RESUMO
INTRODUCTION: Soluble urokinase plasminogen activator receptor (suPAR) is a biologically active protein and increased levels are associated with worse outcomes in critically ill patients. suPAR in bronchoalveolar fluid (BALF) may be helpful to differentiate between types of acute respiratory distress syndrome (ARDS) and may have potential for early detection of fungal infection. METHODS: We prospectively investigated levels of suPAR in BALF and serum in critically ill patients who underwent bronchoscopy for any reason at the ICU of the Department of Internal Medicine, Medical University of Graz, Graz, Austria. RESULTS: Seventy-five patients were available for analyses. Median age was 60 [25th-75th percentile: 50-69] years, 27% were female, and median SOFA score was 12 [11-14] points. Serum suPAR levels were significantly associated with ICU mortality in univariable logistic regression analysis. There was no correlation between BALF and serum suPAR. Serum suPAR was higher in ARDS patients at 11.2 [8.0-17.2] ng/mL compared to those without ARDS at 7.1 [3.7-10.1] (p < 0.001). BALF-suPAR was significantly higher in patients with evidence of fungal lung infection compared to patients without fungal infection both in the general cohort (7.6 [3.2-9.4] vs 2.5 [1.1-5.3], p = 0.013) and in the subgroup of ARDS (7.2 [3.1-39.2] vs 2.5 [1.0-5.2], p = 0.022). All patients were classified as putative/probable invasive aspergillosis. CONCLUSION: We found significant higher levels of serum suPAR in ARDS patients compared to those not fulfilling ARDS criteria. Serum and BALF-suPAR were significantly higher in those patients with evidence for invasive pulmonary aspergillosis. These findings may suggest testing this biomarker for early diagnosis of fungal infection in a greater cohort.
Assuntos
Aspergilose , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Síndrome do Desconforto Respiratório , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Biomarcadores , Estado Terminal , Prognóstico , Estudos Prospectivos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Síndrome do Desconforto Respiratório/diagnósticoRESUMO
Tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are serine proteases and major activators of fibrinolysis in mammalian systems. Because fibrinolysis is an essential component of the response to tissue injury, diverse cells, including cells that participate in the response to injury, have evolved receptor systems to detect tPA and uPA and initiate appropriate cell-signaling responses. Formation of functional receptor systems for the plasminogen activators requires assembly of diverse plasma membrane proteins, including but not limited to: the urokinase receptor (uPAR); integrins; N-formyl peptide receptor-2 (FPR2), receptor tyrosine kinases (RTKs), the N-methyl-d-aspartate receptor (NMDA-R), and low-density lipoprotein receptor-related protein-1 (LRP1). The cell-signaling responses elicited by tPA and uPA impact diverse aspects of cell physiology. This review describes rapidly evolving knowledge regarding the structure and function of plasminogen activator receptor assemblies. How these receptor assemblies regulate innate immunity and inflammation is then considered.
Assuntos
Imunidade Inata , Inflamação/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tecidual/metabolismo , Animais , Ativação Enzimática , Fibrinólise , Humanos , Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Ligantes , Plasminogênio/metabolismo , Conformação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Relação Estrutura-Atividade , Ativador de Plasminogênio Tecidual/químicaRESUMO
Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Biomarcadores Tumorais/genética , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Neoplasias/terapia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Despite being recognized as a therapeutic target in the processes of cancer cell proliferation and metastasis for over 50 years, the interaction of the urokinase plasminogen activator uPA with its receptor uPAR still needs an improved understanding. High resolution crystallographic data (PDB ) of the uPA-uPAR binding geometry was used to perform quantum biochemistry computations within the density functional theory (DFT) framework. A divide to conquer methodology considering a mixed homogeneous/inhomogeneous dielectric model and explicitly taking water molecules into account was employed to obtain a large set of uPA-uPAR residue-residue interaction energies. In order of importance, not only were Phe25 > Tyr24 > Trp30 > Ile28 shown to be the most relevant uPA residues binding it to uPAR, but the residues Lys98 > His87 > Gln40 > Asn22 > Lys23 > Val20 also had significant interaction energies, which helps to explain published experimental mutational data. Furthermore, the results obtained with the uPA-uPAR in/homogeneous dielectric function show that a high dielectric constant value ε = 40 is adequate to take into account the electrostatic environment at the interface between the proteins, while using a smaller value of ε (<10) leads to an overestimation of the uPA-uPAR binding energy. Hot spots of the uPA-uPAR binding domain were identified and a quantum biochemistry description of the uPAR blockers uPA21-30 and cyclo21,29uPA21-29[(S21C;H29C)] was performed, demonstrating that cyclization improves the stability of mimetic peptides without compromising their binding energies to uPAR.
