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1.
Biochem Biophys Res Commun ; 590: 163-168, 2022 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-34979317

RESUMO

Liquid-phase electron microscopy is highly desirable for observing biological samples in their native liquid state at high resolution. We developed liquid imaging approaches for biological cells using scanning electron microscopy. Novel approaches included scanning transmission electron imaging using a liquid-cell apparatus (LC-STEM), as well as correlative cathodoluminescence and electron microscopy (CCLEM) imaging. LC-STEM enabled imaging at a ∼2 nm resolution and excellent contrast for the precise recognition of localization, distribution, and configuration of individually labeled membrane proteins on the native cells in solution. CCLEM improved the resolution of fluorescent images down to 10 nm. Liquid SEM technologies will bring unique and wide applications to the study of the structure and function of cells and membrane proteins in their near-native states at the monomolecular level.


Assuntos
Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica de Varredura , Linhagem Celular Tumoral , Receptores ErbB/ultraestrutura , Fluorescência , Humanos
2.
Sci Rep ; 11(1): 5790, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707468

RESUMO

Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR-528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.


Assuntos
Cetuximab/metabolismo , Epitopos/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Animais , Ligação Competitiva , Células CHO , Cetuximab/química , Cetuximab/ultraestrutura , Cricetulus , Fator de Crescimento Epidérmico/metabolismo , Epitopos/química , Receptores ErbB/ultraestrutura , Temperatura Alta , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
3.
J Biol Chem ; 295(7): 1792-1814, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31879273

RESUMO

Single-pass membrane receptors contain extracellular domains that respond to external stimuli and transmit information to intracellular domains through a single transmembrane (TM) α-helix. Because membrane receptors have various roles in homeostasis, signaling malfunctions of these receptors can cause disease. Despite their importance, there is still much to be understood mechanistically about how single-pass receptors are activated. In general, single-pass receptors respond to extracellular stimuli via alterations in their oligomeric state. The details of this process are still the focus of intense study, and several lines of evidence indicate that the TM domain (TMD) of the receptor plays a central role. We discuss three major mechanistic hypotheses for receptor activation: ligand-induced dimerization, ligand-induced rotation, and receptor clustering. Recent observations suggest that receptors can use a combination of these activation mechanisms and that technical limitations can bias interpretation. Short peptides derived from receptor TMDs, which can be identified by screening or rationally developed on the basis of the structure or sequence of their targets, have provided critical insights into receptor function. Here, we explore recent evidence that, depending on the target receptor, TMD peptides cannot only inhibit but also activate target receptors and can accommodate novel, bifunctional designs. Furthermore, we call for more sharing of negative results to inform the TMD peptide field, which is rapidly transforming into a suite of unique tools with the potential for future therapeutics.


Assuntos
Integrinas/ultraestrutura , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/química , Sequência de Aminoácidos/genética , Receptores ErbB/química , Receptores ErbB/ultraestrutura , Humanos , Integrinas/química , Peptídeos/química , Conformação Proteica , Conformação Proteica em alfa-Hélice/genética , Mapas de Interação de Proteínas , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/ultraestrutura , Transdução de Sinais/genética
4.
Curr Drug Discov Technol ; 17(2): 203-212, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30306874

RESUMO

BACKGROUND: According to WHO, in 2017, about 90.5 million people suffered from cancer and about 8.8 million deaths occurred due to disease. Although the chemotherapeutic agents have decreased the mortality among the cancer patients but high toxicity and non-specific targets are still major drawbacks. Many researchers have identified linomide, a 4-hydroxy-2-quinolone derivative, as a lead molecule for the development of anticancer agents. With this background, we thought of the following objective. OBJECTIVE: The objective of this research work involves the synthesis of a series of N-(2-(4- hydroxy-2-oxo-1-phenyl-1,2-dihydroquinolin-3-yl)-2-oxoethyl)-N-alkyl substituted benzene sulfonamides IVa-d (1-3) by replacing the anilide moiety at the third position of linomide with sulfamoylacyl and also N-methyl by N-phenyl functionality. To perform in silico anticancer activity by using Molegro Virtual Docker (MVD-2013, 6.0) software and in vitro anticancer activity by MTT assay. METHODS: The starting material 4-hydroxy-1-phenylquinolin-2(1H)-one was treated with N-bromosuccinamide to yield compound II. Condensation of compound II with primary amines resulted in compounds IIIa-d, which, on coupling with substituted aromatic sulfonyl chlorides yield the title compounds IVa-d (1-3). RESULTS: All the synthesized compounds were satisfactorily characterized by spectral data. The results of docking revealed that the synthesized compounds exhibited well-conserved hydrogen bonds with one or more amino acid residues in the active pocket of EGFRK tyrosine kinase domain (PDB ID: 1m17). The MolDock Score of compound IVd-1 (-115.503) was the highest amongst those tested. The in vitro anticancer activity results showed that compound IVc-1 (R= - (CH2) 2-CH3 ; R'= -H) and IV d-1 (R= -CH2-C6H5; R'= -H) were found to be most potent against K562 cell line with an IC50 of 0.451 µM/ml and 0.455 µM/ml respectively. Compound IVd-1 also showed better potency against A549 cell line with IC50 value of 0.704 µM/ml. CONCLUSION: The results of in silico and in vitro anticancer activity are in agreement with each other. Compound IV d-1 was found to be most active of the series.


Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Hidroxiquinolinas/farmacologia , Neoplasias/tratamento farmacológico , Células A549 , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , Proliferação de Células , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Receptores ErbB/ultraestrutura , Humanos , Ligação de Hidrogênio , Hidroxiquinolinas/química , Hidroxiquinolinas/uso terapêutico , Concentração Inibidora 50 , Células K562 , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade
5.
J Comput Aided Mol Des ; 33(8): 745-757, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31494804

RESUMO

Protein kinases are an important class of enzymes that play an essential role in virtually all major disease areas. In addition, they account for approximately 50% of the current targets pursued in drug discovery research. In this work, we explore the generation of structure-based quantum mechanical (QM) quantitative structure-activity relationship models (QSAR) as a means to facilitate structure-guided optimization of protein kinase inhibitors. We explore whether more accurate, interpretable QSAR models can be generated for a series of 76 N-phenylquinazolin-4-amine inhibitors of epidermal growth factor receptor (EGFR) kinase by comparing and contrasting them to other standard QSAR methodologies. The QM-based method involved molecular docking of inhibitors followed by their QM optimization within a ~ 300 atom cluster model of the EGFR active site at the M062X/6-31G(d,p) level. Pairwise computations of the interaction energies with each active site residue were performed. QSAR models were generated by splitting the datasets 75:25 into a training and test set followed by modelling using partial least squares (PLS). Additional QSAR models were generated using alignment dependent CoMFA and CoMSIA methods as well as alignment independent physicochemical, e-state indices and fingerprint descriptors. The structure-based QM-QSAR model displayed good performance on the training and test sets (r2 ~ 0.7) and was demonstrably more predictive than the QSAR models built using other methods. The descriptor coefficients from the QM-QSAR models allowed for a detailed rationalization of the active site SAR, which has implications for subsequent design iterations.


Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Quinases/ultraestrutura , Relação Quantitativa Estrutura-Atividade , Domínio Catalítico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/ultraestrutura , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Proteínas Quinases/química , Teoria Quântica
6.
J Struct Biol ; 207(2): 225-233, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31163211

RESUMO

The combinatorial dimerization of the ErbB growth factor receptors (ErbB1- ErbB4) are critical for their function. Here, we have characterized the conformational dynamics of ErbB transmembrane homo-dimers and hetero-dimers by using a coarse-grain simulation framework. All dimers, except ErbB4-4 and ErbB1-4, exhibit at least two conformations. The reported NMR structures correspond to one of these conformations, representing the N-terminal active state in ErbB1-1 (RH2), ErbB2-2 (RH1) and ErbB4-4 (RH) homo-dimers and the LH dimer in ErbB3-3 homo-dimer, validating the computational approach. Further, we predict a right-handed ErbB3-3 dimer conformer that warrants experimental testing. The five hetero-dimers that have not yet been experimentally resolved display prominent right-handed dimers associating by the SmXXXSm motif. Our results provide insights into the constitutive signaling of ErbB4 after cleavage of the extracellular region. The presence of the inactive-like dimer conformers leading to symmetric kinase domains gives clues on the autoinhibition of the receptor dimers. The dimer states characterized here represent an important step towards understanding the combinatorial cross associations in the ErbB family.


