RESUMO
Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a ß-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nucleoplasminas/metabolismo , Multimerização Proteica , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Área Sob a Curva , Dicroísmo Circular , Modelos Moleculares , Peso Molecular , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Espalhamento a Baixo Ângulo , Soluções , Frações Subcelulares/metabolismo , Difração de Raios XRESUMO
Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Ensaios de Triagem em Larga Escala , Humanos , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes de Fusão , Bibliotecas de Moléculas Pequenas , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CGL is a 150 amino-acid residue lectin that was originally isolated from the sea mussel Crenomytilus grayanus. It is specific for binding GalNAc/Gal-containing carbohydrate moieties and in general does not share sequence homology with other known galectins or lectins. Since CGL displays antibacterial, antifungal and antiviral activities, and interacts with high affinity with mucin-type receptors, which are abundant on some cancer cells, knowledge of its structure is of significant interest. Conditions have been established for the expression, purification and crystallization of a recombinant variant of CGL. The crystal structure of recombinant CGL was determined and refined at a resolution of 2.12 Å. The amino-acid sequence of CGL contains three homologous regions (73% similarity) and the folded protein has a ß-trefoil topology. Structural comparison of CGL with the closely related lectin MytiLec allowed description of the glycan-binding pockets.