Mosquito-borne WNV (West Nile virus) is an emerging global threat. The NS3 proteinase, which is essential for the proteolytic processing of the viral polyprotein precursor, is a promising drug target. We have isolated and biochemically characterized the recombinant, highly active NS3 proteinase. We have determined that the NS3 proteinase functions in a manner that is distantly similar to furin in cleaving the peptide and protein substrates. We determined that aprotinin and D-arginine-based 9-12-mer peptides are potent inhibitors of WNV NS3 with K(i) values of 26 nM and 1 nM respectively. Consistent with the essential role of NS3 activity in the life cycle of WNV and with the sensitivity of NS3 activity to the D-arginine-based peptides, we showed that nona-D-Arg-NH2 reduced WNV infection in primary neurons. We have also shown that myelin basic protein, a deficiency of which is linked to neurological abnormalities of the brain, is sensitive to NS3 proteolysis in vitro and therefore this protein represents a convenient test substrate for the studies of NS3. A three-dimensional model of WNV NS3 that we created may provide a structural guidance and a rationale for the subsequent design of fine-tuned inhibitors. Overall, our findings represent a foundation for in-depth mechanistic and structural studies as well as for the design of novel and efficient inhibitors of WNV NS3.

PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP 621 2LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.Secreted Wnt glycoproteins regulate ␤-catenin-dependent (canonical) and ␤-catenin-independent (non-canonical) signaling pathways (1-7). One intriguing and well conserved function of the non-canonical pathway is to control PCP 2 and directional cell motility (8). PCP governs the orientation of cells in a monolayer of a tissue plane (front-back orientation) in such a way that all cells within the monolayer are aligned in the same direction. As a result, PCP is important for the directed collective cell movements and orchestrates the synchronized cell arrangements within the tissue plane in the course of a plethora of biological processes (2, 4, 6 -11).The first PCP signaling events occur at a gastrulation stage of embryogenesis to regulate the polarized cell movement and accomplish convergent extension (CE) for the anterior-posterior body axis elongation, neural tube closure, and craniofacial morphogenesis (8,9,11,12). CE failure results in the multiple severe developmental defects, including a shortened body axis (dwarfism), defective neural system, and craniofacial abnormalities. Defects in the non-canonical Wnt/PCP pathway are linked to a broad range of diseases, including cancer (3, 5). Wnt5a, Wnt5b, and Wnt11, which work through the non-canonical pathway, are often up-regulated in cancer and promote cancer cell motility and invasion (6, 13). Evidently, an in depth mechanistic understanding of the PCP mechanism and its aberrant regulation in disease is required to control tumor progression and metastasis in a clinically advantageous man...

Elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with malignancies. There is a consensus among scientists that cell surface-associated MT1-MMP is a key player in pericellular proteolytic events. Now we have identified an intracellular, hitherto unknown, function of MT1-MMP. We demonstrated that MT1-MMP is trafficked along the tubulin cytoskeleton. A fraction of cellular MT1-MMP accumulates in the centrosomal compartment. MT1-MMP targets an integral centrosomal protein, pericentrin. Pericentrin is known to be essential to the normal functioning of centrosomes and to mitotic spindle formation. Expression of MT1-MMP stimulates mitotic spindle aberrations and aneuploidy in nonmalignant cells. Volumes of data indicate that chromosome instability is an early event of carcinogenesis. In agreement, the presence of MT1-MMP activity correlates with degraded pericentrin in tumor biopsies, whereas normal tissues exhibit intact pericentrin. We believe that our data show a novel proteolytic pathway to chromatin instability and elucidate the close association of MT1-MMP with malignant transformation. Matrix metalloproteinases (MMP(s))1 are a comprehensive family of zinc-enzymes that degrade the extracellular matrix and cell surface molecules (1). Understanding the function of these enzymes in carcinogenesis is critical for the design of anti-cancer pharmaceuticals (2). MT1-MMP is a prototypic member of the membrane-tethered MMP subfamily (3). A transmembrane domain and a cytoplasmic tail (CT) of MT1-MMP associate this abundant membrane-tethered protease with discrete regions of the plasma membrane and the intracellular milieu, respectively. Although MT1-MMP is present in normal tissues, its enhanced expression, unlike of any other of the 23 known human MMPs, is closely associated with aggressive, invasive malignancies (1, 3-5). MT1-MMP transgenic mice displayed mammary gland abnormalities and tumor promotion in mammary gland (6).MT1-MMP functions as one of the main mediators of proteolytic events on the cell surface, and it is directly involved in the pericellular proteolysis of the extracellular matrix, cell surface adhesion, and signaling receptors and in the activation pathway of soluble secretory MMPs (5, 7-9) Cell surface-associated MT1-MMP acts as a growth factor in malignant cells and assumes tumor growth control (4). The conditional expression of MT1-MMP can, by itself, confer tumorigenicity on nonmalignant epithelial cells and cause the formation of invasive tumors (10). MT1-MMP also plays an important role in normal development; MT1-MMP knock-out mice are dwarfs, and they die prematurely (8,11). A loss of the structurally similar primordial At2-MMP induces dwarfism in Arabidopsis plants (12). There is no extracellular matrix in plants, however, that is similar to the collagenous extracellular matrix of mammals. This datum alone is enough to suggest that the protease plays a role in certain functionally relevant intracellular events in addition to its role in pericellular...

