RESUMO
Elevated serum or tissue levels of lectin galactoside-binding soluble 3 binding protein (LGALS3BP) have been associated with short survival and development of metastasis in a variety of human cancers. However, the role of LGALS3BP, particularly in the context of tumor-host relationships, is still missing. Here, we show that LGALS3BP knockdown in MDA-MB-231 human breast cancer cells leads to a decreased adhesion to fibronectin, a reduced transendothelial migration and, more importantly, a reduced expression of vascular endothelial growth factor (VEGF). Production of VEGF, that was restored by exposure of silenced cells to recombinant LGALS3BP, required an intact PI3k/Akt signaling. Furthermore, we show that LGALS3BP was able to directly stimulate HUVEC tubulogenesis in a VEGF-independent, galectin-3-dependent manner. Immunohistochemical analysis of human breast cancer tissues revealed a correlation among LGALS3BP expression, VEGF expression, and blood vessel density. We propose that in addition to its prometastatic role, LGALS3BP secreted by breast cancer cells functions critically as a pro-angiogenic factor through a dual mechanism, i.e by induction of tumor VEGF and stimulation of endothelial cell tubulogenesis.
Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/irrigação sanguínea , Carcinoma Ductal de Mama/irrigação sanguínea , Carcinoma Lobular/irrigação sanguínea , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/metabolismo , Carcinoma Lobular/patologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Feminino , Galectina 3/metabolismo , Técnicas de Silenciamento de Genes , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neovascularização Patológica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c motif ((586)Cys and (589)Cys), while the role of the remaining two residues ((124)Cys and (143)Cys) is unclear. Structural graphic simulation using homology modelling suggested a potential second -S-S- bond, a common feature between adjacent Cys residues in other quinohaemoproteins; however, in LH, these residues are located 18 amino acids apart. Formation of the second disulphide bond was initially chemically confirmed by iodomethane alkylation with 91% loss of enzymic activity, and no significant change was observed with unreduced alkylated protein. Dithiothreitol-induced reduction of LH followed by Cd(2+) treatment also resulted in significant loss of activity in a dose-dependent manner. Subsequent investigation into the role of disulphide bond in LH was performed using engineered (143)CysâSer and (124,143)CysâSer mutants and exhibited 25% and zero activity, respectively, of wild type in the periplasm. Homology structure prediction showed three changes in α-helices and four in ß-pleated sheets in (143)CysâSer mutant, and (124,143)CysâSer mutant had six changes in α-helices and nine in ß-pleated sheets. These mutations resulted in the enlargement of the molecule and affect the enzyme activity because of structural changes in the cytochrome c domain.
Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Mutação de Sentido Incorreto , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Pseudomonas/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Estrutura Terciária de Proteína , Pseudomonas/química , Pseudomonas/genéticaRESUMO
Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors. Increased growth temperatures ranging from 18 to 30 degrees C gradually raised the enzyme production to a peak together with its concomitant aggregation as red solid particles, readily activatable in a fully functional form by mild chaotropic treatment. Here, we demonstrate that the exported lupanine hydroxylase undergoes a cascade transition from a soluble to "non-classical" inclusion body form when build-up in the periplasm exceeded a basal threshold concentration. These periplasmic aggregates were distinct from the non-secreted, signal-sequenceless counterpart that occurred as misfolded, non-functional concatamers in the form of classical inclusion bodies. We discuss our findings in the light of current models of how aggregation of lupanine hydroxylase arises in the periplasmic space.