RESUMEN
The macrophage elastase enzyme (MMP-12) expressed mainly in alveolar macrophages has been identified in the mouse lung as the main destructive agent associated with cigarette smoking, which gives rise to emphysema, both directly via elastin degradation and indirectly by disturbing the proteinase/antiproteinase balance via inactivation of the alpha1-proteinase inhibitor (alpha1-PI), the antagonist of the leukocyte elastase. The catalytic domain of human recombinant MMP-12 has been crystallized in complex with the broad-specificity inhibitor batimastat (BB-94). The crystal structure analysis of this complex, determined using X-ray data to 1.1 A and refined to an R-value of 0.165, reveals an overall fold similar to that of other MMPs. However, the S-shaped double loop connecting strands III and IV is fixed closer to the beta-sheet and projects its His172 side-chain further into the rather hydrophobic active-site cleft, defining the S3 and the S1-pockets and separating them from each other to a larger extent than is observed in other MMPs. The S2-site is planar, while the characteristic S1'-subsite is a continuous tube rather than a pocket, in which the MMP-12-specific Thr215 replaces a Val residue otherwise highly conserved in almost all other MMPs. This alteration might allow MMP-12 to accept P1' Arg residues, making it unique among MMPs. The active-site cleft of MMP-12 is well equipped to bind and efficiently cleave the AlaMetPhe-LeuGluAla sequence in the reactive-site loop of alpha1-PI, as occurs experimentally. Similarities in contouring and particularly a common surface hydrophobicity both inside and distant from the active-site cleft explain why MMP-12 shares many substrates with matrilysin (MMP-7). The MMP-12 structure is an excellent template for the structure-based design of specific inhibitors for emphysema therapy and for the construction of mutants to clarify the role of this MMP.
Asunto(s)
Macrófagos/enzimología , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fenilalanina/metabolismo , Inhibidores de Proteasas/metabolismo , Tiofenos/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes/metabolismo , Cristalización , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/genética , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Inhibidores de Proteasas/química , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Tiofenos/químicaRESUMEN
A full-length cDNA encoding a novel human protein was cloned from placenta cDNA. The corresponding 1542 amino acid protein sequence was termed 'pregnancy-associated plasma protein-E' (PAPP-E) as it shows a 62% homology to the human pregnancy-associated plasma protein-A (PAPP-A) that is a diagnostic marker for trisomies, especially Down syndrome. The conserved domain structure contains five motifs related to the short consensus repeats of complement proteins and selectins, three motifs related to the lin-notch motifs of proteins regulating early tissue differentiation, and a putative zinc-binding motif and active site of the metzincin-superfamily of metalloproteases. The PAPP-E gene was localized to chromosome 1q23-25. Northern blot analysis showed that PAPP-E is predominantly expressed in placenta.
Asunto(s)
Endopeptidasas , Proteínas Gestacionales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Humanos Par 1 , Clonación Molecular , ADN Complementario/química , Femenino , Humanos , Metaloproteinasa 9 de la Matriz/genética , Datos de Secuencia Molecular , Placenta/química , Placenta/metabolismo , Proteínas Gestacionales/química , Proteína Plasmática A Asociada al Embarazo/genética , Alineación de SecuenciaRESUMEN
The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity.
