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1.
MAbs ; 13(1): 1893427, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33682619

RESUMO

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure-function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure-function relationship of terminal galactose to complement activation in mAb therapeutics.


Assuntos
Anticorpos Monoclonais/farmacologia , Ativação do Complemento/efeitos dos fármacos , Complemento C1q/agonistas , Citotoxicidade Imunológica/efeitos dos fármacos , Galactose/metabolismo , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/farmacologia , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células CHO , Complemento C1q/metabolismo , Cricetulus , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Cinética , Modelos Biológicos , Multimerização Proteica , Relação Estrutura-Atividade
2.
J Pharm Sci ; 110(4): 1652-1660, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33383056

RESUMO

Identification of critical quality attributes (CQAs) is an important step for development of biopharmaceuticals with intended performance. An accurate CQA assessment is needed to ensure product quality and focusing on development efforts where control is needed. The assignment of criticality is based on safety and efficacy. Efficacy is related to PK and bioactivity. Here, we developed a novel approach based on antibody-antigen complex structure and modeling as a complementary method for bioactivity assessment. To validate this approach, common product related quality attributes and mutagenesis data from several IgGs were assessed using available antibody-antigen complex structures, and results were compared with experimental data from bioactivity or binding affinity measurements. A stepwise evaluation scheme for structural based analysis is proposed; based on systematic assessment following the scheme, good correlation has been observed between structural analysis and experimental data. This demonstrates that such an approach can be applied as a complementary tool for bioactivity assessment. Main applications are 1) To decouple multiple attributes to achieve amino acid resolution for bioactivity assessment, 2) To assess bioactivity of attributes that cannot be experimentally generated, 3) To provide molecular mechanism for experimental observation and understand structure function relationship. Examples are provided to illustrate these applications.


Assuntos
Produtos Biológicos , Controle de Qualidade , Projetos de Pesquisa
3.
J Pharm Sci ; 109(12): 3690-3696, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32910947

RESUMO

A localized positive charge on IgG (referred to as a "charge patch") shows an adverse effect on pharmacokinetics (PK), so it would seem to be best practice to avoid charge patches during the discovery stage and closely monitor charge interactions during the development process. In certain circumstances, however, charge patches are required for target binding, in which case completely removing charge patches is not feasible. Therefore, quantitative measurement of a charge patch and its impact on PK is critical to the success of therapeutic antibody development. In this article, we generated mutations of a recombinant human antibody (referred to as mAb1) with disrupted charge patches to investigate how charge patches on IgG antibodies impact both target-binding affinity and PK-related factors. We conclude that it is important to modulate the size of the charge patch in order to balance target-binding affinity and PK.


Assuntos
Regiões Determinantes de Complementaridade , Imunoglobulina G , Regiões Determinantes de Complementaridade/genética , Humanos
4.
Anal Chem ; 92(1): 1582-1588, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31815436

RESUMO

Bispecific antibodies (BsAbs) have drawn increasing interest in the biopharmaceutical industry due to their advantage to bind two distinct antigens simultaneously. The knob-into-hole approach is an effective way to produce bispecific antibodies by driving heterodimerization with mutations in the CH3 domain of each half antibody. To better understand the conformational impact by the knob and hole mutations, we combined size-exclusion chromatography (SEC), differential scanning calorimetry (DSC), and hydrogen-deuterium exchange mass spectrometry (H/D exchange MS), to characterize the global and peptide-level conformational changes. We found no significant alteration in structure or conformational dynamics induced by the knob-into-hole framework, and the conformational stability is similar to the wild-type (WT) IgG4 molecules (except for some small difference in the CH3 domain) expressed in E. coli. Functional studies including antigen-binding and neonatal fragment crystallizable (Fc) receptor (FcRn) binding demonstrated no difference between the knob-into-hole and WT IgG4 molecules in E. coli.


Assuntos
Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Varredura Diferencial de Calorimetria , Cromatografia em Gel , Escherichia coli/genética , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Imunoglobulina G/química , Modelos Moleculares , Mutação , Conformação Proteica
5.
MAbs ; 11(7): 1245-1253, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31348721

RESUMO

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients' T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.