Assuntos
Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/química , Sequência de Aminoácidos , Aminoácidos/química , Teoria da Densidade Funcional , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvß3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.
Assuntos
Proliferação de Células , Receptores ErbB/metabolismo , Integrina alfaVbeta3/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases , Modelos Moleculares , Fosforilação , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.
Assuntos
Receptores ErbB/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células , Humanos , Cinética , Proteínas dos Microfilamentos , Metástase Neoplásica , Fosforilação , Domínios Proteicos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/químicaAssuntos
Corantes Fluorescentes , Meningioma , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Humanos , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/cirurgia , Meningioma/diagnóstico por imagem , Meningioma/cirurgia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.
Assuntos
Pirazóis/síntese química , Pirróis/síntese química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Cristalografia por Raios X , Corantes Fluorescentes/química , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica/efeitos dos fármacos , Pirazóis/farmacologia , Pirróis/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Coloração e Rotulagem/métodos , Relação Estrutura-Atividade , Termodinâmica , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
The discovery in higher animals of proteins from the Ly6/uPAR family, which have structural homology with snake "three-finger" neurotoxins, has generated great interest in these molecules and their role in the functioning of the organism. These proteins have been found in the nervous, immune, endocrine, and reproductive systems of mammals. There are two types of the Ly6/uPAR proteins: those associated with the cell membrane by GPI-anchor and secreted ones. For some of them (Lynx1, SLURP-1, SLURP-2, Lypd6), as well as for snake α-neurotoxins, the target of action is nicotinic acetylcholine receptors, which are widely represented in the central and peripheral nervous systems, and in many other tissues, including epithelial cells and the immune system. However, the targets of most proteins from the Ly6/uPAR family and the mechanism of their action remain unknown. This review presents data on the structural and functional properties of the Ly6/uPAR proteins, which reveal a variety of functions within a single structural motif.
Assuntos
Antígenos Ly/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Animais , Glicosilfosfatidilinositóis , Humanos , Neurotoxinas/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/fisiologia , Relação Estrutura-AtividadeRESUMO
Urokinase plasminogen activator receptor (uPAR) is a membrane protein which is attached to the cellular external membrane. The uPAR expression can be observed both in tumor cells and in tumor-associated stromal cells. Thus, in the present study, the human amino-terminal fragment (hATF), as a targeting element to uPAR, is used to conjugate to the surface of superparamagnetic iron nanoparticle (SPIO). Flowcytometry was used to examine the uPAR expression in different tumor cell lines. The specificity of hATF-SPIO was verified by Prussian blue stain and cell phantom test. The imaging properties of hATF-SPIO were confirmed in vivo magnetic resonance imaging (MRI) of uPAR-elevated colon tumor. Finally, the distribution of hATF-SPIO in tumor tissue was confirmed by pathological staining. Results showed that the three cells in which we screened, presented different expression characteristics, i. e., Hela cells strongly expressed uPAR, HT29 cells moderately expressed uPAR, but Lovo cells didn't express uPAR. In vitro, after incubating with Hela cells, hATF-SPIO could specifically combined to and be subsequently internalized by uPAR positive cells, which could be observed via Prussian blue staining. Meanwhile T2WI signal intensity of Hela cells, after incubation with targeted probe, significantly decreased, and otherwise no obvious changes in Lovo cells both by Prussian blue staining and MRI scans. In vivo, hATF-SPIO could be systematically delivered to HT29 xenograft and accumulated in the tumor tissue which was confirmed by Prussian Blue stain compared to Lovo xenografts. Twenty-four hours after injection of targeting probe, the signal intensity of HT29 xenografts was lower than Lovo ones which was statistically significant. This targeting nanoparticles enabled not only in vitro specifically combining to uPAR positive cells but also in vivo imaging of uPAR moderately elevated colon cancer lesions.
Assuntos
Neoplasias do Colo/diagnóstico , Nanopartículas de Magnetita/química , Imagem Molecular/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Linhagem Celular Tumoral , Compostos Férricos , Humanos , Imageamento por Ressonância Magnética , Coloração e RotulagemRESUMO
Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvß6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvß6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvß6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo ß6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvß6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, ß1, ß3, and ß6 alone). Our data suggest that interaction with uPAR requires expression of the complete αß heterodimer (e.g., αvß6), not individual subunits (i.e., αv, ß1, ß3, or ß6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvß6 and uPAR are located in uPAR domains II and III.
