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1.
Stem Cells ; 35(3): 626-640, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28009074

RESUMO

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Membrana/imunologia , Células-Tronco Pluripotentes/metabolismo , Animais , Antígenos de Superfície/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Autorrenovação Celular , Regulação para Baixo/genética , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Fator 3 de Transcrição de Octâmero/metabolismo
2.
J Biol Chem ; 288(1): 59-68, 2013 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-23166326

RESUMO

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.


Assuntos
Fator de Crescimento Insulin-Like II/química , Neoplasias/metabolismo , Animais , Proliferação de Células , Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Glicosilação , Células HEK293 , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/química , Fator de Crescimento Insulin-Like I/química , Espectrometria de Massas/métodos , Camundongos , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/química , Transdução de Sinais
3.
J Proteome Res ; 12(7): 3104-16, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23692254

RESUMO

Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bisanilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose-supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad-spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high-confidence phosphosites on 183 kinases, ∼10% of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2, and PRKDC. Therefore, CTx-0294885 represents a powerful new reagent for analysis of kinome signaling networks that may facilitate development of targeted therapeutic strategies. Proteomics data have been deposited to the ProteomeXchange Consortium ( http://proteomecentral.proteomexchange.org ) via the PRIDE partner repository with the data set identifier PXD000239.


Assuntos
Fosfotransferases/isolamento & purificação , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Pirimidinas/química , ortoaminobenzoatos/química , Linhagem Celular , Cromatografia Líquida/métodos , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Espectrometria de Massas em Tandem/métodos
4.
Growth Factors ; 30(5): 310-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22856597

RESUMO

Ligand-mediated activation of ErbB3 and ErbB4 is implicated in the pathogenesis of several human malignancies including cancer of the ovary and melanoma. We have used the broad ErbB ligand specificity of ErbB4 to assemble and express an ErbB4 fusion protein comprising the first 497 amino acids of the mature ErbB4 ectodomain fused to the human IgG Fc constant region. The purified fusion protein, designated sErbB4.497.Fc, binds the ErbB receptor ligands betacellulin and heregulin-ß1 (HRG-ß1) with high affinity (K(D) = 130 pM), an increase in affinity of 10- to 20-fold, respectively, compared with sErbB4.615.Fc. sErbB4.497.Fc inhibited ligand-stimulated phosphorylation of epidermal growth factor receptor and ErbB2, and blocked HRG-ß1 activation of the IKB/MAP/JNK/AKT signalling pathways. sErbB4.497.Fc inhibited HRG-ß1-stimulated proliferation in MCF7 cells. In a mouse tumour xenograft model, sErbB4.497.Fc as a monotherapy modestly inhibited the growth of MDA-MB-231 breast cancer cells. sErbB4.497.Fc may be useful in an adjuvant setting in combination with conventional therapeutic agents.


Assuntos
Receptores ErbB/metabolismo , Neuregulina-1/antagonistas & inibidores , Neuregulina-1/metabolismo , Receptores Fc/metabolismo , Animais , Betacelulina , Neoplasias da Mama/tratamento farmacológico , Células CHO , Linhagem Celular , Cricetinae , Receptores ErbB/genética , Receptores ErbB/uso terapêutico , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células MCF-7 , Melanoma/patologia , Camundongos , Neoplasias Ovarianas/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-4 , Receptores Fc/genética , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nature ; 443(7108): 218-21, 2006 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-16957736

RESUMO

The insulin receptor is a phylogenetically ancient tyrosine kinase receptor found in organisms as primitive as cnidarians and insects. In higher organisms it is essential for glucose homeostasis, whereas the closely related insulin-like growth factor receptor (IGF-1R) is involved in normal growth and development. The insulin receptor is expressed in two isoforms, IR-A and IR-B; the former also functions as a high-affinity receptor for IGF-II and is implicated, along with IGF-1R, in malignant transformation. Here we present the crystal structure at 3.8 A resolution of the IR-A ectodomain dimer, complexed with four Fabs from the monoclonal antibodies 83-7 and 83-14 (ref. 4), grown in the presence of a fragment of an insulin mimetic peptide. The structure reveals the domain arrangement in the disulphide-linked ectodomain dimer, showing that the insulin receptor adopts a folded-over conformation that places the ligand-binding regions in juxtaposition. This arrangement is very different from previous models. It shows that the two L1 domains are on opposite sides of the dimer, too far apart to allow insulin to bind both L1 domains simultaneously as previously proposed. Instead, the structure implicates the carboxy-terminal surface of the first fibronectin type III domain as the second binding site involved in high-affinity binding.