Assuntos
Anticorpos Biespecíficos/metabolismo , Técnicas Imunológicas , Imunoterapia/métodos , Neoplasias/terapia , Linfócitos T/imunologia , Anticorpos Biespecíficos/uso terapêutico , Antígenos/metabolismo , Citotoxicidade Imunológica , Genes Reporter , Humanos , Células Jurkat , Luciferases/genética , Ativação Linfocitária , Neoplasias/imunologia
6.
Sci Rep ; 9(1): 3900, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846832

RESUMO

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit a patient's T cells to kill cancer cells. There are increasing numbers of TBDs in clinical trials, demonstrating their widely recognized therapeutic potential. Due to the fact that TDBs engage and activate T cells via an anti-CD3 (aCD3) arm, aCD3 homodimer (aCD3 HD) and high-molecular-weight species (HMWS) are product-related impurities that pose a potential safety risk by triggering off-target T-cell activation through bivalent engagement and dimerization of T-cell receptors (TCRs). To monitor and control the level of unspecific T-cell activation, we developed a sensitive and quantitative T-cell-activation assay, which can detect aCD3 HD in TDB drug product by exploiting its ability to activate T cells in the absence of target cells. This assay provides in-vivo-relevant off-target T-cell-activation readout. Furthermore, we have demonstrated that this assay can serve as a platform assay for detecting T-cell-activating impurities across a broad spectrum of aCD3 bispecific molecules. It therefore has the potential to significantly benefit many T-cell-recruiting bispecific programs.


Assuntos
Anticorpos Biespecíficos/imunologia , Bioensaio , Linfócitos T/imunologia , Humanos , Ativação Linfocitária/imunologia
7.
J Immunol Methods ; 448: 74-79, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28579366

RESUMO

Dual specific antibodies and bispecific antibodies that recognize two different antigen targets are currently being regarded as very effective therapeutics for complex human diseases. While effective, designing and developing a bioassay strategy for dual specific antibodies that is reflective of the mechanism of action (MoA) and also measures the dual activities of antibodies pose unique and exciting challenges. An important question asked while developing a bioassay for dual specific antibodies is, "How many bioassays will be needed, one bioassay or two separate bioassays?" Here we present an approach of using one bioassay for a dual specific antibody that targets two receptors in signaling pathways. The presented assay is able to measure the antibody effects on both target bindings, which would not be achievable using two separate assays. Furthermore, this assay can detect changes in the binding of either target, which impact overall efficacy of the antibody. Its improved sensitivity enables substituting two binding assays with this one bioassay for lot release and stability testing to measure any changes on either target binding, ensuring consistency between lots. This is a single-bioassay approach for a dual specific antibody that is MoA reflective of the intended therapeutic indication. The demonstrated assay development and bridging study strategy for this bioassay for a dual specific mAb1 could be applicable to the other dual specific, bispecific antibodies, and antibodies used for combination therapy.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos , Bioensaio/métodos , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/metabolismo , Especificidade de Anticorpos , Antígenos/imunologia , Antígenos/metabolismo , Sítios de Ligação de Anticorpos , Linhagem Celular , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Humanos , Mutação , Ligação Proteica , Receptor ErbB-3/genética , Receptor ErbB-3/imunologia , Receptor ErbB-3/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos
8.
Biotechnol Prog ; 33(3): 786-794, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28188688

RESUMO

Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786-794, 2017.


Assuntos
Anticorpos Monoclonais/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Glicina/metabolismo , Glicosilação , Cadeias Pesadas de Imunoglobulinas/metabolismo , Lisina/metabolismo , Prolina/metabolismo , RNA Mensageiro/metabolismo
9.
J Pharm Sci ; 105(7): 2066-72, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27262204

RESUMO

Due to their potential influence on stability, pharmacokinetics, and product consistency, antibody charge variants have attracted considerable attention in the biotechnology industry. Subtle to significant differences in the level of charge variants and new charge variants under various cell culture conditions are often observed during routine manufacturing or process changes and pose a challenge when demonstrating product comparability. To explore potential solutions to control charge heterogeneity, monoclonal antibodies (mAbs) with native, wild-type C-termini, and mutants with C-terminal deletions of either lysine or lysine and glycine were constructed, expressed, purified, and characterized in vitro and in vivo. Analytical and physiological characterization demonstrated that the mAb mutants had greatly reduced levels of basic variants without decreasing antibody biologic activity, structural stability, pharmacokinetics, or subcutaneous bioavailability in rats. This study provides a possible solution to mitigate mAb heterogeneity in C-terminal processing, improve batch-to-batch consistency, and facilitate the comparability study during process changes.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Animais , Anticorpos Monoclonais/genética , Disponibilidade Biológica , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Estabilidade de Medicamentos , Glicina/química , Injeções Subcutâneas , Focalização Isoelétrica , Lisina/química , Masculino , Mutação , Ratos , Ratos Sprague-Dawley
10.
J Immunol Methods ; 420: 31-7, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25837414

