RESUMO
The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.
Assuntos
Terapia com Luz de Baixa Intensidade/métodos , Melanoma/terapia , Nanopartículas Multifuncionais/uso terapêutico , Neoplasias Cutâneas/terapia , Animais , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/química , Ouro/química , Humanos , Terapia com Luz de Baixa Intensidade/efeitos adversos , Masculino , Melanoma/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos SCID , Nanopartículas Multifuncionais/química , Ácido Oleico/química , Estudo de Prova de Conceito , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The biophysical mechanism of magnetic fields (MFs) acting on living systems is not clear. Previous research showed that, similar to epidermal growth factor (EGF), MF exposure induced EGF receptor (EGFR) clustering and activated EGFR signaling. In this study, we investigated whether MF exposure induced the changes in physical characteristics of EGF and downstream effects of EGF and EGFR interaction. The phase-interrogation surface plasmon resonance (SPR) sensing analyses showed that 50 Hz MF exposure at 4.0 mT for 1 h induced reversible relative permittivity changes of EGF solution. However, compared with sham-exposed EGF solution, the MF-exposed EGF solution did not affect the binding of EGF to EGFR, nor the cell viability and EGFR clustering in human amniotic epithelial cells (FL cells). Our data suggest that cellular EGFR clustering response to MF exposure might not be a result of changes in relative permittivity of EGF in cell culture solution. Bioelectromagnetics. © 2020 Bioelectromagnetics Society.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Campos Magnéticos , Âmnio/citologia , Sistema Livre de Células , Células Cultivadas , Fator de Crescimento Epidérmico/química , Células Epiteliais/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Dispositivos Lab-On-A-Chip , Soluções , Ressonância de Plasmônio de SuperfícieRESUMO
BACKGROUND: Epidermal growth factor (EGF) can promote cell proliferation as well as migration, which is feasible in tissue wound healing. Oil bodies have been exploited as an important platform to produce exogenous proteins. The exogenous proteins were expressed in oil bodies from plant seeds. The process can reduce purification steps, thereby significantly reducing the purification cost. Mostly, the diameter of oil body particle ranges between 1.0 and 1.5 µm in the safflower seeds, however, it reduces to 700-1000 nm in the transgenic safflower seeds. The significant reduction of particle size in transgenic seeds is extremely beneficial to skin absorption. RESULTS: The diameter of oil body in the transgenic safflower seeds was recorded in the range of 700-1000 nm. The smaller particle size improved their skin absorption. The expression level of oleosin-hEGF-hEGF in T3 transgenic seeds was highest at 69.32 mg/g of seeds. The oil body expressing oleosin-hEGF-hEGF had significant proliferative activity on NIH/3T3 cells and improved skin regeneration thereby accelerating wound healing in rats. The wound coverage rate exceeded 98% after treatment for 14 days with oil body expressing oleosin-hEGF-hEGF, while the saline without EGF group and wild type oil body group both showed less than 80%. The neonatal fibroblast and collagen were found to be increased in the safflower oil body expressing oleosin-hEGF-hEGF treatment group. TGF-ß1, bFGF and VEGF were noted as important growth factors in the repair of cutaneous wounds. Their expression level increased after 4 and 7 day treatment, but decreased after 14 days. Therefore, it can promote skin regeneration to accelerate wounds healing. CONCLUSIONS: The expression of oleosin-hEGF-hEGF in T3 transgenic seeds was 80.43 ng/µL oil body. It had significant proliferative activity on NIH/3T3 cells and improved skin regeneration to accelerate wound healing in rats. The expression process of TGF-ß1, bFGF and VEGF increased at first and then gradually declined.
Assuntos
Fator de Crescimento Epidérmico/química , Gotículas Lipídicas/química , Proteínas de Plantas/química , Pele/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Células NIH 3T3 , Tamanho da Partícula , Óleos de Plantas/química , Ratos , Ratos Wistar , Regeneração/efeitos dos fármacos , Sementes/química , Propriedades de Superfície , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/imunologiaRESUMO
Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.