Assuntos
Dobramento de Proteína , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Cristalografia por Raios X , Dimerização , Fragmentos Fab das Imunoglobulinas/imunologia , Microscopia Eletrônica , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor de Insulina/imunologia , Receptor de Insulina/ultraestrutura
6.
Growth Factors ; 27(3): 141-54, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19333814

RESUMO

A number of therapeutic strategies including small molecule tyrosine kinase inhibitors and monoclonal antibodies have been developed to target the epidermal growth factor receptor (EGFR) signalling axis for the treatment of cancer. To date, the focus of therapeutic intervention has been the EGFR itself. In the current study, we have assembled and expressed in mammalian cells a soluble, EGFR ligand trap comprising the first 501 amino acids of the mature EGFR sequence fused in-frame with a human IgG Fc domain. The fusion protein, designated sEGFR501.Fc, was secreted as a 220 kDa disulphide-linked homodimer that exhibited high affinity (0.4-8 nM) in competition assays for a number of EGFR ligands including EGF and transforming growth factor-alpha (TGF-alpha). sEGFR501.Fc inhibited EGF-stimulated tyrosine phosphorylation of the EGFR of the lung cancer cell lines A549 and H1437, and inhibited and blocked the proliferation of H1437 cells. Administration of sEGFR501.Fc to mice bearing human tumour xenografts derived from A431 (epidermoid carcinoma) and DU145 (androgen-independent prostate cancer) tumour cell lines resulted in modest retardation of tumour growth. These results provide proof-in-principle that using high affinity soluble receptors is a viable method for inhibiting multi-ligand systems, and the impetus to optimize this approach and develop reagents with greater affinity and broader specificity.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Fosforilação/efeitos dos fármacos , Tirosina/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
J Immunol Methods ; 444: 29-35, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28216237

RESUMO

The ferret is an established animal model for a number of human respiratory viral infections, such as influenza virus and more recently, Ebola virus. However, a paucity of immunological reagents has hampered the study of cellular immune responses. Here we describe the development and characterisation of a novel monoclonal antibody (mAb) against the ferret CD4 antigen and the characterisation of ferret CD4 T lymphocytes. Recombinant production and purification of the ferret CD4 ectodomain soluble protein allowed hybridoma generation and the generation of a mAb (FeCD4) showing strong binding to ferret CD4 protein and lymphoid cells by flow cytometry. FeCD4 bound to its cognate antigen post-fixation with paraformaldehyde (PFA) which is routinely used to inactivate highly pathogenic viruses. We have also used FeCD4 in conjunction with other immune cell markers to characterise ferret T cells in both primary and secondary lymphoid organs. In summary, we have developed an important reagent for the study of cellular immunological responses in the ferret model of infectious disease.


Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Furões/imunologia , Imunidade Celular , Tecido Linfoide/imunologia , Animais , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Separação Celular/métodos , Concanavalina A/farmacologia , ELISPOT , Furões/genética , Furões/metabolismo , Citometria de Fluxo , Hibridomas , Interferon gama/imunologia , Interferon gama/metabolismo , Ativação Linfocitária , Tecido Linfoide/citologia , Tecido Linfoide/metabolismo , Fenótipo , Ligação Proteica , Especificidade da Espécie , Transfecção
8.
J Biomol Screen ; 16(10): 1196-205, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22086725

RESUMO

Epigenetic aberrations are increasingly regarded as key factors in cancer progression. Recently, deregulation of histone acetyltransferases (HATs) has been linked to several types of cancer. Monocytic leukemia zinc finger protein (MOZ) is a member of the MYST family of HATs, which regulate gene expression in cell proliferation and differentiation. Deregulation of these processes through constitutively active MOZ fusion proteins gives rise to the formation of leukemic stem cells, rendering MOZ an excellent target for treating myeloid leukemia. The authors implemented a hit discovery campaign to identify small-molecule inhibitors of MOZ-HAT activity. They developed a robust, homogeneous assay measuring the acetylation of synthetic histone peptides. In a primary screening campaign testing 243 000 lead-like compounds, they identified inhibitors from several chemical classes. Secondary assays were used to eliminate assay-interfering compounds and prioritize confirmed hits. This study establishes a new high-throughput assay for HAT activity and could provide the foundation for the development of a new class of drugs for the treatment of leukemias.