Assuntos
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/química , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Humanos , Integrinas/química , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Proteômica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Integrin αvß6 is an epithelially-restricted heterodimeric transmembrane glycoprotein, known to interact with the urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression. While the X-ray crystallographic structures of segments of other integrin heterodimers are known, there is no structural information for the complete αvß6 integrin to assess its direct interaction with uPAR. We have performed structural analysis of αvß6·uPAR interactions using model data with docking simulations to pinpoint their interface, in accord with earlier reports of the ß-propeller region of integrin α-chain interacting with uPAR. Interaction of αvß6·uPAR was demonstrated by our previous study using immunoprecipitation coupled with proteomic analysis by mass spectrometry. Recently this interaction was validated with proximity ligation assays and peptide arrays. The data suggested that two potential peptide regions from domain II and one peptide region from domain III of uPAR, interact with αvß6 integrin. Only the peptide region from domain III is consistent with the three-dimensional interaction site proposed in this study. The molecular basis of integrin αvß6·uPAR binding using structural data is discussed for its implications as a potential therapeutic target in cancer management.
Assuntos
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Antígenos de Neoplasias/química , Humanos , Integrinas/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteômica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/químicaRESUMO
Characterization of disulfide bond-containing proteins by hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) requires reduction of the disulfide bonds under acidic and cold conditions, where the amide hydrogen exchange reaction is quenched (pH 2.5, 0 °C). The reduction typically requires a high concentration (>200 mM) of the chemical reducing agent Tris(2-carboxyethyl)phosphine (TCEP) as its reduction rate constant is decreased at low pH and temperature. Serious adverse effects on chromatographic and mass spectrometric performances have been reported when using high concentrations of TCEP. In the present study, we explore the feasibility of using electrochemical reduction as a substitute for TCEP in HDX-MS analyses. Our results demonstrate that efficient disulfide bond reduction is readily achieved by implementing an electrochemical cell into the HDX-MS workflow. We also identify some challenges in using electrochemical reduction in HDX-MS analyses and provide possible conditions to attenuate these limitations. For example, high salt concentrations hamper disulfide bond reduction, necessitating additional dilution of the sample with aqueous acidic solution at quench conditions.
Assuntos
Medição da Troca de Deutério/métodos , Dissulfetos/química , Técnicas Eletroquímicas/métodos , Espectrometria de Massas/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Cromatografia Líquida/métodos , Conformação ProteicaRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is implicated in cancer invasion and metastatic development in prostate cancer and provides therefore an attractive molecular target for both imaging and therapy. In this study, we provide the first in vivo data on an antimetastatic effect of uPAR radionuclide targeted therapy in such lesions and show the potential of uPAR positron emission tomography (PET) imaging for identifying small foci of metastatic cells in a mouse model of disseminating human prostate cancer. Two radiolabeled ligands were generated in high purity and specific activity: a uPAR-targeting probe ((177)Lu-DOTA-AE105) and a nonbinding control ((177)Lu-DOTA-AE105mut). Both uPAR flow cytometry and ELISA confirmed high expression levels of the target uPAR in PC-3M-LUC2.luc cells, and cell binding studies using (177)Lu-DOTA-AE105 resulted in a specific binding with an IC50 value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted (177)Lu groups (p < 0.05) using bioluminescence imaging. Moreover, we found a significantly longer metastatic-free survival, with 65% of all mice without any disseminated metastatic lesions present at 65 days after first treatment dose (p = 0.047). In contrast, only 30% of all mice in the combined control groups treated with (177)Lu-DOTA-AE105mut or vehicle were without metastatic lesions. No treatment-induced toxicity was observed during the study as evaluated by observing animal weight and H&E staining of kidney tissue (dose-limiting organ). Finally, uPAR PET imaging using (64)Cu-DOTA-AE105 detected all small, disseminated metastatic foci when compared with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential for identification of small metastatic lesions using uPAR PET imaging in disseminated prostate cancer, illustrating the promising strategy of uPAR theranostics in prostate cancer.
Assuntos
Complexos de Coordenação/química , Lutécio/química , Oligopeptídeos/química , Neoplasias da Próstata/radioterapia , Radioisótopos/uso terapêutico , Animais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Ligantes , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Tomografia por Emissão de Pósitrons , Neoplasias da Próstata/diagnóstico por imagem , Ligação Proteica , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Proteínas Recombinantes/química , Resultado do Tratamento , Microtomografia por Raio-XRESUMO
The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.
