RESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos , Microscopia de Fluorescência/métodos , Inibidor 2 de Ativador de Plasminogênio/administração & dosagem , Trastuzumab/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/química , Química Farmacêutica/métodos , Humanos , Ligantes , Lipossomos , Nanotecnologia/métodos , Inibidor 2 de Ativador de Plasminogênio/química , Reprodutibilidade dos Testes , Tecnologia Farmacêutica/métodos , Trastuzumab/químicaRESUMO
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.
Assuntos
Antineoplásicos/farmacocinética , Portadores de Fármacos , Inibidor 2 de Ativador de Plasminogênio/farmacocinética , Polietilenoglicóis/química , Inibidores de Serina Proteinase/farmacocinética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Química Farmacêutica , Estabilidade de Medicamentos , Feminino , Humanos , Injeções Intravenosas , Taxa de Depuração Metabólica , Camundongos Endogâmicos BALB C , Camundongos Nus , Modelos Moleculares , Mutação , Inibidor 2 de Ativador de Plasminogênio/administração & dosagem , Inibidor 2 de Ativador de Plasminogênio/sangue , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Conformação Proteica , Inibidores de Serina Proteinase/administração & dosagem , Inibidores de Serina Proteinase/sangue , Inibidores de Serina Proteinase/química , Tecnologia Farmacêutica/métodos , Distribuição TecidualRESUMO
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.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/imunologia , Animais , Humanos , Neoplasias/imunologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Processamento de Proteína Pós-Traducional , Células Th1/imunologia , Células Th2/imunologiaRESUMO
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.
Assuntos
Uridina/análogos & derivados , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias/enzimologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Uridina/síntese química , Uridina/química , Uridina/toxicidade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
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.
Assuntos
Neoplasias/fisiopatologia , Inibidor 2 de Ativador de Plasminogênio/fisiologia , Animais , Humanos , Hidrólise , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/metabolismo , Neoplasias/patologia , Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Conformação ProteicaRESUMO
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.
Assuntos
Neoplasias da Mama/metabolismo , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/fisiologia , Transdução de Sinais/fisiologia , Neoplasias da Mama/química , Neoplasias da Mama/patologia , Humanos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de LDL/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Bismuto/farmacologia , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Neoplasias da Próstata/terapia , Radioisótopos/farmacologia , Animais , Quelantes/farmacologia , Cicloeximida/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Ácido Pentético/farmacologia , Inibidor 2 de Ativador de Plasminogênio/química , Coelhos , Inibidores de Serina Proteinase/farmacologiaRESUMO
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.
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Inibidor 2 de Ativador de Plasminogênio/fisiologia , Neoplasias da Próstata/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Sítios de Ligação , Membrana Celular/metabolismo , Clatrina/metabolismo , Eletroforese em Gel de Poliacrilamida , Endocitose , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Cinética , Ligantes , Masculino , Microscopia Confocal , Inibidor 2 de Ativador de Plasminogênio/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Polimorfismo de Nucleotídeo Único , Serpinas/química , Substituição de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Cinética , Peptídeos/química , Inibidor 2 de Ativador de Plasminogênio/fisiologia , Serpinas/genética , Relação Estrutura-Atividade , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
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.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Apoptose , Regulação da Expressão Gênica , Humanos , Metástase Neoplásica/patologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/genética , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genéticaRESUMO
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.
Assuntos
Túnica Conjuntiva/enzimologia , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Biópsia , Diferenciação Celular , Extratos Celulares , Células Cultivadas , Túnica Conjuntiva/citologia , Túnica Conjuntiva/cirurgia , Meios de Cultivo Condicionados/análise , Fluoresceína-5-Isotiocianato , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Queratinócitos/citologia , Queratinócitos/enzimologia , Cinética , Microscopia de Fluorescência , Peso Molecular , Inibidor 2 de Ativador de Plasminogênio/química , Distribuição Tecidual , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
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.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Modelos Moleculares , Mutação/genética , Oxirredução , Inibidor 2 de Ativador de Plasminogênio/genética , Maleabilidade , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vitronectina/química , Vitronectina/metabolismoRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Proteínas/química , Serpinas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Genes Supressores de Tumor , Cavalos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Invasividade Neoplásica , Ovalbumina/química , Inibidor 2 de Ativador de Plasminogênio/química , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Apoptose/fisiologia , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Análise de Sequência de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Células HeLa , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Polímeros/metabolismo , Estrutura Secundária de Proteína , Cisteína/metabolismo , Dissulfetos/metabolismo , Humanos , Modelos Moleculares , Oxirredução , Peptídeos/química , Peptídeos/metabolismo , Inibidor 2 de Ativador de Plasminogênio/genética , Polímeros/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The control of micrometastatic breast cancer remains problematic. To this end, we are developing a new adjuvant therapy based on (213)Bi-PAI2, in which an alpha-emitting nuclide ((213)Bi) is chelated to the plasminogen activator inhibitor-2 (PAI2). PAI2 targets the cell-surface receptor bound urokinase plasminogen activator (uPA), which is involved with the metastatic spread of cancer cells. We have successfully labelled and tested recombinant human PAI2 with the alpha radioisotope (213)Bi to produce (213)Bi-PAI2, which is highly cytotoxic towards breast cancer cell lines. In this study, the 2-day postinoculation model, using MDA-MB-231 breast cancer cells, was shown to be representative of micrometastatic disease. Our in vivo efficacy experiments show that a single local injection of (213)Bi-PAI2 can completely inhibit the growth of tumour at 2 days postcell inoculation, and a single systemic (i.p.) administration at 2 days causes tumour growth inhibition in a dose-dependent manner. The specific role of uPA as the target for (213)Bi-PAI2 therapy was determined by PAI2 pretreatment blocking studies. In vivo toxicity studies in nude mice indicate that up to 100 microCi of (213)Bi-PAI2 is well tolerated. Thus, (213)Bi-PAI2 is successful in targeting isolated breast cancer cells and preangiogenic cell clusters. These results indicate the promising potential of (213)Bi-PAI2 as a novel therapeutic agent for micrometastatic breast cancer.