Assuntos
Sequência de Aminoácidos/genética , Receptores ErbB/ultraestrutura , Multimerização Proteica , Motivos de Aminoácidos/genética , Receptores ErbB/química , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica/genética , Conformação Proteica , Transdução de Sinais/genética
7.
Methods ; 95: 86-93, 2016 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-26484734

RESUMO

The challenge of determining the architecture and geometry of oligomers of the epidermal growth factor receptor (EGFR) on the cell surface has been approached using a variety of biochemical and biophysical methods. This review is intended to provide a narrative of how key concepts in the field of EGFR research have evolved over the years, from the origins of the prevalent EGFR signalling dimer hypothesis through to the development and implementation of methods that are now challenging the conventional view. The synergy between X-ray crystallography and cellular fluorescence microscopy has become particularly important, precisely because the results from these two methods diverged and highlighted the complexity of the challenge. We illustrate how developments in super-resolution microscopy are now bridging this gap. Exciting times lie ahead where knowledge of the nature of the complexes can assist with the development of a new generation of anti-cancer drugs.


Assuntos
Membrana Celular/ultraestrutura , Cristalografia por Raios X/métodos , Receptores ErbB/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Regulação Alostérica , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Drosophila melanogaster/metabolismo , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Multimerização Proteica , Transdução de Sinais
8.
J Struct Biol ; 192(3): 539-544, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26522274

RESUMO

The discovery of genetic drivers of lung cancer in patient sub-groups has led to their use as predictive biomarkers and as targets for selective drug therapy. Some of the most important lung cancer drivers are mutations in the EGFR gene, for example, the exon 19 deletions and the L858R variant that confer sensitivity to the front line drugs erlotinib and gefitinib; the acquired T790M variants confer drug resistance and a poor prognosis. A challenge then in targeting EGFR is to produce drugs that inhibit both sensitising variants and resistance variants, leaving wild type protein in healthy cells unaffected. One such agent is AstraZeneca's "breakthrough" AZD9291 molecule that shows a 200-fold selectivity for T790M/L858R over wild type EGFR. Our X-ray crystal structure reveals the binding mode of AZD9291 to the kinase domain of wild type EGFR.


Assuntos
Acrilamidas/farmacologia , Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Acrilamidas/metabolismo , Compostos de Anilina/metabolismo , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/ultraestrutura , Cloridrato de Erlotinib/farmacologia , Gefitinibe , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Ligação Proteica/fisiologia , Quinazolinas/farmacologia
9.
Nat Commun ; 6: 7874, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26219252

RESUMO

Molecular trafficking within cells, tissues and engineered three-dimensional multicellular models is critical to the understanding of the development and treatment of various diseases including cancer. However, current tracking methods are either confined to two dimensions or limited to an interrogation depth of ∼15 µm. Here we present a three-dimensional tracking method capable of quantifying rapid molecular transport dynamics in highly scattering environments at depths up to 200 µm. The system has a response time of 1 ms with a temporal resolution down to 50 µs in high signal-to-noise conditions, and a spatial localization precision as good as 35 nm. Built on spatiotemporally multiplexed two-photon excitation, this approach requires only one detector for three-dimensional particle tracking and allows for two-photon, multicolour imaging. Here we demonstrate three-dimensional tracking of epidermal growth factor receptor complexes at a depth of ∼100 µm in tumour spheroids.


Assuntos
Receptores ErbB/metabolismo , Imageamento Tridimensional , Imagem Óptica , Transporte Proteico , Linhagem Celular Tumoral , Receptores ErbB/ultraestrutura , Humanos , Modelos Moleculares , Dinâmica não Linear
10.
Mol Cell Biol ; 35(17): 3083-102, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26124280

RESUMO

The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer.