RESUMO

IgG antibodies are important pharmaceutical molecules that successfully treat a variety of human diseases. The neonatal Fc receptor (FcRn) interacts with IgG Fc in the CH2-CH3 domain and plays a key role in IgG antibody homeostasis and affects its pharmacokinetic properties. An in vitro FcRn binding assay could be a highly valuable complementary tool to assess IgG antibody pharmacokinetics in IgG engineering and screening during the early optimization stage. In addition, it could be useful in biological characterization studies for antibody minor variants, process optimization, and comparability study at later stages of antibody development. Here we developed a homogeneous AlphaScreen-based FcRn assay to assess the binding of FcRn to IgG antibody in vitro. The assay is found to be accurate, precise, specific, and simple: donor beads loaded with FcRn and acceptor beads loaded with IgG1 mAb1 are mixed together with sample IgG at various dilutions and incubated for 1h before acquiring data with a fluorescence reader. This assay can run up to four samples per plate in 2h, which is time and cost effective compared with other FcRn binding methods such as cell-based fluorescent-activated cell scan and surface plasma resonance. Our data demonstrated that this assay is suitable for assessing the FcRn binding in vitro and provides a platform approach that can be readily applied to various antibodies.


Assuntos
Anticorpos Monoclonais Murinos/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoglobulina G/imunologia , Receptores Fc/imunologia , Animais , Anticorpos Monoclonais Murinos/química , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoensaio , Imunoglobulina G/química , Camundongos , Receptores Fc/química , Receptores Fc/genética
11.
Nan Fang Yi Ke Da Xue Xue Bao ; 34(8): 1171-5, 2014 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-25176089

RESUMO

OBJECTIVE: To observe the effect of N-acetylcysteine (NAC) on intestine injury induced by cardiopulmonary bypass (CPB) in rats. METHODS: Thirty-two rats were randomly divided into sham-operated group, NAC control group, CPB model group, and CPB plus NAC treatment group (n=8). In the latter two groups, the rats were subjected to CPB for 1 h. The rats received intraperitoneal injections of normal saline or NAC (0.5 g/kg) as appropriate for 3 successive days prior to CPB, and those in CPB plus NAC group were given NAC (100 mg/kg) in CPB prime followed by infusion at 20 mgsol;(kg·h) until the cessation of CPB. Intestinal and blood samples were collected 2 h after CPB for pathological analysis and measurement of intestinal concentrations of malondialdehyde (MDA), tumor necrosis factor (TNF)-α, interlukin (IL)-6 and activity of superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) and serum levels of diamine oxidase (DAO). RESULTS: Evident oxidative stress and pathological damages of the intestines were observed in rats after CPB. NAC treatment obviously alleviated intestinal damages induced by CPB, decreased the levels of intestinal MDA, TNF-α, IL-6 and serum DAO and increased activity of SOD, GSH, and GSH-Px in the intestines. CONCLUSION: Perioperative NAC treatment can alleviate intestinal injury induced by CPB in rats by suppressing oxidative stress and inflammatory response.


Assuntos
Acetilcisteína/farmacologia , Ponte Cardiopulmonar/efeitos adversos , Intestinos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Inflamação/tratamento farmacológico , Interleucina-6/metabolismo , Intestinos/fisiopatologia , Malondialdeído/metabolismo , Ratos , Superóxido Dismutase/metabolismo , Fator de Necrose Tumoral alfa
12.
J Immunol Methods ; 387(1-2): 51-6, 2013 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-23063556

RESUMO

Dual specificity antibodies such as bispecific and Dual Action Fab (DAF) antibodies are emerging therapeutic products with powerful therapeutic potential. New bioassay formats are needed in order to monitor their dual biological activities. Here we describe the optimization and development of a "bridging" binding method in semi-homogenous (SH) assay format for a bi-specific antibody. In the SH assay format, the antigen and secondary antibodies are mixed directly with the sample solution. The bound complex is then captured onto a microtiter plate coated with the other antigen and subsequently detected by colorimetric methods. We demonstrated that an SH assay achieved comparable dynamic range, quantitation, and specificity to the conventional assay format where each reagent is added sequentially followed by separate washing and incubation steps. The SH assay requires fewer wash steps and the overall assay time is shortened by half, which translates to improved efficiency without any requirement for new equipment and reagents. Using the SH assay format, we also demonstrated that a DAF antibody which can bind to two different antigens with either arm could bind both antigens simultaneously in vitro. The SH assay format has broad application as an assay platform for assessing antigen binding properties efficiently.


Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Imunoensaio/métodos , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Reações Antígeno-Anticorpo/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Modelos Imunológicos , Modelos Moleculares , Ligação Proteica/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes
13.
Sci Rep ; 2: 743, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23077727

RESUMO

Germline/embryonic-specific genes have been found to be activated in somatic tumors. In this study, we further showed that cells functioning as germline could be present in mouse fibrosarcoma cells (L929 cell line). Early germline-like cells spontaneously appeared in L929 cells and further differentiated into oocyte-like cells. These germline-like cells can, in turn, develop into blastocyst-like structures in vitro and cause teratocarcinomas in vivo, which is consistent with natural germ cells in function. Generation of germline-like cells from somatic tumors might provide a novel way to understand why somatic cancer cells have strong features of embryonic/germline development. It is thought that the germline traits of tumors are associated with the central characteristics of malignancy, such as immortalization, invasion, migration and immune evasion. Therefore, germline-like cells in tumors might provide potential targets to tumor biology, diagnosis and therapy.


Assuntos
Fibrossarcoma/genética , Células Germinativas/fisiologia , Células-Tronco/fisiologia , Teratocarcinoma/genética , Animais , Linhagem Celular Tumoral , Fibrossarcoma/patologia , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/citologia , Camundongos , Camundongos Endogâmicos C3H , Células-Tronco/citologia
14.
Dev Dyn ; 241(3): 627-37, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22275192

RESUMO

BACKGROUND: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown. RESULTS: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro. CONCLUSIONS: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning.


Assuntos
Padronização Corporal , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/metabolismo , Polidactilia/genética , Extremidade Superior/embriologia , Animais , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transdução de Sinais , Deformidades Congênitas das Extremidades Superiores/genética , Proteína GLI1 em Dedos de Zinco
15.
J Biol Chem ; 287(8): 5891-7, 2012 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-22203673

RESUMO

Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.


Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Estudos de Viabilidade , Humanos , Radical Hidroxila/farmacologia , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Dados de Sequência Molecular , Mutação , Estabilidade Proteica , Proteólise/efeitos dos fármacos
16.
Mol Biol Cell ; 20(23): 4920-31, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19812254

RESUMO

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Membrana/metabolismo , Músculo Esquelético/citologia , Transdução de Sinais/fisiologia , Animais , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Desmina/metabolismo , Embrião de Mamíferos , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Morfogênese , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Mutação , Mioblastos/citologia , Mioblastos/metabolismo , Miogenina/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Netrinas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Nat Cell Biol ; 10(9): 1062-8, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19160486

RESUMO

Cell spreading, adhesion and remodelling of the extracellular matrix (ECM) involve bi-directional signalling and physical linkages between the ECM, integrins and the cell cytoskeleton. The actin-binding proteins talin1 and 2 link ligand-bound integrins to the actin cytoskeleton and increase the affinity of integrin for the ECM. Here we report that depletion of talin2 in talin1-null (talin1(-/-)) cells did not affect the initiation of matrix-activated spreading or Src family kinase (SFK) activation, but abolished the ECM-integrin-cytoskeleton linkage and sustained cell spreading and adhesion. Specifically, focal adhesion assembly, focal adhesion kinase (FAK) signalling and traction force generation on substrates were severely affected. The talin1 head domain restored beta1 integrin activation but only full-length talin1 restored the ECM-cytoskeleton linkage and normal cytoskeleton organization. Our results demonstrate three biochemically distinct steps in fibronectin-activated cell spreading and adhesion: (1) fibronectin-integrin binding and initiation of spreading, (2) fast cell spreading and (3) focal adhesion formation and substrate traction. We suggest that talin is not required for initial cell spreading. However, talin provides the important mechanical linkage between ligand-bound integrins and the actin cytoskeleton required to catalyse focal adhesion-dependent pathways.


Assuntos
Movimento Celular , Fibroblastos/citologia , Integrina beta1/metabolismo , Talina/deficiência , Actomiosina/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibronectinas/farmacologia , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/enzimologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fosfotirosina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
18.
Cell ; 128(3): 561-75, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17289574

RESUMO

Cell motility proceeds by cycles of edge protrusion, adhesion, and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction, and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process.


Assuntos
Actinas/metabolismo , Adesão Celular , Miosinas/metabolismo , Pseudópodes/química , Animais , Movimento Celular , Fibroblastos/citologia , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Periodicidade , Polímeros/metabolismo , Pseudópodes/ultraestrutura
19.
Biophys J ; 91(10): 3907-20, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16920834

RESUMO

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.


Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Fibroblastos/fisiologia , Mecanotransdução Celular/fisiologia , Proteínas Motores Moleculares/fisiologia , Miosina não Muscular Tipo IIA/fisiologia , Animais , Células Cultivadas , Camundongos , Músculo Esquelético/fisiologia , Estresse Mecânico
20.
Biophys J ; 90(5): 1804-9, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16339875

RESUMO

Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.


Assuntos
Matriz Extracelular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Integrina alfaVbeta3/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Elasticidade , Dureza , Camundongos , Proteínas Tirosina Fosfatases Classe 4 Semelhantes a Receptores , Estresse Mecânico
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