Assuntos
Receptores ErbB/genética , Hemangiossarcoma/tratamento farmacológico , Terapia de Alvo Molecular , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , ADP Ribose Transferases/administração & dosagem , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Cães , Doxorrubicina/administração & dosagem , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Receptores ErbB/antagonistas & inibidores , Exotoxinas/administração & dosagem , Exotoxinas/química , Exotoxinas/genética , Hemangiossarcoma/genética , Hemangiossarcoma/patologia , Humanos , Camundongos , Estadiamento de Neoplasias , Receptores de Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/química , Ativador de Plasminogênio Tipo Uroquinase/genética , Fatores de Virulência/administração & dosagem , Fatores de Virulência/química , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100-200 nm) showed a plasmon absorption band located within the near-infrared range (650-900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0-25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue.
Assuntos
Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Fototerapia , Polímeros/química , Células A549 , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos/química , Portadores de Fármacos/toxicidade , Ouro/toxicidade , Humanos , Ácido Hialurônico/química , Luz , Ácido Oleico/química , Estabilidade Proteica/efeitos da radiação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/efeitos da radiação , TemperaturaRESUMO
Myricetin is a naturally omnipresent benzo-α-pyrone flavonoids derivative; has potent anticancer activity. Receptor tyrosine kinases family provides the decisive role in cancer initiation and progression. These receptors have recently caught the attention of the researchers as an attractive target to combat cancer, owing to the evidences endorsed their over-expression on cancer cells. This study is a concerted effort to explore the potent and specific multi-targeted inhibitor against RTKs and AR\ER employing molecular docking approach. IR, IGF1R, EGFR, VEGFR1, VEGFR2, and AR\ER were chosen as a protein and natural compounds as a ligand. Molecular docking procedure followed by using Maestro 9.6 (Schrödinger Inc). All natural compounds were docked with the X-ray crystal structures of selected proteins by employing grid-based ligand docking with energetics Maestro 9.6. IBS natural compounds docked with each selected protein molecules by using GLIDE high throughput virtual screening. On the basis of Gscore, we selected 20 compounds from IBS (50,000 compounds) along with 68 anticancer compounds from published literature for GLIDE extra precision molecular docking. Calculated docking free energy yielded the excellent dock score for the myricetin when docked with proteins EGFR, IR, and AR\ER. Protein-ligand interactions profile highlighted that the lipophilic, hydrogen bonding and π-π stacking interactions play a central role in protein-ligand interactions at the active site. The results of MTT assay reveal that the myricetin inhibit the viability and proliferation of cancer cells in a dose-dependent manner. Treatment with the myricetin led to down-regulation of mRNA expression of EGFR, IR, mTOR, and Bcl-2. Although, further in vitro and in vivo experimental studies are required for the experimental validation of our findings.
Assuntos
Antagonistas de Receptores de Andrógenos/farmacologia , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Flavonoides/farmacologia , Antagonistas da Insulina/farmacologia , Antagonistas de Receptores de Andrógenos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Fator de Crescimento Epidérmico/química , Flavonoides/química , Flavonoides/farmacocinética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/química , Insulina/metabolismo , Antagonistas da Insulina/química , Ligantes , Simulação de Acoplamento Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/antagonistas & inibidores , Receptores de Estrogênio/metabolismo , Software , TermodinâmicaRESUMO
Cancer is one of the most life-threatening diseases, which causes 7.6 million deaths and around 1 trillion dollars economic loss every year. Theranostic materials are expected to improve early detection and safe treatment through personalized medicine. Driven by the needs, we report the development of a theranostic plasmonic shell-magnetic core star shape nanomaterial based approach for the targeted isolation of rare tumor cells from the whole blood sample, followed by diagnosis and photothermal destruction. Experimental data with whole blood sample spiked with SK-BR-3 cancer cell shows that Cy3 attached S6 aptamer conjugated theranostic plasmonic/magnetic nanoparticles can be used for fluorescence imaging and magnetic separation even in 0.001% mixtures. A targeted photothermal experiment using 1064 nm near-IR light at 2-3 W/cm(2) for 10 min resulted in selective irreparable cellular damage to most of the SK-BR-3 cancer cells. We discuss the possible mechanism and operating principle for the targeted imaging, separation, and photothermal destruction using theranostic magnetic/plasmonic nanotechnology. After the optimization of different parameters, this theranostic nanotechnology-driven assay could have enormous potential for applications as contrast agent and therapeutic actuators for cancer.