MT1-MMP is a key enzyme in cancer cell invasion and metastasis. The activity of cellular MT1-MMP is regulated by furin-like proprotein convertases, TIMPs, shedding, autoproteolysis, dimerization, exocytosis, endocytosis, and recycling. Our data demonstrate that, in addition to these already known mechanisms, MT1-MMP is regulated by O-glycosylation of its hinge region. Insignificant autolytic degradation is characteristic for naturally expressed, glycosylated, MT1-MMP. In turn, extensive autolytic degradation, which leads to the inactivation of the protease and the generation of its C-terminal membrane-tethered degraded species, is a feature of overexpressed MT1-MMP. We have determined that incomplete glycosylation stimulates extensive autocatalytic degradation and self-inactivation of MT1-MMP. Self-proteolysis commences during the secretory process of MT1-MMP through the cell compartment to the plasma membrane. The strongly negatively charged sialic acid is the most important functional moiety of the glycopart of MT1-MMP. We hypothesize that sialic acid of the O-glycosylation cassette restricts the access of the catalytic domain to the hinge region and to the autolytic cleavage site and protects MT1-MMP from autolysis. Overall, our results point out that there is a delicate balance between glycosylation and self-proteolysis of MT1-MMP in cancer cells and that when this balance is upset the catalytically potent MT1-MMP pool is self-proteolyzed.Membrane-tethered MT1-MMP, 2 the most abundant member of the membrane-type (MT) matrix metalloproteinase subfamily, is distinguished from soluble MMPs by a short transmembrane domain and a cytoplasmic tail (1, 2). MT1-MMP functions in cancer cells as an important mediator of proteolytic events on the cell surface (3, 4), and it is directly engaged in the cleavage of cell surface receptors and the pericellular proteolysis of the extracellular matrix components (5-7). MT1-MMP expression is associated with a variety of pathophysiological conditions and especially with cell locomotion, tumor growth, and metastasis (4, 5, 8 -11).MT1-MMP and related membrane-tethered MMPs are regulated, both as proteinases and as membrane proteins, at the transcriptional and post-transcriptional levels by multifaceted coordinated mechanisms (12-22). The trafficking and the internalization of MT1-MMP have been identified as two additional mechanisms that regulate its biological functions. Both clathrin-coated pits and caveolae are involved in the internalization of 19,[23][24][25][26][27][28].To exercise its proteolytic activity, MT1-MMP requires the proteolytic removal of its N-terminal prodomain sequence (29). Because the prodomain part of MT1-MMP has the furin-cleavage motif, furin and related proprotein convertases (PCs) are the physiological activators of latent MT1-MMP (13, 30 -32). Proteolytic processing by the PCs leads to the activation of the latent MT1-MMP zymogen, which occurs primarily in the trans-Golgi network during the secretory passage of MT1-MMP (30, 33). The levels of expressio...

