RESUMEN
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
Multiple sclerosis (MS) is a disease of the central nervous system with autoimmune etiology. Susceptibility to MS is linked to viral and bacterial infections. Matrix metalloproteinases (MMPs) play a significant role in the fragmentation of myelin basic protein (MBP) and demyelination. The splice variants of the single MBP gene are expressed in the oligodendrocytes of the central nervous system (classic MBP) and in the immune cells (Golli-MBPs). Our data suggest that persistent inflammation caused by environmental risk factors is a step to MS. We have discovered biochemical evidence suggesting the presence of the inflammatory proteolytic pathway leading to MS. The pathway involves the self-activated furin and PC2 proprotein convertases and membrane type-6 MMP (MT6-MMP/MMP-25) that is activated by furin/PC2. These events are followed by MMP-25 proteolysis of the Golli-MBP isoforms in the immune system cells and stimulation of the specific autoimmune T cell clones. It is likely that the passage of these autoimmune T cell clones through the disrupted blood-brain barrier to the brain and the recognition of neuronal, classic MBP causes inflammation leading to the further up-regulation of the activity of the multiple individual MMPs, the massive cleavage of MBP in the brain, demyelination, and MS. In addition to the cleavage of Golli-MBPs, MMP-25 proteolysis readily inactivates crystallin alphaB that is a suppressor of MS. These data suggest that MMP-25 plays an important role in MS pathology and that MMP-25, especially because of its restricted cell/tissue expression pattern and cell surface/lipid raft localization, is a promising drug target in MS.
Asunto(s)
Células Presentadoras de Antígenos/patología , Encéfalo/inmunología , Inflamación/enzimología , Metaloproteinasas de la Matriz Asociadas a la Membrana/metabolismo , Esclerosis Múltiple/etiología , Proproteína Convertasas/metabolismo , Células Presentadoras de Antígenos/metabolismo , Encéfalo/enzimología , Encéfalo/patología , Proteínas Ligadas a GPI , Humanos , Inflamación/etiología , Metaloproteinasas de la Matriz Asociadas a la Membrana/genética , Microdominios de Membrana , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Proteína Básica de Mielina/metabolismo , Transducción de Señal , Distribución Tisular , Regulación hacia ArribaRESUMEN
Successful cancer therapies aim to induce selective apoptosis in neoplastic cells. The current suboptimal efficiency and selectivity drugs have therapeutic limitations and induce concomitant side effects. Recently, novel cancer therapies based on the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) have emerged. TRAIL, a key component of the natural antitumor immune response, selectively kills many tumor cell types. Earlier studies with recombinant TRAIL, however, revealed its many shortcomings including a short half-life, off-target toxicity, and existence of TRAIL-resistant tumor cells. We improved the efficacy of recombinant TRAIL by redesigning its structure and the expression and purification procedures. The result is a highly stable leucine zipper (LZ)-TRAIL chimera that is simple to produce and purify. This chimera functions as a trimer in a manner that is similar to natural TRAIL. The formulation of the recombinant LZ-TRAIL we have developed has displayed high specific activity in both cell-based assays in vitro and animal tests in vivo. Our results have shown that the half-life of LZ-TRAIL is improved and now exceeds 1 h in mice compared with a half-life of only minutes reported earlier for recombinant TRAIL. We have concluded that our LZ-TRAIL construct will serve as a foundation for a new generation of fully human LZ-TRAIL proteins suitable for use in preclinical and clinical studies and for effective combination therapies to overcome tumor resistance to TRAIL.
Asunto(s)
Leucina Zippers/genética , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Ratones SCID , Neoplasias/metabolismo , Neoplasias/patología , Ingeniería de Proteínas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Metaloproteinasa 14 de la Matriz/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular , Metabolismo Energético , Femenino , Humanos , Péptido Hidrolasas/metabolismo , Mapeo de Interacción de ProteínasRESUMEN
Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. alpha1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH(81)CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.
Asunto(s)
Estrógenos/fisiología , Metaloproteinasas de la Matriz/fisiología , Neoplasias Hormono-Dependientes/enzimología , Neoplasias/enzimología , alfa 1-Antitripsina/metabolismo , Línea Celular Tumoral , Humanos , Macrófagos/enzimología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/aislamiento & purificación , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz Secretadas , Modelos Moleculares , Neoplasias/genética , Neutrófilos/enzimología , Receptores de Estrógenos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.
Asunto(s)
Complemento C3b/metabolismo , Metaloendopeptidasas/fisiología , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , Separación Celular , Sistema Libre de Células , Complemento C4b/metabolismo , Proteínas del Sistema Complemento/metabolismo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoprecipitación , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/metabolismo , Proteínas Opsoninas/metabolismo , Unión Proteica , Factores de Tiempo , TransfecciónRESUMEN
Membrane type-1 matrix metalloproteinase (MT1-MMP), a prototypic member of the membrane-tethered MMP family, is an essential component of a cellular proteolysis apparatus. Recognition of protein cleavage targets followed by proteolysis is a main function of MT1-MMP. For the first time, however, we present evidence that MT1-MMP and other structurally related membrane MMPs bind C1q, the recognition unit of the first component of complement C1 that initiates activation of the classical pathway of complement. These interactions involve the catalytic domain of MT1-MMP and the C1q globular domain. In silico modeling followed by mutagenesis and the in vitro and cell-based binding studies showed that the His(171)-Glu-Lys-Gln-Ala-Asp(176) and Val(223)-Arg-Asn(224) peptide sequences of MT1-MMP are directly involved in the binding with C1q. These sequence regions are spatially distant from the active site of the protease. As a result, the catalytically active and the catalytically latent forms of cellular MT1-MMP are both efficient in binding with C1q. In agreement, despite the MT1-MMP/C1q interactions, C1q is totally resistant to MT1-MMP proteolysis. The discovery of the unconventional, receptor/ligand-like interactions of MT1-MMP with C1q, an essential component of immunity, is a significant step toward a more complete understanding of the role of this membrane-tethered protease in cancer.
Asunto(s)
Membrana Celular/metabolismo , Complemento C1q/metabolismo , Metaloendopeptidasas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/enzimología , Biología Computacional , Humanos , Ligandos , Metaloproteinasas de la Matriz Asociadas a la Membrana , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Matrix metalloproteinases (MMPs) including membrane type 1 MMP (MT1-MMP) can degrade extracellular matrix and cell surface receptor molecules and have an essential function in malignancy. Recently, we established a functional link between MT1-MMP and the receptor of complement component 1q (gC1qR). The gC1qR is known as a compartment-specific regulator of diverse cellular and viral proteins. Once released by proliferating cells, soluble gC1qR may inhibit complement component 1q hemolytic activity and play important roles in vivo in assisting tumor cells to evade destruction by complement. Here, we report that gC1qR is susceptible to MT1-MMP proteolysis in vitro and in cell cultures. The major MT1-MMP cleavage site (Gly(79) down arrow Gln(80)) is localized within the structurally disordered loop connecting the beta(3) and the beta(4) strands of gC1qR. The recombinant MT1-MMP construct that included the catalytic domain but lacked the hemopexin-like domain lost the proteolytic capacity; however, it retained the ability to bind gC1qR. Inhibition of MT1-MMP activity by a hydroxamate inhibitor converted the protease into a cell surface receptor of gC1qR and promoted co-precipitation MT1-MMP with the soluble gC1qR protein. It is tempting to hypothesize that these novel mechanisms may play important roles in vivo and have to be taken into account in designing hydroxamate-based cancer therapy.