Assuntos
Epigênese Genética/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Histona Acetiltransferases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Humanos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas
9.
Mol Cancer Ther ; 9(6): 1809-19, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20515953

RESUMO

Elevated expression of insulin-like growth factor-II (IGF-II) is frequently observed in a variety of human malignancies, including breast, colon, and liver cancer. As IGF-II can deliver a mitogenic signal through both IGF-IR and an alternately spliced form of the insulin receptor (IR-A), neutralizing the biological activity of this growth factor directly is a potential alternative option to IGF-IR-directed agents. Using a Fab-displaying phage library and a biotinylated precursor form of IGF-II (1-104 amino acids) as a target, we isolated Fabs specific for the E-domain COOH-terminal extension form of IGF-II and for mature IGF-II. One of these Fabs that bound to both forms of IGF-II was reformatted into a full-length IgG, expressed, purified, and subjected to further analysis. This antibody (DX-2647) displayed a very high affinity for IGF-II/IGF-IIE (K(D) value of 49 and 10 pmol/L, respectively) compared with IGF-I (approximately 10 nmol/L) and blocked binding of IGF-II to IGF-IR, IR-A, a panel of insulin-like growth factor-binding proteins, and the mannose-6-phosphate receptor. A crystal complex of the parental Fab of DX-2647 bound to IGF-II was resolved to 2.2 A. DX-2647 inhibited IGF-II and, to a lesser extent, IGF-I-induced receptor tyrosine phosphorylation, cellular proliferation, and both anchorage-dependent and anchorage-independent colony formation in various cell lines. In addition, DX-2647 slowed tumor progression in the Hep3B xenograft model, causing decreased tumoral CD31 staining as well as reduced IGF-IIE and IGF-IR phosphorylation levels. Therefore, DX-2647 offers an alternative approach to targeting IGF-IR, blocking IGF-II signaling through both IGF-IR and IR-A.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Fator de Crescimento Insulin-Like II/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Animais , Anticorpos Monoclonais/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Humanos , Imuno-Histoquímica , Camundongos , Transdução de Sinais/efeitos dos fármacos , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de Xenoenxerto
10.
J Mol Biol ; 394(5): 878-92, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19835884

RESUMO

The insulin receptor (IR) and the homologous Type 1 insulin-like growth factor receptor (IGF-1R) are cell-surface tyrosine kinase receptors that effect signaling within the respective pathways of glucose metabolism and normal human growth. While ligand binding to these receptors is assumed to result in a structural transition within the receptor ectodomain that then effects signal transduction across the cell membrane, little is known about the molecular detail of these events. Presented here are small-angle X-ray scattering data obtained from the IR and IGF-1R ectodomains in solution. We show that, in solution, the ectodomains of IR and IGF-1R have a domain disposition that is very similar to that seen in the crystal structure of the ectodomain of IR, despite the constituent domains being in relatively sparse contact and potentially mobile. We also show that the IGF-1R ectodomain is capable of binding up to three molecules of IGF-1 in solution, with surprisingly little apparent change in relative domain disposition compared to the apo form. While the observed 3:1 ligand-binding stoichiometry appears to contradict earlier explanations of the absence of a bell-shaped dose-response curve for IGF-1R in ligand displacement assays, it is readily understood in the context of the harmonic oscillator model of the negative cooperativity of ligand binding to IGF-1R. Taken together, our findings suggest that the structural movements within these receptors upon ligand binding are small and are possibly limited to local rotation of domains.


Assuntos
Antígenos CD/química , Fator de Crescimento Insulin-Like I/metabolismo , Insulina/metabolismo , Receptor IGF Tipo 1/química , Receptor de Insulina/química , Animais , Antígenos CD/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Espalhamento a Baixo Ângulo
11.
Proc Natl Acad Sci U S A ; 103(33): 12429-34, 2006 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16894147

RESUMO

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 A resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) beta-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more alpha-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.


Assuntos
Fator de Crescimento Insulin-Like I/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor de Insulina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cristalografia por Raios X , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptor IGF Tipo 1/química , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Alinhamento de Sequência
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