Assuntos
Fator de Crescimento Epidérmico/química , Magnetismo , Nanopartículas/uso terapêutico , Nanotecnologia/métodos , Neoplasias/terapia , Fototerapia/métodos , Animais , Linhagem Celular Tumoral , Humanos , Nanopartículas/química , CoelhosRESUMO
AIM: Adenoid cystic carcinoma (ACC) is a relatively rare epithelial tumour of the salivary glands in the maxillofacial region. About 40-60% of the patients develop distant metastases, which have been documented most commonly in the lung but also in brain, bone, liver, thyroid, spleen and pancreatic gland. SUMMARY: A 55-year-old women with intraosseous ACC in the mandible mimicking apical periodontitis following curative resection and radiotherapy is presented. Three years later, multiple lung metastases were observed followed by chemotherapy. Five years after curative resection, the patient presented simultaneously with new expansive soft tissue in the pancreas and mammary gland as well as in the kidney found to be metastatic ACC. No case has been reported to date on the manifestation of distant metastases of intraosseous ACC in the breast and the kidney as described by these observations. Metastatic mammary gland ACC stained positive for epithelial growth factor receptor (EGFR) but was negative for HER-2/neu and Cyclooxygenase-2 (COX-2) expression.
Assuntos
Perda do Osso Alveolar/diagnóstico , Carcinoma Adenoide Cístico/secundário , Erros de Diagnóstico , Neoplasias Mandibulares/patologia , Periodontite Periapical/diagnóstico , Neoplasias das Glândulas Salivares/patologia , Perda do Osso Alveolar/etiologia , Neoplasias da Mama/secundário , Carcinoma Adenoide Cístico/patologia , Carcinoma Adenoide Cístico/cirurgia , Ciclo-Oxigenase 2/biossíntese , Diagnóstico Diferencial , Fator de Crescimento Epidérmico/química , Feminino , Humanos , Oxigenoterapia Hiperbárica , Neoplasias Renais/secundário , Neoplasias Mandibulares/cirurgia , Pessoa de Meia-Idade , Osteomielite/diagnóstico , Osteomielite/terapia , Neoplasias Pancreáticas/secundário , Receptor ErbB-2/análise , Neoplasias das Glândulas Salivares/cirurgiaRESUMO
As an alternative to conventional, target-oriented drug discovery, we report a strategy that identifies compounds on the basis of the state that they induce in a signaling network. Immortalized human cells are grown in microtiter plates and treated with compounds from a small-molecule library. The target network is then activated and lysates derived from each sample are arrayed onto glass-supported nitrocellulose pads. By probing these microarrays with antibodies that report on the abundance or phosphorylation state of selected proteins, a global picture of the target network is obtained. As proof of concept, we screened 84 kinase and phosphatase inhibitors for their ability to induce different states in the ErbB signaling network. We observed functional connections between proteins that match our understanding of ErbB signaling, indicating that state-based screens can be used to define the topology of signaling networks. Additionally, compounds sort according to the multidimensional phenotypes they induce, suggesting that state-based screens may inform efforts to identify the targets of biologically active small molecules.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/imunologia , Fator de Crescimento Epidérmico/imunologia , Transdução de Sinais/imunologia , Anticorpos/imunologia , Reações Antígeno-Anticorpo , Linhagem Celular , Análise por Conglomerados , Relação Dose-Resposta a Droga , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/efeitos dos fármacos , Humanos , Biblioteca de Peptídeos , Análise Serial de Proteínas , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
The Tie receptor tyrosine kinases and their angiopoietin (Ang) ligands play central roles in developmental and tumor-induced angiogenesis. Here we present the crystal structures of the Tie2 ligand-binding region alone and in complex with Ang2. In contrast to prediction, Tie2 contains not two but three immunoglobulin (Ig) domains, which fold together with the three epidermal growth factor domains into a compact, arrowhead-shaped structure. Ang2 binds at the tip of the arrowhead utilizing a lock-and-key mode of ligand recognition-unique for a receptor kinase-where two complementary surfaces interact with each other with no domain rearrangements and little conformational change in either molecule. Ang2-Tie2 recognition is similar to antibody-protein antigen recognition, including the location of the ligand-binding site within the Ig fold. Analysis of the structures and structure-based mutagenesis provide insight into the mechanism of receptor activation and support the hypothesis that all angiopoietins interact with Tie2 in a structurally similar manner.
