RESUMO
Missense mutations in the gene, MAP3K1, are a common cause of 46,XY gonadal dysgenesis, accounting for 15-20% of cases [Ostrer, 2014, Disorders of sex development (DSDs): an update. J. Clin. Endocrinol. Metab., 99, 1503-1509]. Functional studies demonstrated that all of these mutations cause a protein gain-of-function that alters co-factor binding and increases phosphorylation of the downstream MAP kinase pathway targets, MAPK11, MAP3K and MAPK1. This dysregulation of the MAP kinase pathway results in increased CTNNB1, increased expression of WNT4 and FOXL2 and decreased expression of SRY and SOX9. Unique and recurrent pathogenic mutations cluster in three semi-contiguous domains outside the kinase region of the protein, a newly identified N-terminal domain that shares homology with the Guanine Exchange Factor (residues Met164 to Glu231), a Plant HomeoDomain (residues Met442 to Trp495) and an ARMadillo repeat domain (residues Met566 to Glu862). Despite the presence of the mutation clusters and clinical data, there exists a dearth of mechanistic insights behind the development imbalance. In this paper, we use structural modeling and functional data of these mutations to understand alterations of the MAP3K1 protein and the effects on protein folding, binding and downstream target phosphorylation. We show that these mutations have differential effects on protein binding depending on the domains in which they occur. These mutations increase the binding of the RHOA, MAP3K4 and FRAT1 proteins and generally decrease the binding of RAC1. Thus, pathologies in MAP3K1 disrupt the balance between the pro-kinase activities of the RHOA and MAP3K4 binding partners and the inhibitory activity of RAC1.
Assuntos
Transtornos do Desenvolvimento Sexual/genética , MAP Quinase Quinase Quinase 1/genética , MAP Quinase Quinase Quinase 4/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Domínio Armadillo/genética , Transtorno 46,XY do Desenvolvimento Sexual , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Proteína Forkhead Box L2/genética , Regulação da Expressão Gênica/genética , Disgenesia Gonadal 46 XY/genética , Disgenesia Gonadal 46 XY/patologia , Humanos , MAP Quinase Quinase Quinase 1/química , MAP Quinase Quinase Quinase 4/química , Sistema de Sinalização das MAP Quinases/genética , Masculino , Mutação de Sentido Incorreto/genética , Ligação Proteica/genética , Proteínas Proto-Oncogênicas/genética , Proteína da Região Y Determinante do Sexo/genética , Proteínas rac1 de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Despite the increasing understanding of female reproduction, the molecular diagnosis of primary ovarian insufficiency (POI) is seldom obtained. The RNA-binding protein NANOS3 poses as an interesting candidate gene for POI since members of the Nanos family have an evolutionarily conserved function in germ cell development and maintenance by repressing apoptosis. We performed mutational analysis of NANOS3 in a cohort of 85 Brazilian women with familial or isolated POI, presenting with primary or secondary amenorrhea, and in ethnically-matched control women. A homozygous p.Glu120Lys mutation in NANOS3 was identified in two sisters with primary amenorrhea. The substituted amino acid is located within the second C2HC motif in the conserved zinc finger domain of NANOS3 and in silico molecular modelling suggests destabilization of protein-RNA interaction. In vitro analyses of apoptosis through flow cytometry and confocal microscopy show that NANOS3 capacity to prevent apoptosis was impaired by this mutation. The identification of an inactivating missense mutation in NANOS3 suggests a mechanism for POI involving increased primordial germ cells (PGCs) apoptosis during embryonic cell migration and highlights the importance of NANOS proteins in human ovarian biology.
Assuntos
Homozigoto , Mutação , Insuficiência Ovariana Primária/genética , Proteínas de Ligação a RNA/genética , Adolescente , Adulto , Amenorreia/genética , Animais , Apoptose , Brasil , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , Feminino , Citometria de Fluxo , Humanos , Microscopia Confocal , Ligação Proteica , RNA/química , Adulto Jovem , Dedos de ZincoRESUMO
Vascular endothelial growth factor (VEGF) and αvß3 integrin are key molecules that actively participate in tumor angiogenesis and metastasis. Some integrin-blocking molecules are currently under clinical trials for cancer and metastasis treatment. However, the mechanism of action of such inhibitors is not completely understood. We have previously demonstrated the anti-angiogenic and anti-metastatic properties of DisBa-01, a recombinant His-tag RGD-disintegrin from Bothrops alternatus snake venom in some experimental models. DisBa-01 blocks αvß3 integrin binding to vitronectin and inhibits integrin-mediated downstream signaling cascades and cell migration. Here we add some new information on the mechanism of action of DisBa-01 in the tumor microenvironment. DisBa-01 supports the adhesion of fibroblasts and MDA-MB-231 breast cancer cells but it inhibits the adhesion of these cells to type I collagen under flow in high shear conditions, as a simulation of the blood stream. DisBa-01 does not affect the release of VEGF by fibroblasts or breast cancer cells but it strongly decreases the expression of VEGF mRNA and of its receptors, vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2) in endothelial cells. DisBa-01 at nanomolar concentrations also modulates metalloprotease 2 (MMP-2) and 9 (MMP-9) activity, the latter being decreased in fibroblasts and increased in MDA-MB-231 cells. In conclusion, these results demonstrate that αvß3 integrin inhibitors may induce distinct effects in the cells of the tumor microenvironment, resulting in blockade of angiogenesis by impairing of VEGF signaling and in inhibition of tumor cell motility.