RESUMO
Glioblastoma multiforme (GBM) is a rapidly progressive disease of morbidity and mortality and is the most common form of primary brain cancer in adults. Lack of appropriate in vivo models has been a major roadblock to developing effective therapies for GBM. A new highly invasive in vivo GBM model is described that was derived from a spontaneous brain tumor (VM-M3) in the VM mouse strain. Highly invasive tumor cells could be identified histologically on the hemisphere contralateral to the hemisphere implanted with tumor cells or tissue. Tumor cells were highly expressive for the chemokine receptor CXCR4 and the proliferation marker Ki-67 and could be identified invading through the pia mater, the vascular system, the ventricular system, around neurons, and over white matter tracts including the corpus callosum. In addition, the brain tumor cells were labeled with the firefly luciferase gene, allowing for non-invasive detection and quantitation through bioluminescent imaging. The VM-M3 tumor has a short incubation time with mortality occurring in 100% of the animals within approximately 15 days. The VM-M3 brain tumor model therefore can be used in a pre-clinical setting for the rapid evaluation of novel anti-invasive therapies.
Assuntos
Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Neurônios/patologia , Animais , Astrocitoma/genética , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adesão Celular , Técnicas Imunoenzimáticas , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Neurônios/metabolismo , RNA Mensageiro/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
The spindle-pole body (SPB), the yeast analog of the centrosome, serves as the major microtubule (MT) organizing center in the yeast cell. In addition to this central function, the SPB organizes and concentrates proteins required for proper coordination between the nuclear-division cycle and cytokinesis. For example, the Schizosaccharomyces pombe septation-initiation network (SIN), which is responsible for initiating actomyosin ring constriction and septation, is assembled at the SPB through its two scaffolding components, Sid4 and Cdc11. In an effort to identify novel SIN interactors, we purified Cdc11 and identified by mass spectrometry a previously uncharacterized protein associated with it, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. A fusion between the N-terminal 300 amino acids of Sid4 and a SPB targeting domain of Ppc89 supplies the essential function of Sid4 in anchoring the SIN. ppc89Delta cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Fuso Acromático/química , Fuso Acromático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Deleção de Genes , Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/química , Ligação Proteica , Transporte Proteico , Schizosaccharomyces/ultraestrutura , Proteínas de Schizosaccharomyces pombe/química , Fuso Acromático/ultraestruturaRESUMO
Prp19 is an essential splicing factor and a member of the U-box family of E3 ubiquitin ligases. Prp19 forms a tetramer via a central coiled-coil domain. Here, we show the U-box domain of Prp19 exists as a dimer within the context of the Prp19 tetramer. A high-resolution structure of the homodimeric state of the Prp19 U-box was determined by X-ray crystallography. Mutation of the U-box dimer interface abrogates U-box dimer formation and is lethal in vivo. The structure of the U-box dimer enables construction of a complete model of Prp19 providing insights into how the tetrameric protein functions as an E3 ligase. Finally, comparison of the Prp19 U-box homodimer with the heterodimeric complex of BRCA1/BARD1 RING-finger domains uncovers a common architecture for a family of oligomeric U-box and RING-finger E3 ubiquitin ligases, which has mechanistic implications for E3 ligase-mediated polyubiquitination and E4 polyubiquitin ligases.
Assuntos
Proteínas de Transporte/química , Enzimas de Conjugação de Ubiquitina/química , Ubiquitina-Proteína Ligases/química , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Enzimas Reparadoras do DNA , Dimerização , Humanos , Modelos Químicos , Dados de Sequência Molecular , Proteínas Nucleares , Poliubiquitina/química , Poliubiquitina/metabolismo , Estrutura Terciária de Proteína , Fatores de Processamento de RNA , RNA Nuclear Pequeno , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
U-box-containing Prp19p is an integral component of the Prp19p-associated complex (the nineteen complex, or NTC) that is essential for activation of the spliceosome. Prp19p makes numerous protein-protein contacts with other NTC components and is required for NTC stability. Here we show that Prp19p forms a tetramer in vitro and in vivo and we map the domain required for its oligomerization to a central tetrameric coiled-coil. Biochemical and in vivo analyses are consistent with Prp19p tetramerization providing an interaction surface for a single copy of its binding partner, Cef1p. Electron microscopy showed that the isolated Prp19p tetramer is an elongated particle consisting of four globular WD40 domains held together by a central stalk consisting of four N-terminal U-boxes and four coiled-coils. These structural and functional data provide a basis for understanding the role of Prp19p as a key architectural component of the NTC.
Assuntos
RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromatografia em Gel , Dicroísmo Circular , Análise Mutacional de DNA , Dimerização , Genótipo , Processamento de Imagem Assistida por Computador , Immunoblotting , Imunoprecipitação , Microscopia Eletrônica , Modelos Biológicos , Reação em Cadeia da Polimerase , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Splicing de RNA , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe , Spliceossomos/metabolismo , Relação Estrutura-Atividade , Temperatura , Técnicas do Sistema de Duplo-Híbrido , UltracentrifugaçãoRESUMO
The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the cross-brace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the U-box. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val-->Ile point mutation in the Prp19p U-box domain leads to pre-mRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.