Membrane type-1 matrix metalloproteinase (MT1-MMP) degrades the extracellular matrix, initiates the activation pathway of soluble MMPs and regulates the functionality of cell adhesion signaling receptors, thus playing an important role in many cell functions. Intracellular transport mechanisms, currently incompletely understood, regulate the presentation of MT1-MMP at the cell surface. We have focused our efforts on identifying these mechanisms. To understand the transport of MT1-MMP across the cell, we used substitution and deletion mutants, the trafficking of which was examined using antibody uptake and Chariot delivery experiments. Our experiments have demonstrated that the microtubulin cytoskeleton and the centrosomes (the microtubulin cytoskeleton-organizing centers) are essential for the trafficking and the internalization of MT1-MMP. We determined that after reaching the plasma membrane, MT1-MMP is internalized in the Rab-4-positive recycling endosomes and the Rab-11-positive pericentrosomal recycling endosomes. The microtubular trafficking causes the protease to accumulate in the pericentrosomal region of the cell. We believe that the presence of the transmembrane domain is required for the microtubular vesicular trafficking of MT1-MMP because the soluble mutants are not presented at the cell surface and they are not delivered to the centrosomes. The observed transport mechanisms provide a vehicle for the intracellular targets and, accordingly, for an intracellular cleavage function of MT1-MMP in malignant cells, which routinely overexpress this protease.

An elevated expression of membrane type-1 matrix metalloproteinase (MT1-MMP) is closely associated with multiple malignancies. Recently, we discovered that recycled MT1-MMP was trafficked along the tubulin cytoskeleton into the centrosomal compartment and cleaved the integral centrosomal protein pericentrin-2. These events correlated with the induction of chromosome instability and aneuploidy in nonmalignant Madine-Darby canine kidney cells. Accordingly, we hypothesized that MT1-MMP is an oncogene that promotes malignant transformation of normal cells rather than just an enzyme that supports growth of preexisting tumors. To prove our hypothesis, we transfected normal 184B5 human mammary epithelial cells with MT1-MMP (184B5-MT1 cells). MT1-MMP was colocalized with pericentrin in the centrosomal compartment and especially in the midbody of dividing cells. 184B5-MT1 cells acquired the ability to activate MMP-2, to cleave pericentrin, and to invade the Matrigel matrix. 184B5-MT1 cells exhibited aneuploidy, and they were efficient in generating tumors in the orthotopic xenograft model in immunodeficient mice. Because of the absence of tumor angiogenesis and the resulting insufficient blood supply, the tumors then regressed with significant accompanying necrosis. Gene array studies confirmed a significant upregulation of oncogenes and tumorigenic genes but not the angiogenesis-promoting genes in 184B5-MT1 cells. We believe that our data point to a novel function of MT1-MMP in the initial stages of malignant transformation and to new and hitherto unknown transition mechanism from normalcy to malignancy. (Cancer Res 2006; 66(21): 10460-5)