Assuntos
Receptores ErbB/genética , Receptores ErbB/ultraestrutura , Sequência de Aminoácidos , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Cristalografia por Raios X , Ativação Enzimática/genética , Receptores ErbB/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Estrutura Terciária de Proteína , Deleção de Sequência/genética
11.
BMC Genomics ; 16 Suppl 5: S8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26041145

RESUMO

BACKGROUND: The epidermal growth factor receptor (EGFR) is a member of the ErbB family that is involved in a number of processes responsible for cancer development and progression such as angiogenesis, apoptosis, cell proliferation and metastatic spread. Malfunction in activation of protein tyrosine kinases has been shown to result in uncontrolled cell growth. The EGFR TK domain has been identified as suitable target in cancer therapy and tyrosine kinase inhibitors such as erlotinib have been used for treatment of cancer. Mutations in the region of the EGFR gene encoding the tyrosine kinase (TK) domain causes altered responses to EGFR TK inhibitors (TKI). In this paper we perform molecular dynamics simulations and PCA analysis on wild-type and mutant (T854A) structures to gain insight into the structural changes observed in the target protein upon mutation. We also report two novel inhibitors identified by combined approach of QSAR model development. RESULTS: The wild-type and mutant structure was observed to be stable for 26 ns and 24 ns respectively. In PCA analysis, the mutant structure proved to be more flexible than wild-type. We developed a 3D-QSAR model using 38 thiazolyl-pyrazoline compounds which was later used for prediction of inhibitory activity of natural compounds of ZINC library. The 3D-QSAR model was proved to be robust by the statistical parameters such as r2 (0.9751), q2(0.9491) and pred_r2(0.9525). CONCLUSION: Analysis of molecular dynamics simulations results indicate stability loss and increased flexibility in the mutant structure. This flexibility results in structural changes which render the mutant protein drug resistant against erlotinib. We report two novel compounds having high predicted inhibitory activity to EGFR TK domain with both wild-type and mutant structure.


Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/ultraestrutura , Cloridrato de Erlotinib/farmacologia , Humanos , Indóis/farmacologia , Isoflavonas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Simulação de Dinâmica Molecular , Mutação/genética , Naftiridinas/farmacologia , Análise de Componente Principal , Pirazóis/química , Relação Quantitativa Estrutura-Atividade
12.
Gene ; 539(1): 82-90, 2014 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-24491504

RESUMO

Epidermal growth factor receptor tyrosine kinase (EGFR-TK) is an attractive target for cancer therapy. Despite a number of effective EGFR inhibitors that are constantly expanding and different methods being employed to obtain novel compounds, the search for newer EGFR inhibitors is still a major scientific challenge. In the present study, a molecular docking and molecular dynamics investigation has been carried out with an ensemble of EGFR-TK structures against a synthetically feasible library of curcumin analogs to discover potent EGFR inhibitors. To resolve protein flexibility issue we have utilized 5 EGFR wild type crystal structures during docking as this gives improved possibility of identifying an active compound as compared to using a single crystal structure. We then identified five curcumin analogs representing different scaffolds that can serve as lead molecules. Finally, the 5 ns molecular dynamics simulation shows that knoevenagel condensate of curcumin specifically C29 and C30 can be used as starting blocks for developing effective leads capable of inhibiting EGFR.


Assuntos
Curcumina/análogos & derivados , Curcumina/metabolismo , Receptores ErbB/antagonistas & inibidores , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Antineoplásicos/farmacologia , Domínio Catalítico/genética , Cristalografia por Raios X , Descoberta de Drogas , Receptores ErbB/ultraestrutura , Humanos , Neoplasias/tratamento farmacológico , Relação Estrutura-Atividade
13.
Artigo em Inglês | MEDLINE | ID: mdl-24370847

RESUMO

Signal transduction is regulated by protein-protein interactions. In the case of the ErbB family of receptor tyrosine kinases (RTKs), the precise nature of these interactions remains a topic of debate. In this review, we describe state-of-the-art imaging techniques that are providing new details into receptor dynamics, clustering, and interactions. We present the general principles of these techniques, their limitations, and the unique observations they provide about ErbB spatiotemporal organization.


Assuntos
Receptores ErbB/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Transdução de Sinais/fisiologia , Receptores ErbB/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos
14.
Methods Cell Biol ; 117: 1-19, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24143969

RESUMO

This chapter presents a general approach for the application of spatial intensity distribution analysis (SpIDA) to pharmacodynamic quantification of receptor tyrosine kinase homodimerization in response to direct ligand activation or transactivation by G protein-coupled receptors. A custom graphical user interface developed for MATLAB is used to extract quantal brightness and receptor density information from intensity histograms calculated from single fluorescence microscopy images. This approach allows measurement of monomer/oligomer protein mixtures within subcellular compartments using conventional confocal laser scanning microscopy. Application of quantitative pharmacological analysis to data obtained using SpIDA provides a universal method for comparing studies between cell lines and receptor systems. In addition, because of its compatibility with conventional immunostaining approaches, SpIDA is suitable not only for use in recombinant systems but also for the characterization of mechanisms involving endogenous proteins. Therefore, SpIDA enables these biological processes to be monitored directly in their native cellular environment.


