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1.
J Biol Chem ; 289(37): 25750-63, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25074926

RESUMO

TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.


Assuntos
Apoptose/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Sanguíneas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Jurkat , Proteína S , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Estomatite Vesicular/genética , c-Mer Tirosina Quinase , Receptor Tirosina Quinase Axl
2.
J Biol Chem ; 289(37): 25737-49, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25074939

RESUMO

MERTK, a member of the TAM (TYRO3, AXL, and MERTK) receptor tyrosine kinases, has complex and diverse roles in cell biology. On the one hand, knock-out of MERTK results in age-dependent autoimmunity characterized by failure of apoptotic cell clearance, while on the other, MERTK overexpression in cancer drives classical oncogene pathways leading to cell transformation. To better understand the interplay between cell transformation and efferocytosis, we stably expressed MERTK in human MCF10A cells, a non-tumorigenic breast epithelial cell line devoid of endogenous MERTK. While stable expression of MERTK in MCF10A resulted in enhanced motility and AKT-mediated chemoprotection, MERTK-10A cells did not form stable colonies in soft agar, or enhance proliferation compared with parental MCF10A cells. Concomitant to chemoresistance, MERTK also stimulated efferocytosis in a gain-of-function capacity. However, unlike AXL, MERTK activation was highly dependent on apoptotic cells, suggesting MERTK may preferentially interface with phosphatidylserine. Consistent with this idea, knockdown of MERTK in breast cancer cells MDA-MB 231 reduced efferocytosis, while transient or stable expression of MERTK stimulated apoptotic cell clearance in all cell lines tested. Moreover, human breast cancer cells with higher endogenous MERTK showed higher levels of efferocytosis that could be blocked by soluble TAM receptors. Finally, through MERTK, apoptotic cells induced PD-L1 expression, an immune checkpoint blockade, suggesting that cancer cells may adopt MERTK-driven efferocytosis as an immune suppression mechanism for their advantage. These data collectively identify MERTK as a significant link between cancer progression and efferocytosis, and a potentially unrealized tumor-promoting event when MERTK is overexpressed in epithelial cells.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Células Epiteliais/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Apoptose/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fagocitose/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , c-Mer Tirosina Quinase , Receptor Tirosina Quinase Axl
3.
Mol Cancer Ther ; 22(2): 155-167, 2023 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-36459691

RESUMO

STRO-002 is a novel homogeneous folate receptor alpha (FolRα) targeting antibody-drug conjugate (ADC) currently being investigated in the clinic as a treatment for ovarian and endometrial cancers. Here, we describe the discovery, optimization, and antitumor properties of STRO-002. STRO-002 was generated by conjugation of a novel cleavable 3-aminophenyl hemiasterlin linker-warhead (SC239) to the nonnatural amino acid para-azidomethyl-L-phenylalanine incorporated at specific positions within a high affinity anti-FolRα antibody using Sutro's XpressCF+, which resulted in a homogeneous ADC with a drug-antibody ratio (DAR) of 4. STRO-002 binds to FolRα with high affinity, internalizes rapidly into target positive cells, and releases the tubulin-targeting cytotoxin 3-aminophenyl hemiasterlin (SC209). SC209 has reduced potential for drug efflux via P-glycoprotein 1 drug pump compared with other tubulin-targeting payloads. While STRO-002 lacks nonspecific cytotoxicity toward FolRα-negative cell lines, bystander killing of target negative cells was observed when cocultured with target positive cells. STRO-002 is stable in circulation with no change in DAR for up to 21 days and has a half-life of 6.4 days in mice. A single dose of STRO-002 induced significant tumor growth inhibition in FolRα-expressing xenograft models and patient-derived xenograft models. In addition, combination treatment with carboplatin or Avastin further increased STRO-002 efficacy in xenograft models. The potent and specific preclinical efficacy of STRO-002 supports clinical development of STRO-002 for treating patients with FolRα-expressing cancers, including ovarian, endometrial, and non-small cell lung cancer. Phase I dose escalation for STRO-002 is in progress in ovarian cancer and endometrial cancer patients (NCT03748186 and NCT05200364).


