RESUMEN
PURPOSE: Protease nexin-1 (PN-1), a serpin encoded by the SERPINE2 gene, has serine protease inhibitory activity and neurotrophic properties in the brain. PN-1 inhibits retinal angiogenesis; however, PN-1's neurotrophic capacities in the retina have not yet been evaluated. Pigment epithelium-derived factor (PEDF) is a serpin that exhibits neurotrophic and antiangiogenic activities but lacks protease inhibitory properties. The aim of this study is to compare PN-1 and PEDF. METHODS: Sequence comparisons were performed using computer bioinformatics programs. Mouse and bovine eyes, human retina tissue, and ARPE-19 cells were used to prepare RNA and protein samples. Interphotoreceptor matrix lavage was obtained from bovine eyes. Gene expression and protein levels were evaluated with reverse-transcription PCR (RT-PCR) and western blotting, respectively. Recombinant human PN-1, a version of PN-1 referred to as PN-1[R346A] lacking serine protease inhibitory activity, and PEDF proteins were used, as well as synthetic peptides designed from PEDF and PN-1 sequences. Survival activity in serum-starved, rat-derived retinal precursor (R28) cells was assessed with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) cell death assays. Bcl2 levels were measured with RT-PCR. RESULTS: PN-1 is analogous in primary and tertiary structure to PEDF. A region in PN-1 shares homology with the neurotrophic active region of PEDF, a 17-residue region within alpha helix C. The native human retina, ARPE-19 cells, and murine RPE and retina expressed the gene for PN-1 (SERPINE2 and Serpine2 mRNA). The retina, ARPE-19 cell lysates, and bovine interphotoreceptor matrix contained PN-1 protein. The addition of PN-1, PN-1[R346A], or the 17mer peptide of PN-1 to serum-starved retina cells decreased the number of TUNEL-positive nuclei relative to the untreated cells, such as PEDF. PN-1, PN-1[R346A], and PN-1-17mer treatments increased the Bcl2 transcript levels in serum-starved cells, as seen with PEDF. CONCLUSIONS: PN-1 and PEDF share structural and functional features, and expression patterns in the retina. These serpins' mechanisms of action as cell survival factors are independent of serine protease inhibition. We have identified PN-1 as a novel factor for the retina that may play a neuroprotective role in vivo, and small peptides as relevant candidates for preventing retinal degeneration.
Asunto(s)
Proteínas del Ojo/química , Proteínas del Ojo/farmacología , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/farmacología , Serpina E2/química , Serpina E2/farmacología , Serpinas/química , Serpinas/farmacología , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Bovinos , Línea Celular , Supervivencia Celular , Citoprotección/efectos de los fármacos , Ojo/metabolismo , Humanos , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Serpina E2/genética , Serpina E2/metabolismo , Homología Estructural de ProteínaRESUMEN
Pro-coagulant, anti-coagulant and fibrinolytic pathways are responsible for maintaining hemostatic balance under physiological conditions. Any deviation from these pathways would result in hypercoagulability leading to life threatening diseases like myocardial infarction, stroke, portal vein thrombosis, deep vein thrombosis (DVT) and pulmonary embolism (PE). Angiogenesis is the process of sprouting of new blood vessels from pre-existing ones and plays a critical role in vascular repair, diabetic retinopathy, chronic inflammation and cancer progression. Serpins; a superfamily of protease inhibitors, play a key role in regulating both angiogenesis and coagulation. They are characterized by the presence of highly conserved secondary structure comprising of 3 ß-sheets and 7-9 α-helices. Inhibitory role of serpins is modulated by binding to cofactors, specially heparin and heparan sulfate proteoglycans (HSPGs) present on cell surfaces and extracellular matrix. Heparin and HSPGs are the mainstay of anti-coagulant therapy and also have therapeutic potential as anti-angiogenic inhibitors. Many of the heparin binding serpins that regulate coagulation cascade are also potent inhibitors of angiogenesis. Understanding the molecular mechanism of the switch between their specific anti-coagulant and anti-angiogenic role during inflammation, stress and regular hemostasis is important. In this review, we have tried to integrate the role of different serpins, their interaction with cofactors and their interplay in regulating coagulation and angiogenesis.
Asunto(s)
Coagulación Sanguínea , Heparina/química , Neovascularización Patológica , Serpinas/fisiología , Angiotensinógeno/química , Animales , Anticoagulantes/química , Antitrombinas/química , Proteínas Sanguíneas/química , Vasos Sanguíneos/fisiopatología , Progresión de la Enfermedad , Matriz Extracelular/química , Proteínas del Ojo/química , Fibrinólisis , Proteoglicanos de Heparán Sulfato/química , Heparina de Bajo-Peso-Molecular/química , Homeostasis , Humanos , Inflamación , Conformación Molecular , Factores de Crecimiento Nervioso/química , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor de Proteína C/química , Serpina E2/química , Serpinas/química , Transducción de SeñalRESUMEN
Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with ß-strand 3A and the loop connecting α-helix A with ß-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.
Asunto(s)
Anticuerpos Monoclonales/química , Serpina E2/antagonistas & inhibidores , Serpina E2/química , Afinidad de Anticuerpos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Mapeo Epitopo , Escherichia coli/metabolismo , Glicosilación , Heparina/química , Humanos , Cinética , Mutagénesis Sitio-Dirigida , Neoplasias/metabolismo , Péptido Hidrolasas/química , Conformación Proteica , Transporte de Proteínas , Serpina E2/genética , Resonancia por Plasmón de Superficie , Factores de TiempoRESUMEN
Protease nexin-1 (PN1) is a specific and extremely efficient inhibitor of thrombin. However, unlike other thrombin inhibitors belonging to the serpin family, PN1 is not synthesized in the liver and does not circulate in the blood. Rather, PN1 is expressed by multiple cell types, including macrophages, smooth muscle cells, and platelets, and it is on the surface of these cells, bound to glycosaminoglycans, that PN1 inhibits the signaling functions of thrombin. PN1 sets the threshold for thrombin-induced platelet activation and has been implicated in atherosclerosis. However, in spite of the emerging importance of PN1 in thrombosis and atherosclerosis, little is know about how it associates to cells and how it inhibits thrombin at rates that surpass the diffusion limit. To address these issues, we determined the crystal structures of PN1 in complex with heparin, and in complex with catalytically inert thrombin. The crystal structures suggest a unique 2-step mechanism of thrombin recognition involving rapid electrostatics-driven association to form an initial glycosaminoglycan-bridged complex, followed by a large conformational rearrangement to form the productive Michaelis complex.
Asunto(s)
Heparina/química , Serpina E2/química , Trombina/química , Sitios de Unión , Conformación de Carbohidratos , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares/química , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Serpinas/química , Electricidad EstáticaRESUMEN
Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that plays an important role in cardiovascular disorders and tumor development. The potential role of PAI-1 as a drug target has been evaluated in various animal models (e.g. mouse and rat). Sensitivity to PAI-1 inhibitory agents varied in different species. To date, absence of PAI-1 structures from species other than human hampers efforts to reveal the molecular basis for the observed species differences. Here we describe the structure of latent mouse PAI-1. Comparison with available structures of human PAI-1 reveals (1) a differential positioning of α-helix A; (2) differences in the gate region; and (3) differences in the reactive center loop position. We demonstrate that the optimal binding site of inhibitors may be dependent on the orthologs, and our results affect strategies in the rational design of a pharmacologically active PAI-1 inhibitor.