Assuntos
Angiopoietina-2/química , Receptor TIE-2/química , Sequência de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Fator de Crescimento Epidérmico/química , Fibrinogênio/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Receptor TIE-2/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Integrins are modular (alphabeta) heterodimeric proteins that mediate cell adhesion and convey signals across the plasma membrane. Interdomain motions play a key role in signal transduction by propagating structural changes through the molecule, thus controlling the activation state and adhesive properties of the integrin. We expressed a soluble fragment of the human integrin beta2 subunit comprising the plexin-semaphorin-integrin domain (PSI)/hybrid domain/I-EGF1 fragment and present its crystal structure at 1.8-A resolution. The structure reveals an elongated molecule with a rigid architecture stabilized by nine disulfide bridges. The PSI domain is located centrally and participates in the formation of extended interfaces with the hybrid domain and I-EGF1 domains, respectively. The hybrid domain/PSI interface involves the burial of an Arg residue, and contacts between PSI and I-EGF1 are mainly mediated by well conserved Arg and Trp residues. Conservation of key interacting residues across the various integrin beta subunits sequences suggests that our structure represents a good model for the entire integrin family. Superposition with the integrin beta3 receptor in its bent conformation suggests that an articulation point is present at the linkage between its I-EGF1 and I-EGF2 modules and underlines the importance of this region for the control of integrin-mediated cell adhesion.
Assuntos
Antígenos CD18/química , Fator de Crescimento Epidérmico/química , Sequência de Aminoácidos , Arginina/química , Adesão Celular , Moléculas de Adesão Celular/química , Cristalografia por Raios X , DNA Complementar/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Cadeias beta de Integrinas/química , Integrinas/química , Modelos Moleculares , Modelos Estatísticos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Semaforinas/química , Homologia de Sequência de Aminoácidos , Triptofano/químicaRESUMO
We present and test two methods to use quantum chemical calculations to improve standard protein structure refinement by molecular dynamics simulations restrained to experimental NMR data. In the first, we replace the molecular mechanics force field (employed in standard refinement to supplement experimental data) for a site of interest by quantum chemical calculations. This way, we obtain an accurate description of the site, even if a molecular-mechanics force field does not exist for this site, or if there is little experimental information about the site. Moreover, the site may change its bonding during the refinement, which often is the case for metal sites. The second method is to extract a molecular mechanics potential for the site of interest from a quantum chemical geometry optimisation and frequency calculation. We apply both methods to the two Ca2+ sites in the epidermal growth factor-like domains 3 and 4 in the vitamin K-dependent protein S and compare them to various methods to treat these sites in standard refinement. We show that both methods perform well and have their advantages and disadvantages. We also show that the glutamate Ca2+ ligand is unlikely to bind in a bidentate mode, in contrast to the crystal structure of an EGF domain of factor IX.
Assuntos
Cálcio/metabolismo , Fator de Crescimento Epidérmico/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteína S/química , Sítios de Ligação , Calibragem , Cátions Bivalentes/química , Ligantes , Modelos Moleculares , Nitrogênio/química , Oxigênio/química , Estrutura Terciária de ProteínaRESUMO
Marfan Syndrome (MFS) is an autosomal dominant disorder caused by mutations in the fibrillin-1 gene (FBN1). Several calves, all sired by a phenotypically normal bull, were found to exhibit the major clinical and pathological characteristics of human MFS (aortic dissection, joint laxity, lens dislocation), and were recognized as potential models of the human disease. In this study, Fbn1 cDNA from affected animals was sequenced and a heterozygous c.3598G > A transition was detected in exon 29, which predicted the substitution of an evolutionarily conserved glutamic acid by lysine at position 1200 (p.E1200K). This residue is part of a calcium-binding epidermal growth factor-like (cbEGF-like) module, a domain that is frequently altered in human MFS. Analysis of genomic DNA from the original bull's sperm showed that less than 20% of the sperm harbored the mutation, consistent with the presence of germline mosaicism. This study validates the use of these animals as models of human MFS. These cows will be valuable for investigations into the molecular pathogenesis of MFS, and may lead to better therapeutic testing and evaluation of human Marfan patients.