Assuntos
Antineoplásicos , Carcinoma Pulmonar de Células não Pequenas , Neoplasias do Endométrio , Imunoconjugados , Neoplasias Pulmonares , Feminino , Humanos , Animais , Camundongos , Imunoconjugados/química , Tubulina (Proteína)/metabolismo , Receptor 1 de Folato , Antineoplásicos/farmacologia , Neoplasias do Endométrio/tratamento farmacológico , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
FASEB J ; 22(5): 1380-92, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18198210

RESUMO

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.


Assuntos
Anticorpos Monoclonais/metabolismo , Epitopos/química , Anticorpos Anti-HIV/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/metabolismo , Mimetismo Molecular , Peptídeos/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes , Cristalização , Cristalografia por Raios X , Proteína gp120 do Envelope de HIV/antagonistas & inibidores , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Oligossacarídeos/química , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/imunologia , Coelhos
5.
ACS Chem Biol ; 11(7): 1844-51, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27064299

RESUMO

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound-enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization-mass spectrometry.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Animais , Linhagem Celular , Humanos
9.
Proc Natl Acad Sci U S A ; 102(38): 13372-7, 2005 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-16174734

RESUMO

Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man(9)GlcNAc(2) highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man(4), Man(5), Man(7), and Man(8)) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man(9)GlcNAc(2) moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine.


Assuntos
Anticorpos Monoclonais/química , Epitopos/química , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV , HIV-1 , Manose/química , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Sítios de Ligação de Anticorpos/imunologia , Sequência de Carboidratos , Cristalografia por Raios X , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/química , HIV-1/imunologia , Manose/imunologia , Dados de Sequência Molecular , Oligossacarídeos de Cadeias Ramificadas/química , Oligossacarídeos de Cadeias Ramificadas/imunologia
10.
J Am Chem Soc ; 126(28): 8640-1, 2004 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-15250702

RESUMO

A covalent array for the display of complex oligosaccharides in microtiter plates has been developed. This strategy is conducive to the display of carbohydrates to proteins of interest such as lectins and antibodies, including the broadly neutralizing antibody 2G12 against HIV envelope oligomannose and can be cleaved from the surface for further characterization by mass spectrometry. The system was used to probe the multivalent interaction of 2G12 with an optimal epitope (Kd 0.1 muM).


Assuntos
Técnicas de Química Combinatória , Oligossacarídeos/química , Sequência de Carboidratos , Ditiotreitol/química , Dados de Sequência Molecular , Estrutura Molecular , Oligossacarídeos/síntese química , Espectrometria de Massas por Ionização por Electrospray , Triazóis/química
11.
Science ; 300(5628): 2065-71, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12829775

RESUMO

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.


Assuntos
Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Oligossacarídeos/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Especificidade de Anticorpos , Sítios de Ligação de Anticorpos , Moléculas de Adesão Celular/metabolismo , Centrifugação com Gradiente de Concentração , Cristalização , Cristalografia por Raios X , Dimerização , Dissacarídeos/química , Dissacarídeos/metabolismo , Epitopos , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Humanos , Ligação de Hidrogênio , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/imunologia , Lectinas/química , Lectinas/imunologia , Lectinas/metabolismo , Lectinas Tipo C/metabolismo , Ligantes , Mananas/química , Mananas/metabolismo , Manosídeos/química , Manosídeos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo
12.
Proc Natl Acad Sci U S A ; 101(49): 17033-8, 2004 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-15563589

RESUMO

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.


Assuntos
Polissacarídeos/química , Análise Serial de Proteínas/métodos , Proteínas/química , Sequência de Carboidratos , Reagentes de Ligações Cruzadas , Ligantes , Ligação Proteica , Robótica
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