Assuntos
Receptores ErbB/metabolismo , Imagem Molecular/métodos , Neurônios/metabolismo , Receptor trkB/metabolismo , Receptores Dopaminérgicos/metabolismo , Software , Apomorfina/farmacologia , Linhagem Celular , Receptores ErbB/genética , Receptores ErbB/ultraestrutura , Imunofluorescência , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligantes , Microscopia Confocal , Microscopia de Fluorescência , Imagem Molecular/estatística & dados numéricos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Multimerização Proteica , Quinazolinas/farmacologia , Receptor trkB/genética , Receptor trkB/ultraestrutura , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/ultraestrutura , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , Ativação Transcricional , Tirfostinas/farmacologia
15.
Biochem J ; 453(3): 337-44, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23734957

RESUMO

Formation of an asymmetric dimer by the EGFR (epidermal growth factor receptor) kinase domains results in allosteric activation. Since this dimer does not readily form in solution, the EGFR kinase domain phosphorylates most peptide substrates with a relatively low catalytic efficiency. Peptide C is a synthetic peptide substrate of EGFR developed by others that is phosphorylated with a significantly higher catalytic efficiency, and we sought to understand the basis for this. Peptide C was found to increase EGFR kinase activity by promoting formation of the EGFR kinase domain asymmetric dimer. Activation of the kinase domain by Peptide C also enhances phosphorylation of other substrates. Aggregation of the EGFR kinase domain by Peptide C probably underlies activation, and Peptide C precipitates several other proteins. Peptide C was found to form fibrils independent of the presence of EGFR, and these fibrils may facilitate aggregation and activation of the kinase domain. These results establish that a peptide substrate of EGFR may increase catalytic activity by promoting kinase domain dimerization by an aggregation-mediated mechanism.


Assuntos
Receptores ErbB/metabolismo , Peptídeos/metabolismo , Cromatografia Líquida de Alta Pressão , Dimerização , Receptores ErbB/ultraestrutura , Immunoblotting , Microscopia Eletrônica de Transmissão , Fosforilação , Especificidade por Substrato
16.
Nat Struct Mol Biol ; 18(9): 984-9, 2011 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-21822280

RESUMO

To our knowledge, no structural study to date has characterized, in an intact receptor, the coupling of conformational change in extracellular domains through a single-pass transmembrane domain to conformational change in cytoplasmic domains. Here we examine such coupling, and its unexpected complexity, using nearly full-length epidermal growth factor receptor (EGFR) and negative-stain EM. The liganded, dimeric EGFR ectodomain can couple both to putatively active, asymmetrically associated kinase dimers and to putatively inactive, symmetrically associated kinase dimers and monomers. Inhibitors that stabilize the active or inactive conformation of the kinase active site, as well as mutations in the kinase dimer interface and a juxtamembrane phosphorylation site, shift the equilibrium among the three kinase association states. This coupling of one conformation of an activated receptor ectodomain to multiple kinase-domain arrangements reveals previously unanticipated complexity in transmembrane signaling and facilitates regulation of receptor function in the juxtamembrane and cytoplasmic environments.


Assuntos
Receptores ErbB/química , Domínio Catalítico , Dimerização , Receptores ErbB/genética , Receptores ErbB/ultraestrutura , Humanos , Ligantes , Mutação , Estrutura Terciária de Proteína
17.
Methods Mol Biol ; 680: 153-64, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21153380