Assuntos
Cálcio/química , Fator de Crescimento Epidérmico/química , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Proteínas dos Microfilamentos/genética , Sequência de Aminoácidos , Animais , Bovinos , DNA Complementar/metabolismo , Modelos Animais de Doenças , Fibrilina-1 , Fibrilinas , Mutação em Linhagem Germinativa , Humanos , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
The protein avidin found in egg white seems optimized for binding the small vitamin biotin as a stable homotetramer. Indeed, along with its streptavidin ortholog in the bacterium Streptomyces avidinii, this protein shows the strongest known noncovalent bond of a protein with a small ligand. A third known member of the avidin family, as similar to avidin as is streptavidin, is found at the C-terminal ends of the multidomain fibropellin proteins found in sea urchin. The fibropellins form a layer known as the apical lamina that surrounds the sea urchin embryo throughout development. Based upon the structure of avidin, we deduced a structural model for the avidin-like domain of the fibropellins and found that computational modeling predicts a lack of biotin binding and the preservation of tetramerization. To test this prediction we expressed and purified the fibropellin avidin-like domain and found it indeed to be a homotetramer incapable of binding biotin. Several lines of evidence suggest that the avidin-like domain causes the entire fibropellin protein to tetramerize. We suggest that the presence of the avidin-like domain serves a structural (tetrameric form) rather than functional (biotin-binding) role and may therefore be a molecular instance of exaptation-the modification of an existing function toward a new function. Finally, based upon the oligomerization of the avidin-like domain, we propose a model for the overall structure of the apical lamina.
Assuntos
Avidina/química , Biotina/química , Fator de Crescimento Epidérmico/química , Proteínas da Matriz Extracelular/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dicroísmo Circular , DNA Complementar/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Vetores Genéticos , Ligantes , Substâncias Macromoleculares/química , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Ouriços-do-Mar , Homologia de Sequência de Aminoácidos , Software , Estreptavidina/química , Streptomyces/metabolismo , TermodinâmicaRESUMO
Patients with glioblastoma multiforme have a poor prognosis due to recurrences originating from spread cells. The use of radionuclide targeting might increase the chance of inactivating single tumor cells with minimal damage to surrounding healthy tissue. As a target, overexpressed epidermal growth factor receptors (EGFR) may be used. A natural ligand to EGFR, the epidermal growth factor (EGF) is an attractive targeting agent due to its low molecular weight (6 kDa) and high affinity for EGFR. 177Lu (T(1/2) = 6.7 days) is a radionuclide well suited for treatment of small tumor cell clusters, since it emits relatively low-energy beta particles. The goal of this study was to prepare and preclinically evaluate both in vitro and in vivo the [177Lu]Bz-DTPA-EGF conjugate. The conjugate was characterized in vitro for its cell-binding properties, and in vivo for its pharmacokinetics and ability to target EGFR. [177Lu]Bz-DTPA-EGF bound to cultured U343 glioblastoma cells with an affinity of 1.9 nM. Interaction with EGFR led to rapid internalization, and more than 70% of the cell-associated radioactivity was internalized after 30 minutes of incubation. The retention of radioactivity was good, with more than 65% of the 177Lu still cell-associated after 2 days. Biodistribution studies of i.v. injected [177Lu]Bz-DTPA-EGF in NMRI mice demonstrated a rapid blood clearance. Most of the radioactivity was found in the liver and kidneys. The liver uptake was receptor-mediated, since it could be significantly reduced by preinjection of unlabeled EGF. In conclusion, [177Lu]Bz-DTPA-EGF seems to be a promising candidate for locoregional treatment of glioblastoma due to its high binding affinity, low molecular weight, and ability to target EGFR in vivo.
