RESUMEN
Despite the longstanding existence of liposome technology in drug delivery applications, there have been no ligand-directed liposome formulations approved for clinical use to date. This lack of translation is due to several factors, one of which is the absence of molecular tools for the robust quantification of ligand density on the surface of liposomes. We report here for the first time the quantification of proteins attached to the surface of small unilamellar liposomes using single-molecule fluorescence imaging. Liposomes were surface-functionalized with fluorescently labeled human proteins previously validated to target the cancer cell surface biomarkers plasminogen activator inhibitor-2 (PAI-2) and trastuzumab (TZ, Herceptin®). These protein-conjugated liposomes were visualized using a custom-built wide-field fluorescence microscope with single-molecule sensitivity. By counting the photobleaching steps of the fluorescently labeled proteins, we calculated the number of attached proteins per liposome, which was 11⯱â¯4 proteins for single-ligand liposomes. Imaging of dual-ligand liposomes revealed stoichiometries of the two attached proteins in accordance with the molar ratios of protein added during preparation. Preparation of PAI-2/TZ dual-ligand liposomes via two different methods revealed that the post-insertion method generated liposomes with a more equal representation of the two differently sized proteins, demonstrating the ability of this preparation method to enable better control of liposome protein densities. We conclude that the single-molecule imaging method presented here is an accurate and reliable quantification tool for determining ligand density and stoichiometry on the surface of liposomes. This method has the potential to allow for comprehensive characterization of novel ligand-directed liposomes that should facilitate the translation of these nanotherapies through to the clinic.
Asunto(s)
Sistemas de Liberación de Medicamentos , Microscopía Fluorescente/métodos , Inhibidor 2 de Activador Plasminogénico/administración & dosificación , Trastuzumab/administración & dosificación , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/química , Química Farmacéutica/métodos , Humanos , Ligandos , Liposomas , Nanotecnología/métodos , Inhibidor 2 de Activador Plasminogénico/química , Reproducibilidad de los Resultados , Tecnología Farmacéutica/métodos , Trastuzumab/químicaRESUMEN
Pregnancy zone protein (PZP) and plasminogen activator inhibitor type 2 (PAI-2) are two multifunctional proteins that are elevated in normal pregnancy and numerous other inflammatory states. Both proteins were originally identified as protease inhibitors, but current evidence supports the notion that they may also function as modulators of T-helper cells and/or extracellular chaperones. Exacerbated inflammation, fibrinolytic disturbances and misfolded proteins are all implicated in the pathology of preeclampsia, a leading cause of maternal and foetal mortality and morbidity. Notably, reduced levels of PZP or PAI-2 are associated with preeclampsia and clarification of their diverse functions in normal pregnancy could provide much needed insight regarding the pathogenesis of this disorder. Given that inflammation and protein misfolding underlie the pathology of a very large number of disorders, the contributions of PZP and PAI-2 to extracellular proteostasis and immunoregulation could be broad-reaching.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/metabolismo , Proteínas Gestacionales/química , Proteínas Gestacionales/metabolismo , Animales , Regulación de la Expresión Génica , HumanosRESUMEN
PURPOSE: Overexpression of the serine protease urokinase (uPA) is recognised as an important biomarker of metastatic disease and a druggable anticancer target. Plasminogen activator inhibitor type-2 (PAI-2/SerpinB2) is a specific uPA inhibitor with proven potential for use in targeted therapy. However, PAI-2 is rapidly cleared via the renal system which impairs tumor uptake and efficacy. Here we aimed to improve the pharmacological properties of PAI-2 by site-specific PEGylation. METHODS: Several cysteine to serine substitution mutants were generated for PEGylation with PEG-maleimide (size range 12-30 kDa) and the physico-chemical and biochemical properties of the PEG-PAI-2 conjugates characterised. Radiolabeled proteins were used for evaluation of blood clearance and tissue uptake profiles in an orthotopic breast tumor xenograft mouse model. RESULTS: PEGylation of the PAI-2(C161S) mutant gave a predominant mono-PEGylated-PAI-2 product (~90%) with full uPA inhibitory activity, despite a significant increase in hydrodynamic radius. Compared to un-PEGylated protein the plasma half-life and AUC for PEG20-PAI-2(C161S) were significantly increased. This translated to a 10-fold increase in tumor retention after 24 h compared to PAI-2(C161S), an effect not seen in non-target organs. CONCLUSIONS: Our data underscores the potential for PEG20-PAI-2(C161S) drug conjugates to be further developed as anti-uPA targeted therapeutics with enhanced tumor retention.