Assuntos
Matriz Extracelular , Proteólise , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Vitronectina , Regulação Alostérica , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Medição da Troca de Deutério , Drosophila melanogaster , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Invasividade Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Espalhamento de Radiação , Relação Estrutura-Atividade , Vitronectina/química , Vitronectina/metabolismo , Raios XAssuntos
Nefropatias/sangue , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Biomarcadores/sangue , Humanos , Imunoensaio , Valor Preditivo dos Testes , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Sensibilidade e Especificidade , Homologia Estrutural de ProteínaRESUMO
In this work the use of a high-throughput nanomechanical detection system based on a DVD-ROM optical drive and cantilever sensors is presented for the detection of urokinase plasminogen activator receptor inflammatory biomarker (uPAR). Several large scale studies have linked elevated levels of soluble uPAR (suPAR) to infectious diseases, such as HIV, and certain types of cancer. Using hundreds of cantilevers and a DVD-based platform, cantilever deflection response from antibody-antigen recognition is investigated as a function of suPAR concentration. The goal is to provide a cheap and portable detection platform which can carry valuable prognostic information. In order to optimize the cantilever response the antibody immobilization and unspecific binding are initially characterized using quartz crystal microbalance technology. Also, the choice of antibody is explored in order to generate the largest surface stress on the cantilevers, thus increasing the signal. Using optimized experimental conditions the lowest detectable suPAR concentration is currently around 5 nM. The results reveal promising research strategies for the implementation of specific biochemical assays in a portable and high-throughput microsensor-based detection platform.
Assuntos
Nanotecnologia/métodos , Dispositivos de Armazenamento Óptico , Óptica e Fotônica/métodos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Especificidade de Anticorpos , Biomarcadores/metabolismo , Humanos , Fator de Crescimento Derivado de Plaquetas/imunologia , Prognóstico , Técnicas de Microbalança de Cristal de Quartzo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila/metabolismoRESUMO
Urokinase-type plasminogen activator (uPA) is a serine protease that converts the plasminogen zymogen into the enzymatically active plasmin. uPA is synthesized and secreted as the single-chain molecule (scuPA) composed of an N-terminal domain (GFD) and kringle (KD) and C-terminal proteolytic (PD) domains. Earlier, the structure of ATF (which consists of GFD and KD) was solved by NMR (A. P. Hansen et al. (1994) Biochemistry, 33, 4847-4864) and by X-ray crystallography alone and in a complex with the soluble form of the urokinase receptor (uPAR, CD87) lacking GPI (C. Barinka et al. (2006) J. Mol. Biol., 363, 482-495). According to these data, GFD contains two ß-sheet regions oriented perpendicularly to each other. The area in the GFD responsible for binding to uPAR is localized in the flexible Ω-loop, which consists of seven amino acid residues connecting two strings of antiparallel ß-sheet. It was shown by site-directed mutagenesis that shortening of the Ω-loop length by one amino acid residue leads to the inability of GFD to bind to uPAR (V. Magdolen et al. (1996) Eur. J. Biochem., 237, 743-751). Here we show that, in contrast to the above-mentioned studies, we found no sign of the ß-sheet regions in GFD in our uPA preparations either free or in a complex with uPAR. The GFD seems to be a rather flexible and unstructured domain, demonstrating in spite of its apparent flexibility highly specific interaction with uPAR both in vitro and in cell culture experiments. Circular dichroism, tryptophan fluorescence during thermal denaturation of the protein, and heteronuclear NMR spectroscopy of ¹5N/¹³C-labeled ATF both free and in complex with urokinase receptor were used to judge the secondary structure of GFD of uPA.
Assuntos
Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.
Assuntos
Pseudópodes/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/química , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Animais , Linhagem Celular , Cromatografia em Gel , Dicroísmo Circular , Drosophila , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Somatomedinas/química , Somatomedinas/metabolismo , Ressonância de Plasmônio de Superfície , Vitronectina/química , Vitronectina/metabolismoRESUMO
The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein with an established role in focalizing uPA-mediated plasminogen activation on cell surfaces. Distinct from this function, uPAR also modulates cell adhesion and migration on vitronectin-rich matrices. Although uPA and vitronectin engage structurally distinct binding sites on uPAR, they nonetheless cooperate functionally, as uPA binding potentiates uPAR-dependent induction of lamellipodia on vitronectin matrices. We now present data advancing the possibility that it is the burial of the ß-hairpin in uPA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized by engagement of the ß-hairpin in uPA, favors the proper assembly of an active, compact receptor structure that stimulates lamellipodia induction on vitronectin. This molecular model has wide implications for drug development targeting uPAR function.