Assuntos
Fator de Crescimento Epidérmico/uso terapêutico , Glioblastoma/radioterapia , Lutécio/uso terapêutico , Radioisótopos/uso terapêutico , Compostos Radiofarmacêuticos/uso terapêutico , Animais , Linhagem Celular Tumoral , Quelantes/química , Quelantes/farmacocinética , Avaliação Pré-Clínica de Medicamentos , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacocinética , Humanos , Lutécio/química , Lutécio/farmacocinética , Camundongos , Camundongos Endogâmicos , Ácido Pentético/química , Ácido Pentético/farmacocinética , Ligação Proteica , Radioisótopos/química , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Sensibilidade e Especificidade , Distribuição TecidualRESUMO
The genes coding for human and mouse AMACO, an extracellular matrix protein containing VWA-like domains related to those in MAtrilins and COllagens, were detected in databases, the cDNAs were cloned, and the primary structures were deduced from the nucleotide sequences. The genes consist of 14 exons and have a similar exon/intron organization. The protein consists of a signal peptide sequence, an N-terminal VWA domain connected to two additional, tandem VWA domains by a cysteine-rich sequence and an epidermal growth factor (EGF)-like domain. The C terminus is made up of another EGF-like domain followed by a unique sequence present in mouse, but absent in human. The predicted molecular weight of the proteins is 79,485 in human and 83,024 in mouse. Full-length AMACO was expressed in 293-EBNA cells, purified by use of an affinity tag and subjected to biochemical characterization. Both monomers and aggregates of AMACO were recovered, as shown by electron microscopy and SDS-PAGE. AMACO was found in the media of a variety of established cell lines of both fibroblast and epithelial origin. In the matrix formed by 293-EBNA cells overexpressing the protein, AMACO was deposited in patchy structures that were often cell-associated. Affinity-purified antibodies detect expression in cartilage and expression associated with certain basement membranes. In the kidney of adult mice, a second promoter located in intron 4 is active. If the resulting transcript is translated it could not yield a secreted protein because of the lack of a signal peptide sequence. The developmental switch from an AMACO mRNA, expressed by the newborn kidney, to the truncated transcript found in the adult kidney indicates an unusual regulation of AMACO expression.
Assuntos
Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/química , Rim/metabolismo , Fator de von Willebrand/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Basal/metabolismo , Biomarcadores Tumorais , Northern Blotting , Proteínas de Ligação ao Cálcio , Cartilagem/metabolismo , Linhagem Celular , Condrócitos/metabolismo , Cisteína/química , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/química , Células Epiteliais , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Hibridização In Situ , Queratinócitos/metabolismo , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Família Multigênica , Oligonucleotídeos Antissenso/metabolismo , Peptídeos/química , Plasmídeos/metabolismo , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Fatores de Transcrição , Fator de von Willebrand/metabolismoRESUMO
Epidermal growth factor receptor (EGFR) ligands are synthesized as type I membrane protein precursors exposed at the cell surface. Shedding of the ectodomain of these proteins is the way cells regulate the equilibrium between cell-associated and diffusible forms of these growth factors. Whereas the regulated shedding of transforming growth factor-alpha, HB-EGF, and amphiregulin precursors have been clearly established, regulation of full-length pro-EGF shedding has not been clearly demonstrated. Here, using both wild-type and M2 mutant CHO-K1 as well as HeLa cell lines transiently transfected with epitope-tagged rat pro-EGF expression plasmid, we demonstrate that these cells synthesize EGF as a high molecular weight membrane-associated precursor glycoprotein expressed at the cell surface. All cell lines are able to release the entire ectodomain of pro-EGF in the extracellular medium following juxtamembrane cleavage of the precursor once it is present at the cell surface. More significantly we clearly established that CHO-M2 and HeLa cells only constitutively release low levels of pro-EGF. This shedding is a regulated phenomenon in wild-type CHO cells where it can be induced by different agents such as phorbol 12-myristate 13-acetate (PMA), pervanadate, and serum but not by calcium ionophores. Using specific inhibitors as well as protein kinase C (PKC) depletion, PMA stimulation was shown to be completely dependent on PKC activation whereas pervanadate and serum stimulation were not. Regulated ectodomain shedding involves the activity of a zinc metalloprotease as determined by inhibition with phenantrolin and TAPI-2 and by the results obtained with the CHO-M2 shedding defective mutant cell line. Comparison of the ability of CHO and HeLa cell lines to shed pro-EGF and pro-TNF-alpha upon stimulation greatly suggests that TACE (ADAM 17) may not be the ectoprotease involved in the secretion of pro-EGF ectodomain and that this protease, which remains to be identified, shows a restricted cellular expression pattern.