Asunto(s)
Antineoplásicos/farmacocinética , Portadores de Fármacos , Inhibidor 2 de Activador Plasminogénico/farmacocinética , Polietilenglicoles/química , Inhibidores de Serina Proteinasa/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Química Farmacéutica , Estabilidad de Medicamentos , Femenino , Humanos , Inyecciones Intravenosas , Tasa de Depuración Metabólica , Ratones Endogámicos BALB C , Ratones Desnudos , Modelos Moleculares , Mutación , Inhibidor 2 de Activador Plasminogénico/administración & dosificación , Inhibidor 2 de Activador Plasminogénico/sangre , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Conformación Proteica , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/sangre , Inhibidores de Serina Proteinasa/química , Tecnología Farmacéutica/métodos , Distribución TisularRESUMEN
Plasminogen activator inhibitor (PAI)-2 expression is acutely upregulated in pregnancy, inflammation, infection, and other pathophysiological conditions. Circumstances that prevent PAI-2 upregulation are associated with chronic pathology. Altogether this strongly suggests that PAI-2 is one of the many proteins that maintain homeostasis during damage or stress. However, several functions ranging from a classical serpin to various intracellular roles have been ascribed to PAI-2 and, because none of these have been definitively proven in vivo, to this day its precise role or roles remains an enigma. This review readdresses the evidence supporting a role for PAI-2 in fibrinolysis and proteolysis within extracellular environments and includes a review of the many potential intracellular functions attributed to PAI-2.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/fisiología , Animales , Femenino , Humanos , Inhibidor 2 de Activador Plasminogénico/química , EmbarazoRESUMEN
SerpinB2 or plasminogen activator inhibitor type 2 (PAI-2) is widely described as an inhibitor of extracellular urokinase plasminogen activator. However, the evidence that this represents the physiological role of SerpinB2 is not overly compelling. SerpinB2 is substantially up-regulated under multiple inflammatory conditions, and dysregulated expression and polymorphisms are associated with several human inflammatory diseases. A bewildering array of diverse functions and activities have been attributed to SerpinB2, but despite ≈900 publications in the field, no coherent view of what SerpinB2 does in vivo has emerged. Although SerpinB2 is abundantly expressed by activated macrophages and a range of other haematopoietic and non-haematopoietic cells, SerpinB2(-/-) mice have surprisingly few phenotypes. However, SerpinB2(-/-) mice were recently shown to generate increased Th1 responses, suggesting that at least one function of SerpinB2 is sculpting of the adaptive immune response.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/inmunología , Animales , Humanos , Neoplasias/inmunología , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/metabolismo , Procesamiento Proteico-Postraduccional , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
A urokinase targeting conjugate of 2'-deoxy-5-fluorouridine (5-FUdr) was synthesized and tested for tumor-cell selective cytotoxicity in vitro. The 5-FUdr prodrug 2'-deoxy-5-fluoro-3'-O-(3-carboxypropanoyl)uridine (5-FUdrsuccOH) containing an ester-labile succinate linker was attached to the specific urokinase inhibitor plasminogen activator inhibitor type II (PAI-2) and was found to preferentially kill urokinase-over expressing cancer cells. Up to 7 molecules of 5-FUdr were incorporated per PAI-2 molecule without affecting protein activity. This is the first time a small organic cytotoxin has been conjugated to PAI-2.
Asunto(s)
Uridina/análogos & derivados , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias/enzimología , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/metabolismo , Uridina/síntesis química , Uridina/química , Uridina/toxicidad , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis-signalling co-receptors.
Asunto(s)
Neoplasias/fisiopatología , Inhibidor 2 de Activador Plasminogénico/fisiología , Animales , Humanos , Hidrólisis , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias/metabolismo , Neoplasias/patología , Plasminógeno/metabolismo , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/metabolismo , Conformación ProteicaRESUMEN
PAI-1 and PAI-2 (plasminogen-activator inibitor types 1 and 2) are inhibitors of cell surface uPA (urokinase plasminogen activator). However, tumour expression of PAI-1 and PAI-2 correlates with poor compared with good patient prognosis in breast cancer respectively. This biological divergence may be related to additional functional roles of PAI-1. For example, the inhibition of uPA by PAI-1 reveals a cryptic high-affinity site within the PAI-1 moiety for the VLDLr (very-low-density-lipoprotein receptor), which sustains cell signalling events initiated by binding of uPA to its receptor. These interactions and subsequent signalling events promote proliferation of breast cancer cells. Biochemical and structural analyses show that, unlike PAI-1, the PAI-2 moiety of uPA-PAI-2 does not contain a high-affinity-binding site for VLDLr, although uPA-PAI-2 is still efficiently endocytosed via this receptor in breast cancer cells. Furthermore, global protein tyrosine phosphorylation events were not sustained by uPA-PAI-2 and cell proliferation was not affected. We thus propose a structurally based mechanism for these differences between PAI-1 and PAI-2 and suggest that PAI-2 is able to inhibit and clear uPA activity without initiating mitogenic signalling events through VLDLr.