Assuntos
Bismuto/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Inibidor 2 de Ativador de Plasminogênio/farmacologia , Inibidores de Serina Proteinase/farmacologia , Partículas alfa , Animais , Bismuto/química , Bismuto/farmacocinética , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Neoplasias Experimentais , Inibidor 2 de Ativador de Plasminogênio/química , Inibidores de Serina Proteinase/química , Células Tumorais CultivadasRESUMO
Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that is subject to regulation at the post-transcriptional level. At least two mRNA instability elements reside within the PAI-2 transcript; one in the coding region and another within the 3'-untranslated region (UTR). For the latter, a functional AU-rich motif (ARE) has been identified that provides a binding site for a number of cellular proteins, including the mRNA stability protein, HuR. In this study, we used the yeast three-hybrid system to screen a human leukocyte cDNA library to identify other proteins that associate with the PAI-2 ARE. This screen identified tristetraprolin (TTP) as a PAI-2 mRNA ARE-binding protein. UV cross-linking and immunoprecipitation experiments showed that TTP expressed in HEK293 cells could associate with the PAI-2 ARE in vitro. Co-transfection of plasmids expressing TTP and PAI-2 in HEK293 cells resulted in an increase in the decay rate of PAI-2 mRNA and loss of PAI-2 protein in a process that was dependent upon the PAI-2 3'-UTR. The 29-nt PAI-2 AU-rich element alone was also capable of conferring TTP-dependent mRNA instability to a reporter transcript. The extent of PAI-2 mRNA stability was remarkably sensitive to TTP since TTP-dependent PAI-2 mRNA decay occurred at TTP levels that were below Western blot detection limits. This study identifies TTP as a functional PAI-2 ARE-binding protein that modulates the post-transcriptional regulation of the PAI-2 gene.
Assuntos
Antígenos de Superfície , Proteínas de Ligação a DNA , Proteínas Imediatamente Precoces/farmacologia , Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Northern Blotting , Western Blotting , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Proteínas Fúngicas/metabolismo , Biblioteca Gênica , Globinas/química , Globinas/metabolismo , Humanos , Modelos Genéticos , Testes de Precipitina , Ligação Proteica , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Transfecção , Tristetraprolina , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.
Assuntos
Inibidor 2 de Ativador de Plasminogênio/química , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Aminoácidos/química , Eletroforese em Gel de Poliacrilamida , Humanos , Ligação de Hidrogênio , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Treonina/química , Ureia/farmacologiaRESUMO
The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.
Assuntos
Cristalografia por Raios X , Peptídeos/química , Inibidor 2 de Ativador de Plasminogênio/química , Elétrons , Escherichia coli/metabolismo , Deleção de Genes , Glicina/química , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serpinas/química , Treonina/químicaRESUMO
Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor that inhibits urokinase. Constitutive and regulated PAI-2 gene expression involves post-transcriptional events, and an AU-rich mRNA instability motif within the 3'-untranslated region of PAI-2 mRNA is required for this process (Maurer, F., Tierney, M., and Medcalf, R. L. (1999) Nucleic Acids Res. 27, 1664-1673). Here we show that instability determinants are present within various exons of the PAI-2 coding region, most notably within exon 4. Deletion of exon 4 from the full-length PAI-2 cDNA results in a doubling in the half-life of PAI-2 mRNA, whereas a 28-nucleotide region within exon 4 contains binding sites for cytoplasmic proteins. Inducible stabilization of PAI-2 mRNA in HT-1080 cells treated with phorbol ester and tumor necrosis factor does not alter the binding of proteins to the exon 4 instability determinant, but resulted in a transient increase in the binding of factors to the AU-rich RNA instability element. Hence, PAI-2 mRNA stability is influenced by elements located within both the coding region and the 3'-untranslated region and that cytoplasmic mRNA binding factors may influence steady state and inducible PAI-2 mRNA expression. Finally a 10-nucleotide region flanking the exon 4 protein-binding site is homologous to instability elements within five other transcripts, suggesting that a common coding region determinant may exist.