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1.
Cancer Sci ; 110(12): 3773-3787, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31578782

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis, and it is also found on the surface of tumor cells. Currently, there are no Ab therapeutics targeting VEGFR2 approved for the treatment of prostate cancer or leukemia. Therefore, development of novel efficacious anti-VEGFR2 Abs will benefit cancer patients. We used the Institute of Cellular and Organismic Biology human Ab library and affinity maturation to develop a fully human Ab, anti-VEGFR2-AF, which shows excellent VEGFR2 binding activity. Anti-VEGFR2-AF bound Ig-like domain 3 of VEGFR2 extracellular region to disrupt the interaction between VEGF-A and VEGFR2, neutralizing downstream signaling of the receptor. Moreover, anti-VEGFR2-AF inhibited capillary structure formation and exerted Ab-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity in vitro. We found that VEGFR2 is expressed in PC-3 human prostate cancer cell line and associated with malignancy and metastasis of human prostate cancer. In a PC-3 xenograft mouse model, treatment with anti-VEGFR2-AF repressed tumor growth and angiogenesis as effectively and safely as US FDA-approved anti-VEGFR2 therapeutic, ramucirumab. We also report for the first time that addition of anti-VEGFR2 Ab can enhance the efficacy of docetaxel in the treatment of a prostate cancer mouse model. In HL-60 human leukemia-xenografted mice, anti-VEGFR2-AF showed better efficacy than ramucirumab with prolonged survival and reduced metastasis of leukemia cells to ovaries and lymph nodes. Our findings suggest that anti-VEGFR2-AF has strong potential as a cancer therapy that could directly target VEGFR2-expressing tumor cells in addition to its anti-angiogenic action.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Leucemia/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos/uso terapêutico , Linhagem Celular Tumoral , Epitopos de Linfócito B , Humanos , Masculino , Camundongos , Fosforilação , Fator A de Crescimento do Endotélio Vascular/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Proteins ; 63(4): 777-86, 2006 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-16544327

RESUMO

Vigna radiata plant defensin 1 (VrD1) is the first reported plant defensin exhibiting insecticidal activity. We report herein the nuclear magnetic resonance solution structure of VrD1 and the implication on its insecticidal activity. The root-mean-square deviation values are 0.51 +/- 0.35 and 1.23 +/- 0.29 A for backbone and all heavy atoms, respectively. The VrD1 structure comprises a triple-stranded antiparallel beta-sheet, an alpha-helix, and a 3(10) helix stabilized by four disulfide bonds, forming a typical cysteine-stabilized alphabeta motif. Among plant defensins of known structure, VrD1 is the first to contain a 3(10) helix. Glu26 is highly conserved among defensins; VrD1 contains an arginine at this position, which may induce a shift in the orientation of Trp10, thereby promoting the formation of this 3(10) helix. Moreover, VrD1 inhibits Tenebrio molitor alpha-amylase. Alpha-amylase has an essential role in the digestion of plant starch in the insect gut, and expression of the common bean alpha-amylase inhibitor 1 in transgenic pea imparts complete resistance against bruchids. These results imply that VrD1 insecticidal activity has its basis in the inhibition of a polysaccharide hydrolase. Sequence and structural comparisons between two groups of plant defensins having different specificity toward insect alpha-amylase reveal that the loop between beta2 and beta3 is the probable binding site for the alpha-amylase. Computational docking experiments were used to study VrD1-alpha-amylase interactions, and these results provide information that may be used to improve the insecticidal activity of VrD1.


Assuntos
Defensinas/química , Defensinas/farmacologia , Fabaceae/química , Inseticidas/química , Inseticidas/farmacologia , Proteínas de Plantas/química , Tenebrio/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência Conservada , Defensinas/metabolismo , Fabaceae/metabolismo , Fabaceae/parasitologia , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Tenebrio/enzimologia , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo
3.
Peptides ; 24(12): 1853-61, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15127937

RESUMO

Interactions between proteins and heparin play a crucial role in most of the cellular process. Unraveling the forces that govern the formation of these complexes is vital for understanding the specificities involved in these biomolecular events. In the present study, a detailed analysis has been undertaken to evaluate the effect(s) of peptide conformation on heparin-binding, using a chimeric peptide, apaK6--a chimera of a highly stable neurotoxic peptide from honey-bee venom and a de novo designed lysine-rich peptide. The dissociation constants of these peptide-heparin complexes were found to be in the submicromolar range. Comparison of the results obtained from the titration of the disulfide-reduced and disulfide-intact chimeric peptide with various sulfated oligosaccharides, derived from heparin, suggest that the initial structure of the peptide has pronounced effect on the binding affinity, binding modes and also on binding preferences. The results of this study indicate that the heparin-binding specificity of an isolated peptide and that exhibited by the same peptide when present in a globular protein could be significantly different, especially if the isolated peptide undergoes conformational change(s) upon binding to the sulfated oligosaccharides. In addition, such dependency of the binding specificity on the preformed structures could be utilized for the design of high-affinity and sequence-specific heparin-binding polypeptides.


Assuntos
Apamina/química , Oligossacarídeos/química , Peptídeos/química , Proteínas Recombinantes de Fusão/química , Dicroísmo Circular , Heparina/química , Concentração Osmolar , Conformação Proteica
4.
Biochem Biophys Res Commun ; 294(3): 535-40, 2002 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-12056799

RESUMO

A novel 7-kDa non-specific lipid transfer protein-2 (nsLTP2) has been isolated from rice (Oryza sativa) seeds. In contrast to nsLTP1s, few nsLTP2s have been purified and characterized. Complete amino acid sequence of rice nsLTP2 was determined by N-terminal Edman degradation of the intact protein as well as the peptide fragments resulted from trypsin digestions. Rice nsLTP2 consists of 69 amino acid residues with eight conserved cysteines forming four disulfide bonds. The secondary structure of rice nsLTP2 is predominantly alpha-helical as determined by circular dichroism spectroscopy. Cysteine pairings of nsLTP2 have one miss match at Cys(35)-X-Cys(37) motif compared to nsLTP1. Primary structure analysis of various plant nsLTP2s revealed an interesting conservation of sequence features among nsLTP2 family.


Assuntos
Metabolismo dos Lipídeos , Proteínas de Membrana Transportadoras/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Proteínas de Membrana Transportadoras/análise , Proteínas de Membrana Transportadoras/isolamento & purificação , Dados de Sequência Molecular , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Alinhamento de Sequência
5.
J Biol Chem ; 277(38): 35267-73, 2002 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-12011089

RESUMO

The three-dimensional structure of rice nonspecific lipid transfer protein (nsLTP2) has been solved for the first time. The structure of nsLTP2 was obtained using 813 distance constraints, 30 hydrogen bond constraints, and 19 dihedral angle constraints. Fifteen of the 50 random simulated annealing structures satisfied all of the constraints and possessed good nonbonded contacts. The novel three-dimensional fold of rice nsLTP2 contains a triangular hydrophobic cavity formed by three prominent helices. The four disulfide bonds required for stabilization of the nsLTP2 structure show a different pattern of cysteine pairing compared with nsLTP1. The C terminus of the protein is very flexible and forms a cap over the hydrophobic cavity. Molecular modeling studies suggested that the hydrophobic cavity could accommodate large molecules with rigid structures, such as sterols. The positively charged residues on the molecular surface of nsLTP2 are structurally similar to other plant defense proteins.


Assuntos
Proteínas de Transporte/química , Oryza/química , Proteínas de Plantas , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Soluções
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