Assuntos
Membrana Celular/metabolismo , Fator de Crescimento Epidérmico/química , Zinco/química , Proteínas ADAM , Proteína ADAM17 , Animais , Células CHO , Cálcio/metabolismo , Clonagem Molecular , Cricetinae , DNA Complementar/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Epitopos/química , Células HeLa , Humanos , Ionóforos/farmacologia , Cinética , Metaloendopeptidases/metabolismo , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Radioimunoensaio , Ratos , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/metabolismo , Vanadatos/farmacologiaRESUMO
Bone morphogenetic protein-1 (BMP-1) is a shorter spliced variant of mammalian tolloid (mTld), both of which cleave the C-propeptides of type I procollagen during the synthesis of extracellular matrix collagen fibrils. The fact that BMP-1 and mTld both exhibit procollagen C-proteinase (PCP) activity and that BMP-1 is the smaller variant might indicate that BMP-1 comprises the minimal required sequences for PCP activity. BMP-1 comprises a metalloproteinase domain, three CUB domains, and an epidermal growth factor (EGF)-like domain, which is located between the second and third CUB (complement components C1r/C1s, the sea urchin protein Uegf, and BMP-1) domains. In this study we showed the following. 1) The CUB1 domain is required for secretion of the molecule. Domain swapping experiments, in which CUB1 and other CUB domains were interchanged, resulted in retention of the proteins by cells. Therefore, CUB1 and its location immediately adjacent to the metalloproteinase domain are essential for secretion of the protein. 2) Mutants lacking the EGF-like and CUB3 domains exhibited full C-proteinase activity. In contrast, mutants lacking the CUB2 domain were poor C-proteinases. 3) Further studies showed that Glu-483 on the beta4-beta5 loop of CUB2 is essential for C-proteinase activity of BMP-1. In conclusion, the study showed that the minimal domain structure for PCP activity is considerably shorter than expected and comprises the metalloproteinase domain and the CUB1 and CUB2 domains of BMP-1.
Assuntos
Proteínas Morfogenéticas Ósseas/química , Proteínas Morfogenéticas Ósseas/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Western Blotting , Proteína Morfogenética Óssea 1 , Linhagem Celular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Fator de Crescimento Epidérmico/química , Deleção de Genes , Biblioteca Gênica , Ácido Glutâmico/química , Humanos , Mutagênese Sítio-Dirigida , Mutação , Placenta/metabolismo , Mutação Puntual , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de TempoRESUMO
Epidermal growth factor (EGF) regulates cell proliferation and differentiation by binding to the EGF receptor (EGFR) extracellular region, comprising domains I-IV, with the resultant dimerization of the receptor tyrosine kinase. In this study, the crystal structure of a 2:2 complex of human EGF and the EGFR extracellular region has been determined at 3.3 A resolution. EGFR domains I-III are arranged in a C shape, and EGF is docked between domains I and III. The 1:1 EGF*EGFR complex dimerizes through a direct receptor*receptor interaction, in which a protruding beta-hairpin arm of each domain II holds the body of the other. The unique "receptor-mediated dimerization" was verified by EGFR mutagenesis.
Assuntos
Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Aminoácidos Aromáticos/química , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cristalização , Cristalografia por Raios X , Dimerização , Dissulfetos/química , Humanos , Ligantes , Estrutura Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Selênio/química , Análise de Sequência de DNARESUMO
PURPOSE: To isolate mono-PEGylated epidermal growth factor (EGF) isoforms, identify the site of PEGylation, and evaluate the biologic activity of each isoform. METHODS: EGF was PEGylated with an NHS-PEG derivative (Mw 3,400). Mono-PEGylated EGF fraction was separated by gel-filtration HPLC and three mono-PEGylated EGF isoforms were purified by RP-HPLC. Tryptic digestion mapping of both EGF and mono-PEGylated EGF isoforms was performed to identify the PEGylation sites using RP-HPLC. The digested fragments were also analyzed by matrix-assisted laser desorption and ionization time of flight (MALDI-TOF) mass spectroscopy for further verification of the three PEG conjugation sites. The biologic activity of positional isoforms was evaluated by a cell proliferation assay and a receptor tyrosine kinase activity assay to determine the effect of PEGylation site on its activity. RESULTS: Mono-PEGylated EGF was composed of three positional isomers. Tryptic digestion mapping and MALDI-TOF analysis permitted the identification of the PEGylated site of the three isoforms at N-terminus. Lysine 28, and Lysine 48. PEG-N-terminus EGF, among the three positional isomers, showed the highest activity in a cell proliferation assay and in a receptor-binding assay. CONCLUSION: This study demonstrates that biologic activities of mono-PEGylated EGF isomers are highly dependent upon the site of PEGylation in EGF.