Asunto(s)
Neoplasias de la Mama/metabolismo , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/fisiología , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/fisiología , Transducción de Señal/fisiología , Neoplasias de la Mama/química , Neoplasias de la Mama/patología , Humanos , Inhibidor 1 de Activador Plasminogénico/metabolismo , Inhibidor 2 de Activador Plasminogénico/metabolismo , Unión Proteica/fisiología , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Receptores de Superficie Celular/metabolismo , Receptores de LDL/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Células Tumorales Cultivadas , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
OBJECTIVES: The key objective of the study was to determine the single and multiple dose toxicity and efficacy of Bismuth-213 labeled PAI2 based targeted alpha therapy by selectively targeting uPA and uPAR in regressing prostate cancer in a nude mouse model. Targeted alpha therapy (TAT) is an experimental therapeutic modality for cancer. Another objective was to compare the in vivo pharmacokinetics using two different chelators to form the radioisotope-protein construct. METHODS: The single and multiple intra-peritoneal (IP) dose toxicity and efficacy of 213BiPAI2 was investigated in nude mice and its toxicity in rabbits. CD 31 staining for vasculature and uPA expression were measured at different stages of tumor growth. The pharmacokinetics of the chelators cDTPA and CHX-A'' were measured. RESULTS: All TAT regimes were well tolerated in mice and rabbits on biochemical and haematological examination. Capillaries became evident at six days post-cell inoculation. uPA expression was positive at all stages of tumour growth. No significant differences were observed between cDTPA and CHX-A. '' Inhibition of tumour growth was observed at 947 and 1421 MBq/kg single dose injection at three days post-PC3 cell inoculation. The three day post-inoculation multiple dose regime gave complete tumour growth inhibition at a total dose of 947 MBq/kg given on five successive days. Mice treated at 6, 12 and 18 days post-inoculation showed significantly slower tumour growth compared to controls. CONCLUSIONS: The efficacy of single and multiple dose TAT in mice was demonstrated within tolerance limits, the multiple dose regime being no more toxic than the single dose. Either of the two chelators could be used for 213Bi studies.
Asunto(s)
Bismuto/farmacología , Inhibidor 2 de Activador Plasminogénico/metabolismo , Neoplasias de la Próstata/terapia , Radioisótopos/farmacología , Animales , Quelantes/farmacología , Cicloheximida/farmacología , Humanos , Masculino , Ratones , Ratones Desnudos , Metástasis de la Neoplasia , Trasplante de Neoplasias , Ácido Pentético/farmacología , Inhibidor 2 de Activador Plasminogénico/química , Conejos , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.
Asunto(s)
Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Inhibidor 2 de Activador Plasminogénico/fisiología , Neoplasias de la Próstata/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/fisiología , Sitios de Unión , Membrana Celular/metabolismo , Clatrina/metabolismo , Electroforesis en Gel de Poliacrilamida , Endocitosis , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Cinética , Ligandos , Masculino , Microscopía Confocal , Inhibidor 2 de Activador Plasminogénico/química , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/química , Resonancia por Plasmón de Superficie , Factores de Tiempo , Activador de Plasminógeno de Tipo Uroquinasa/química , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
The serine protease inhibitor (serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis, inflammation, metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of serpins through saturation mutagenesis of the P8 residue for a typical family member, plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the serpin inhibitory mechanism. The importance of this residue for inhibition of the PAI-2 protease target urinary plasminogen activator (urokinase, uPA) is confirmed, although a high degree of tolerance to P8 substitution is observed. Out of 19 possible PAI-2 P8 mutants, 16 display inhibitory activities within an order of magnitude of the wild-type P8 Thr species. Crystal structures of complexes between PAI-2 and RCL-mimicking peptides with P8 Met or Asp mutations are determined, and structural comparison with the wild-type complex substantiates the ability of the S8 pocket to accommodate disparate side-chains. These data indicate that the identity of the P8 residue is not a determinant of efficient RCL insertion, and provide further evidence for functional plasticity of key residues within enzyme structures. Poor correlation of observed PAI-2 P8 mutant activities with a range of physicochemical, evolutionary and thermodynamic predictive indices highlights the practical limitations of existing approaches to predicting the molecular phenotype of protein variants.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Polimorfismo de Nucleótido Simple , Serpinas/química , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Cinética , Péptidos/química , Inhibidor 2 de Activador Plasminogénico/fisiología , Serpinas/genética , Relación Estructura-Actividad , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidoresRESUMEN
Plasminogen activator inhibitor type-2 (PAI-2) is a nonconventional serine protease inhibitor (serpin) with unique and tantalizing properties that is generally considered to be an authentic and physiological inhibitor of urokinase. However, the fact that only a small percentage of PAI-2 is secreted has been a long-standing argument for alternative roles for this serpin. Indeed, PAI-2 has been shown to have a number of intracellular roles: it can alter gene expression, influence the rate of cell proliferation and differentiation, and inhibit apoptosis in a manner independent of urokinase inhibition. Despite these recent advances in defining the intracellular function of PAI-2, it still remains one of the most mysterious and enigmatic members of the serpin superfamily.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Animales , Apoptosis , Regulación de la Expresión Génica , Humanos , Metástasis de la Neoplasia/patología , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genéticaRESUMEN
The purpose was to characterize plasminogen activator inhibitor type 2 (PAI-2) expression in normal human conjunctiva in vivo and in vitro. PAI-2 antigen was assayed by immunostaining and immunoblotting of extracts from normal human conjunctival epithelial lysates and conditioned media (CM) of cultured human conjunctival keratinocytes. Immunostaining of normal human conjunctival epithelia revealed that PAI-2 was found consistently in the superficial keratinocytes and, in some biopsies, also in the lower keratinocyte layers. In all cases, PAI-2 was concentrated around the cell periphery. In extracts of conjunctival epithelia and cultured conjunctival keratinocytes, PAI-2 had an apparent molecular weight of 45 kDa, consistent with the non-glycosylated form. The majority of PAI-2, approximately 90%, was cell associated, however, a small percentage of PAI-2 was released into the CM in a linear manner with time. PAI-2 in the conditioned medium had a higher molecular weight, consistent with a glycosylated form. Conjunctival PAI-2 was active, as shown by its ability to complex with a target enzyme, urokinase plasminogen activator (uPA). Although PAI-2 was detectable both in monolayer (i.e., relatively undifferentiated) conjunctival keratinocyte cultures as well as in stratified (i.e., more differentiated) cultures, steady state levels of PAI-2 were greater in the latter. PAI-2 is constitutively expressed by normal human conjunctival epithelial cells. The expression of PAI-2 throughout all epithelial layers in some biopsies of conjunctiva in vivo contrasts with the previously established distribution of PAI-2 in corneal epithelia, where it is present exclusively in the most superficial (i.e. most highly differentiated) cells. The role of PAI-2 in either tissue is unclear. However, we speculate that its distinct distribution in conjunctival versus corneal epithelia underscores inherent differences between these tissues, and may reflect specific functions of this proteinase inhibitor in both conjunctival and corneal epithelial cells.
Asunto(s)
Conjuntiva/enzimología , Inhibidor 2 de Activador Plasminogénico/metabolismo , Biopsia , Diferenciación Celular , Extractos Celulares , Células Cultivadas , Conjuntiva/citología , Conjuntiva/cirugía , Medios de Cultivo Condicionados/análisis , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente Indirecta , Colorantes Fluorescentes , Humanos , Inmunohistoquímica , Queratinocitos/citología , Queratinocitos/enzimología , Cinética , Microscopía Fluorescente , Peso Molecular , Inhibidor 2 de Activador Plasminogénico/química , Distribución Tisular , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.
Asunto(s)
Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/metabolismo , Cisteína/genética , Cisteína/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Modelos Moleculares , Mutación/genética , Oxidación-Reducción , Inhibidor 2 de Activador Plasminogénico/genética , Docilidad , Unión Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vitronectina/química , Vitronectina/metabolismoRESUMEN
Maspin, a member of the serpin superfamily, has tumor suppressing activity against breast and prostate cancer. Maspin inhibits tumor growth by blocking cell invasion, and its reactive center loop (RCL) is thought to mediate this activity. To understand this function on the molecular level, we have solved the three-dimensional structure of Maspin to 3.1 A resolution. The molecular structure shows the characteristic features of the serpin fold, but the RCL of Maspin is unique in length, composition, and placement. Although the RCL of Maspin is accessible and cleavable by some proteinases, it functions in the uncleaved, constrained conformation observed here. These structural results will contribute to our understanding of the mechanism by which Maspin suppresses tumors.
