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...