RESUMO

A number of altered pathways in cancer cells depend on growth factor receptors. The amplification/alteration of the epidermal growth factor receptor (EGFR) has been shown to play a significant role in enhancing tumor burden in a number of tumors, including malignant glioblastomas (GBM). To dissect the role of EGFR expression in tumor progression in mouse models of cancer and ultimately evaluate targeted therapies, it is necessary to visualize the dynamics of EGFR in real time in vivo. Non-invasive imaging based on quantitative and qualitative changes in light emission by fluorescent and bioluminescent markers offers a huge potential to facilitate drug development. Multiple approaches could be used to follow a molecular target or pathway with the fusion of a bioluminescent-fluorescent marker. This unit describes a protocol for simultaneously imaging EGFR activity and progression of GBM in a mouse model. Human glioma cells transduced with lentiviral vectors bearing different combinations of fluorescent and bioluminescent proteins either fused to EGFR or expressed alone can be grown as monolayers and maintained over several passages. The unit begins with a method for transducing glioma cells with lentiviral vectors for stable expression of these fluorescent and bioluminescent markers in vitro, followed by transplantation of engineered glioma cells in mice, and, finally, sequential bioluminescent imaging of EGFR expression and GBM progression in mice. The protocol details characterization of engineered glioma cells in culture, surgical preparation, craniotomy, cell implantation, animal recovery, and imaging procedures to study kinetics of EGFR expression and GBM progression.


Assuntos
Receptores ErbB/metabolismo , Receptores ErbB/ultraestrutura , Glioblastoma/patologia , Microscopia de Fluorescência/métodos , Engenharia Tecidual/métodos , Animais , Transplante de Células/métodos , Vetores Genéticos/genética , Humanos , Lentivirus , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Nus , Transdução Genética
18.
Acta Biochim Biophys Sin (Shanghai) ; 42(11): 763-70, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20929928

RESUMO

The molecular mechanism underlying epidermal growth factor receptor (EGFR) localization in mitochondria remains largely unknown. Using immune electron microscopy, we validated that EGFR could be localized on either the outer or the inner membrane of mitochondria. Mutant receptor lacked amino acids 646-660 was flawed in migration onto the organelles, whereas the mutated receptor with a defective endocytosis showed a greater capability of moving onto mitochondria upon stimulation of epidermal growth factor (EGF). Gefitinib, an inhibitor of EGFR kinase, inhibited the receptor endocytosis after short time of treatment, yet, only reduced cell viability as well as the amount of mitochondrial EGFR after longer time of exposure. Moreover, the content of mitochondrial EGFR transfer was decreased when the cells were exposed to the apoptotic inducer etoposide. EGF-induced programmed cell death usually coincided with a decline in mitochondrial EGFR. These data indicated that the mitochondrial-localized EGFR is independent of its internalization and may be correlated with cell survival and participate in the ligand-induced programmed cell death.


Assuntos
Endocitose , Receptores ErbB/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/genética , Receptores ErbB/ultraestrutura , Etoposídeo/farmacologia , Gefitinibe , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Immunoblotting , Microscopia Imunoeletrônica , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/ultraestrutura , Mutação , Transporte Proteico/efeitos dos fármacos , Quinazolinas/farmacologia
19.
Opt Express ; 16(25): 20258-65, 2008 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-19065164

RESUMO

We combine single molecule fluorescence orientation imaging with single-pair fluorescence resonance energy transfer microscopy, using a total internal reflection microscope. We show how angles and FRET efficiencies can be determined for membrane proteins at the single molecule level and provide data from the epidermal growth factor receptor system in cells.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Membrana Celular/metabolismo , Cristalografia/métodos , Receptores ErbB/metabolismo , Receptores ErbB/ultraestrutura , Transferência Ressonante de Energia de Fluorescência/instrumentação , Técnicas de Sonda Molecular/instrumentação , Linhagem Celular Tumoral , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Conformação Proteica
20.
Annu Rev Biophys ; 37: 353-73, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18573086

RESUMO

High-resolution X-ray crystal structures determined in the past six years dramatically influence our view of ligand-induced activation of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. Ligand binding to the extracellular region of EGFR promotes a major domain reorganization, plus local conformational changes, that are required to generate an entirely receptor-mediated dimer. In this activated complex the intracellular kinase domains associate to form an asymmetric dimer that supports the allosteric activation of one kinase. These models are discussed with emphasis on recent studies that add details or bolster the generality of this view of activation of this family of receptors. The EGFR family is implicated in several disease states, perhaps most notably in cancers. Activating tumor mutations have been identified in the intracellular and extracellular regions of EGFR. The impact of these tumor mutations on the understanding of EGFR activation and of its inhibition is discussed.


Assuntos
Regulação da Expressão Gênica/fisiologia , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Sítios de Ligação , Simulação por Computador , Receptores ErbB/química , Receptores ErbB/metabolismo , Receptores ErbB/ultraestrutura , Ligação Proteica , Relação Estrutura-Atividade
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