Asunto(s)
Antineoplásicos/farmacología , Proteínas/química , Serpinas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Genes Supresores de Tumor , Caballos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Invasividad Neoplásica , Ovalbúmina/química , Inhibidor 2 de Activador Plasminogénico/química , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de ProteínaRESUMEN
We review here the molecular mechanisms that underlie alpha1-antitrypsin deficiency and show how an understanding of this mechanism has allowed us to explain the deficiency of other members of the serine proteinase inhibitor or serpin superfamily. These include the deficiency of antithrombin, C1-inhibitor and alpha1-antichymotrypsin in association with thrombosis, angio-oedema and emphysema respectively. Moreover the accumulation of mutant neuroserpin within neurones causes the novel dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). We have grouped these conditions together as the serpinopathies as recognition of their common pathophysiology provides a platform to develop strategies to treat the associated clinical syndromes.
Asunto(s)
Demencia/patología , Deficiencia de alfa 1-Antitripsina/patología , alfa 1-Antitripsina/química , Alelos , Animales , Antitrombinas/química , Antitrombinas/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Enfisema/patología , Humanos , Hígado/metabolismo , Enfermedades Pulmonares/patología , Mutación , Neuropéptidos/química , Inhibidor 1 de Activador Plasminogénico/química , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/genética , Polímeros/química , Estructura Terciaria de Proteína , Serpinas/química , Trombosis/patología , alfa 1-Antitripsina/genética , NeuroserpinaRESUMEN
The plasminogen activator inhibitor type-2 (PAI-2) dependent apoptosis protection is due to the 33 amino acids fragment located between helix C and D of PAI-2, this fragment may interact with some unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2 as a bait to perform a yeast two-hybrid screen using a cDNA library constructed with HeLa cells during apoptosis, and retrieved a clone encoding 94 amino acid residues of C-terminus of pre-mRNA processing factor 8 (PRPF8). Co-immunoprecipitation experiments confirmed that PAI-2 could interact with PRPF8 in vivo. PAI-2 could bind PRPF8 C-terminal in both the inside and outside of nuclear. These results suggested that the interaction between these two proteins might not be involved in the apoptosis process.
Asunto(s)
Proteínas Portadoras/metabolismo , Inhibidor 2 de Activador Plasminogénico/metabolismo , Precursores del ARN/metabolismo , Secuencia de Aminoácidos , Apoptosis , Secuencia de Bases , Western Blotting , Núcleo Celular/metabolismo , Células Clonales , Biblioteca de Genes , Células HeLa , Humanos , Inhibidor 2 de Activador Plasminogénico/química , Pruebas de Precipitina , Unión Proteica , Estructura Secundaria de Proteína , Precursores del ARN/química , Proteínas de Unión al ARN , Técnicas del Sistema de Dos HíbridosRESUMEN
The apoptosis protection by plasminogen activator inhibitor -2(PAI-2) is dependent on a 33 amino acids fragment between helix C and D of PAI-2 which is probably may be due to the interaction of PAI-2 with unknown intracellular proteins. In this study we used the fragment between helix C and D of PAI-2 as bait to screen a HeLa cells cDNA library constructed during apoptosis in a yeast two-hybrid system and retrieved a clone that encodes 241 amino acids of proteasome (prosome, macropain) subunit, beta type 1(PSMbeta1) which plays important roles in NF-kappaB activation. GST-pulldown experiments confirmed the interaction between PAI-2 and PSMB1 in vitro. These data suggest that the antiapoptosis activity of PAI-2 is probably related to its interaction with PSMbeta1.
Asunto(s)
Apoptosis/fisiología , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Inhibidor 2 de Activador Plasminogénico/química , Inhibidor 2 de Activador Plasminogénico/metabolismo , Análisis de Secuencia de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Plasminogen activator inhibitor type 2 (PAI-2) is the only wild-type serpin that polymerizes spontaneously under physiological conditions. We show that PAI-2 loses its ability to polymerize following reduction of thiol groups, suggesting that an intramolecular disulfide bond is essential for the polymerization. A novel disulfide bond was identified between C79 (in the CD-loop) and C161 (at the bottom of helix F). Substitution mutants in which this disulfide bond was broken did not polymerize. Reactive center loop peptide insertion experiments and binding of bis-ANS to hydrophobic cavities indicate that the C79-C161 disulfide bond stabilizes PAI-2 in a polymerogenic conformation with an open A-beta-sheet. Elimination of this disulfide bond causes A-beta-sheet closure and abrogates the polymerization. The finding that cytosolic PAI-2 is mostly monomeric, whereas PAI-2 in the secretory pathway is prone to polymerize, suggests that the redox status of the cell could regulate PAI-2 polymerization. Taken together, our data suggest that the CD-loop functions as a redox-sensitive switch that converts PAI-2 between an active stable monomeric and a polymerogenic conformation, which is prone to form inactive polymers.