RESUMEN
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)↓LI 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.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Embrión no Mamífero/embriología , Metaloproteinasa 14 de la Matriz/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Sitios de Unión/genética , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Polaridad Celular , Citoesqueleto/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Hibridación in Situ , Metaloproteinasa 14 de la Matriz/genética , Datos de Secuencia Molecular , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transfección , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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 PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(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.
Asunto(s)
Furina/metabolismo , Metaloproteinasa 14 de la Matriz/química , Metaloproteinasa 14 de la Matriz/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Humanos , Metaloproteinasa 14 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Estructura Terciaria de ProteínaRESUMEN
Membrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD downward arrowL50 or P58QS downward arrowL61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111 downward arrowY112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells.
Asunto(s)
Precursores Enzimáticos/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Procesamiento Proteico-Postraduccional/fisiología , Línea Celular Tumoral , Activación Enzimática/fisiología , Precursores Enzimáticos/química , Femenino , Humanos , Metaloproteinasa 14 de la Matriz/química , Proteínas de Neoplasias/química , Neoplasias/química , Estructura Terciaria de Proteína/fisiología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismoRESUMEN
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.
Asunto(s)
Metaloproteinasas de la Matriz/química , Animales , Células CHO , Dominio Catalítico , Línea Celular Tumoral , Membrana Celular/metabolismo , Cricetinae , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glicosilación , Humanos , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Ratones , Ratones Endogámicos BALB C , Modelos Genéticos , Trasplante de NeoplasiasRESUMEN
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.
Asunto(s)
Arginina/análisis , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Virus del Nilo Occidental/enzimología , Secuencia de Aminoácidos , Animales , Arginina/química , Células Cultivadas , Secuencia Conservada , Furina/química , Furina/metabolismo , Ratones , Datos de Secuencia Molecular , Vaina de Mielina/metabolismo , Neuronas/metabolismo , Péptidos/química , Péptidos/farmacología , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/genética , ARN Helicasas/aislamiento & purificación , ARN Helicasas/metabolismo , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Serpinas/farmacología , Especificidad por Sustrato , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/aislamiento & purificación , Virus del Nilo Occidental/genéticaRESUMEN
Anthrax toxin consists of protective antigen (PA), and lethal (LF) and edema (EF) factors. A 83 kDa PA monomer (PA83) precursor binds to the cell receptor. Furin-like proprotein convertases (PCs) cleave PA83 to generate cell-bound 63 kDa protein (PA63). PA63 oligomerizes to form a ring-shaped heptamer that binds LF-EF and facilitates their entry into the cells. Several additional PCs, as opposed to furin alone, are capable of processing PA83. Following the incomplete processing of the available pool of PA83, the functional heptamer includes both PA83 and PA63. The available structures of the receptor-PA complex imply that the presence of either one or two molecules of PA83 will not impose structural limitations on the formation of the heptamer and the association of either the (PA83)(1)(PA63)(6) or (PA83)(2)(PA63)(5) heteroheptamer with LF-EF. Our data point to the intriguing mechanism of anthrax that appears to facilitate entry of the toxin into the cells which express limiting amounts of PCs and an incompletely processed PA83 pool.
Asunto(s)
Antígenos Bacterianos/metabolismo , Bacillus anthracis/metabolismo , Toxinas Bacterianas/metabolismo , Endocitosis , Furina/metabolismo , Animales , Antibacterianos/farmacología , Bacillus anthracis/química , Sitios de Unión , Células Cultivadas , Dimerización , Furina/deficiencia , Ratones , Modelos Moleculares , Peso Molecular , Proproteína Convertasas/metabolismo , Receptores de Superficie Celular/metabolismo , Venenos de VíborasRESUMEN
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.
Asunto(s)
Membrana Celular/enzimología , Metaloendopeptidasas/metabolismo , Microtúbulos/enzimología , Animales , Sitios de Unión de Anticuerpos , Células CHO , Línea Celular , Línea Celular Tumoral , Centrosoma/enzimología , Centrosoma/metabolismo , Cricetinae , Citoesqueleto/fisiología , Perros , Humanos , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/química , Metaloendopeptidasas/genética , Metaloendopeptidasas/inmunología , Microtúbulos/metabolismo , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Eliminación de Secuencia , Vesículas Transportadoras/enzimología , Vesículas Transportadoras/genética , Tubulina (Proteína)/fisiologíaRESUMEN
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 ALRRLLG1156 downward arrow L1157FG and ALRRLLS2068 downward arrow L2069FG, respectively. The presence of Asp-948 at the P1 position inactivated the corresponding site (ALRRLLD948-L949FGD) 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.
Asunto(s)
Antígenos/metabolismo , Transformación Celular Neoplásica , Centrosoma/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Animales , Secuencia de Bases , Línea Celular Tumoral , Cartilla de ADN , Humanos , Hidrólisis , Metaloproteinasa 14 de la Matriz , Metaloproteinasas de la Matriz Asociadas a la Membrana , RatonesRESUMEN
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 non-malignant 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.