE cells. Unlike the heparin from which it was derived, however, the hexasaccharide cannot promote the binding of 125I-bFGF to a recombinant high affinity bFGF receptor (flg) or restore the bFGF-dependent proliferative response to ACE cells grown in the presence of 5 mM sodium chlorate. Collectively, these data indicate that a hexasaccharide can be as effective as heparin as an antagonist of bFGF-mediated cell mitogenesis.

Homogeneously sized, heparin-derived oligosaccharides were prepared from heparin following partial depolymerization with nitrous acid, reduction with sodium borohydride, and fractionation by gel permeation chromatography. The resulting pools of di-, tetra-, hexa-, octa-, and decasaccharides were sequentially applied to an affinity column of human recombinant basic fibroblast growth factor (bFGF) covalently attached to Sepharose 4B and further fractionated into subpools based on their elution from this column in response to gradients of sodium chloride. In general, pools of smaller heparin-derived oligosaccharides required relatively lower salt concentration for complete elution, and pools of larger oligosaccharides required higher salt concentration. The homogeneously sized pools and affinity-fractionated subpools of heparin-derived oligosaccharides were quantitatively assessed as inhibitors or enhancers of specific bFGF-mediated biological activities in five separate assay systems as follows: assay 1, to compete with human lymphoblastoid cells expressing syndecan (RO-12 UC cells) for binding to bFGF-coated wells (Ishihara, M., Tyrrell, D.J., Kiefer, M.C., Barr, P.J., and Swiedler, S.J. (1992) Anal. Biochem. 202, 310-315); assay 2, to inhibit 125I-bFGF binding to "low affinity sites" of adrenocortical endothelial (ACE) cells; assay 3, to inhibit bFGF-induced proliferation of ACE cells; assay 4, to support mitogenic activity of bFGF in a growth stimulation assay of chlorate-treated ACE cells; and assay 5, to enhance the in vitro interaction between 125I-bFGF and the recombinant extra-cellular domain of FGF high affinity receptor. The data derived from the five assay systems demonstrated that heparin-derived hexa- and octasaccharides inhibited the interaction between cell surface heparan sulfate proteoglycan and bFGF (assays 1 and 2) and bFGF-induced proliferation of ACE cells (assay 3) but were unable to enhance the binding of bFGF to its high affinity receptor in vitro (assay 5) or to support bFGF-induced mitogenesis in ACE cells (assay 4). These two activities required at least a decasaccharide with high affinity for bFGF.

To define the inhibitory requirements of mammalian collagenase, several N-substituted amide and peptide derivatives of the mercaptomethyl analogue of leucine, 2-[(R,S)mercaptomethyl]-4-methylpentanoic acid (H psi[SCH2]-DL-leucine), were synthesized and tested as inhibitors of pig synovial collagenase with soluble type I collagen as substrate. H psi[SCH2]-DL-leucine (IC50 = 320 microM) was about 10 times more potent than the beta-mercaptomethyl compound, N-acetylcysteine. The amide of H psi[SCH2]-DL-leucine was six times more potent than the parent thiol acid. Aliphatic N-substituted amides were less potent than the unsubstituted amide, whereas the N-benzyl amide was slightly more potent. Dipeptides, particularly those with an aromatic group at P2', were up to 20-fold more potent, while tripeptides with an aromatic L-amino acid at P2' and Ala-NH2 at P3' were up to 2200 times more potent than H psi[SCH2]-DL-leucine. The resolved diastereomers of H psi[SCH2]-DL-Leu-Phe-Ala-NH2 inhibited by 50% at 0.3 and 0.04 microM, respectively. The most potent inhibitor synthesized, an isomer of H psi[SCH2]-DL-Leu-L-3-(2'-naphthyl)alanyl-Ala-NH2, exhibited an IC50 of 0.014 microM, a value about 300 times less than similar thiol-based analogues of the P'-cleavage sequence of type I collagen, H psi[SCH2]-DL-Leu-Ala-Gly-Gln-. These structure-function studies establish within the present series of compounds that the most effective inhibitors of mammalian collagenase are not closely related to the P2'-P3' elements of the cleavage site of the natural substrate but rather have an aromatic group at the P2' position and Ala-NH2 at the P3' position.