Membrane type-1 matrix metalloproteinase (MT1-MMP) exhibits distinctive and important pericellular cleavage functions. Recently, we determined that MT1-MMP was trafficked to the centrosomes in the course of endocytosis. Our data suggested that the functionally important, integral, centrosomal protein, pericentrin-2, was a cleavage target of MT1-MMP in human and in canine cells and that the sequence of the cleavage sites were ALRRLLG 1156 2L 1157 FG and ALRRLLS 2068 2L 2069 FG, respectively. The presence of Asp-948 at the P1 position inactivated the corresponding site (ALRRLLD 948 -L 949 FGD) in murine pericentrin. To confirm that MT1-MMP itself cleaves pericentrin directly, rather than indirectly, we analyzed the cleavage of the peptides that span the MT1-MMP cleavage site. In addition, we analyzed glioma U251 cells, which co-expressed MT1-MMP with the wild type murine pericentrin and the D948G mutant. We determined that the D948G mutant that exhibited the cleavage sequence of human pericentrin was sensitive to MT1-MMP, whereas unmodified murine pericentrin was resistant to proteolysis. Taken together, our results confirm that MT1-MMP cleaves pericentrin-2 in humans but not in mice and that mouse models of cancer probably cannot be used to critically examine MT1-MMP functionality. MT1-MMP2 /MMP-14 is a prototypic member of the membranetethered matrix metalloproteinases (1). Although MT1-MMP is present in normal tissues, its enhanced expression is directly linked to tumor progression and metastasis (2-6). Cell surface-associated MT1-MMP is a multifunctional enzyme (7), and it is involved in the pericellular proteolysis of the extracellular matrix, the activation of soluble MMPs, and the cleavage of adhesion and signaling cell receptors (8 -10). The functional activity of MT1-MMP is regulated by its activation by furinlike proprotein convertases, by its inhibition by TIMPs, and by its selfproteolysis and shedding (11-13). Evidence is also emerging that exocytosis, endocytosis, and recycling also regulate the presentation of MT1-MMP on the cell surface and, consequently, its cell surface-associated proteolytic activity (14 -17).Recently, we determined that functionally active MT1-MMP, which was presented on the cell surface, was internalized, trafficked alongside the microtubular cytoskeleton, and delivered to the centrosomal compartment (16,18). The presence of MT1-MMP in the pericentrosomal space correlated with the cleavage of human pericentrin-2 (kendrin), an integral and functionally important centrosomal, 3336-amino-acid residue long, protein (19 -22), and chromosome instability in non-malignant epithelial Madin-Darby canine kidney cells (18,23). Centrosomes, spindle pole bodies, are cellular organelles that exhibit an ability to organize microtubules and to nucleate (24). The normal functionality of centrosomes is essential to the organization of the cytoskeleton and the mitotic spindle, self-duplication, and cell cycle progression (25, 26). Conversely, centrosomal abnormalities, early predictors of carcinog...

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD2L 50 site initiates the MT1-MMP activation, whereas the 108 RRKR 111 2Y 112 cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.Cells rely on pericellular proteolysis to penetrate through the tissue (1, 2). The initiation of the invasive phenotype most frequently represents a matrix metalloproteinase (MMP) 2 -dependent process (3). Twenty four individual MMPs are encoded by the human genome (4, 5). MMPs are secreted (soluble MMP) or membrane-bound endoproteases (membrane-type MMPs) (6). They include a signal peptide, an ϳ80-residue prodomain, an ϳ170-residue zinc-and calcium-dependent catalytic domain, a short hinge region, and a four-bladed ␤-propeller hemopexinlike C-terminal domain (7). MT1-MMP is distinguished from soluble MMPs by a transmembrane domain and a cytoplasmic tail.The prodomain maintains the MMP proenzymes in their latent state (8). In the proenzyme, the catalytic zinc ion is chelated by the three His residues of the conserved HEXX-HXXGXXH active site motif and by the conserved Cys residue of the cysteine-switch motif (PRC 93 GVPD in MT1-MMP) from the C-terminal portion of the prodomain (7, 9, 10). When the interaction of the Cys residue with the active site Zn 2ϩ is disrupted by proteolytic removal of the prodomain (8, 10, 11), the active site becomes accessible to water. The coordination of the catalytic zinc with a water molecule is essential for the catalysis (12).Since the discovery of MT1-MMP in 1994 -1995 and the findings showing its role in the activation of MMP-2 (13, 14) the following question remains. What is the mechanism of the MT1-MMP activation? In contrast to soluble MMPs, which are secreted as inactive proenzymes and activated extracellularly, the latent MT1-MMP proenzyme is processed intracellularly by the furin-like proprotein convertases at the 108 RRKR 111 2Y 112 sequence (the furin site) in the linker motif connecting the prodomain and the catalytic domain sequences (13,15,16). The cleavage by furin takes place in the course of the secretion pathway (17, 18) and results in the mature enzyme sequence commencing from the N-terminal Tyr 112 (8). We recently demonstrated, however, that the activatio...