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1.
PLoS One ; 6(3): e17692, 2011 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-21408192

RESUMO

BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.


Assuntos
Compostos Azabicíclicos/farmacologia , Ciclo Celular , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Ácidos Ftálicos/farmacologia , Benzoquinonas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Tempo , Quinase 1 Polo-Like
2.
Hum Mol Genet ; 17(3): 367-75, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-17959936

RESUMO

Charcot-Marie-Tooth (CMT) disease type 2A is a progressive, neurodegenerative disorder affecting long peripheral motor and sensory nerves. The most common clinical sign is weakness in the lower legs and feet, associated with muscle atrophy and gait defects. The axonopathy in CMT2A is caused by mutations in Mitofusin 2 (Mfn2), a mitochondrial GTPase necessary for the fusion of mitochondria. Most Mfn2 disease alleles dominantly aggregate mitochondria upon expression in cultured fibroblasts and neurons. To determine whether this property is related to neuronal pathogenesis, we used the HB9 promoter to drive expression of a pathogenic allele, Mfn2(T105M), in the motor neurons of transgenic mice. Transgenic mice develop key clinical signs of CMT2A disease in a dosage-dependent manner. They have a severe gait defect due to an inability to dorsi-flex the hindpaws. Consequently, affected animals drag their hindpaws while walking and support themselves on the hind knuckles, rather than the soles. This distal muscle weakness is associated with reduced numbers of motor axons in the motor roots and severe reduction of the anterior calf muscles. Many motor neurons from affected animals show improper mitochondrial distribution, characterized by tight clusters of mitochondria within axons. This transgenic line recapitulates key motor features of CMT2A and provides a system to dissect the function of mitochondria in the axons of mammalian motor neurons.


Assuntos
Doença de Charcot-Marie-Tooth/patologia , Doença de Charcot-Marie-Tooth/fisiopatologia , Animais , Axônios/patologia , Sequência de Bases , Doença de Charcot-Marie-Tooth/genética , Primers do DNA/genética , GTP Fosfo-Hidrolases , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Membro Posterior/fisiopatologia , Homozigoto , Humanos , Coxeadura Animal/genética , Coxeadura Animal/patologia , Coxeadura Animal/fisiopatologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Mitocôndrias/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Neurônios Motores/patologia , Mutagênese Sítio-Dirigida , Fenótipo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
3.
Nat Rev Mol Cell Biol ; 8(11): 870-9, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17928812

RESUMO

Recent findings have sparked renewed appreciation for the remarkably dynamic nature of mitochondria. These organelles constantly fuse and divide, and are actively transported to specific subcellular locations. These dynamic processes are essential for mammalian development, and defects lead to neurodegenerative disease. But what are the molecular mechanisms that control mitochondrial dynamics, and why are they important for mitochondrial function? We review these issues and explore how defects in mitochondrial dynamics might cause neuronal disease.


Assuntos
Mitocôndrias/metabolismo , Mitocôndrias/patologia , Animais , Humanos , Fusão de Membrana , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia
4.
J Cell Biol ; 176(4): 405-14, 2007 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-17296794

RESUMO

Mfn2, an oligomeric mitochondrial protein important for mitochondrial fusion, is mutated in Charcot-Marie-Tooth disease (CMT) type 2A, a peripheral neuropathy characterized by axonal degeneration. In addition to homooligomeric complexes, Mfn2 also associates with Mfn1, but the functional significance of such heterooligomeric complexes is unknown. Also unknown is why Mfn2 mutations in CMT2A lead to cell type-specific defects given the widespread expression of Mfn2. In this study, we show that homooligomeric complexes formed by many Mfn2 disease mutants are nonfunctional for mitochondrial fusion. However, wild-type Mfn1 complements mutant Mfn2 through the formation of heterooligomeric complexes, including complexes that form in trans between mitochondria. Wild-type Mfn2 cannot complement the disease alleles. Our results highlight the functional importance of Mfn1-Mfn2 heterooligomeric complexes and the close interplay between the two mitofusins in the control of mitochondrial fusion. Furthermore, they suggest that tissues with low Mfn1 expression are vulnerable in CMT2A and that methods to increase Mfn1 expression in the peripheral nervous system would benefit CMT2A patients.


Assuntos
Doença de Charcot-Marie-Tooth/genética , GTP Fosfo-Hidrolases/genética , Fusão de Membrana/genética , Mitocôndrias/genética , Mutação/genética , Animais , Axônios/metabolismo , Axônios/patologia , Células Cultivadas , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/fisiopatologia , Fibroblastos , GTP Fosfo-Hidrolases/metabolismo , Predisposição Genética para Doença/genética , Substâncias Macromoleculares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Degeneração Walleriana/fisiopatologia
5.
Biochim Biophys Acta ; 1763(5-6): 482-9, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16571363

RESUMO

Mitochondrial fusion requires coordinated fusion of the outer and inner membranes. This process leads to exchange of contents, controls the shape of mitochondria, and is important for mitochondrial function. Two types of mitochondrial GTPases are essential for mitochondrial fusion. On the outer membrane, the fuzzy onions/mitofusin proteins form complexes in trans that mediate homotypic physical interactions between adjacent mitochondria and are likely directly involved in outer membrane fusion. Associated with the inner membrane, the OPA1 dynamin-family GTPase maintains membrane structure and is a good candidate for mediating inner membrane fusion. In yeast, Ugo1p binds to both of these GTPases to form a fusion complex, although a related protein has yet to be found in mammals. An understanding of the molecular mechanism of fusion may have implications for Charcot-Marie-Tooth subtype 2A and autosomal dominant optic atrophy, neurodegenerative diseases caused by mutations in Mfn2 and OPA1.


Assuntos
Fusão de Membrana , Membranas Mitocondriais/metabolismo , Animais , Ergosterol/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas SNARE/metabolismo , Fenômenos Fisiológicos Virais
6.
Science ; 305(5685): 858-62, 2004 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-15297672

RESUMO

Vesicle fusion involves vesicle tethering, docking, and membrane merger. We show that mitofusin, an integral mitochondrial membrane protein, is required on adjacent mitochondria to mediate fusion, which indicates that mitofusin complexes act in trans (that is, between adjacent mitochondria). A heptad repeat region (HR2) mediates mitofusin oligomerization by assembling a dimeric, antiparallel coiled coil. The transmembrane segments are located at opposite ends of the 95 angstrom coiled coil and provide a mechanism for organelle tethering. Consistent with this proposal, truncated mitofusin, in an HR2-dependent manner, causes mitochondria to become apposed with a uniform gap. Our results suggest that HR2 functions as a mitochondrial tether before fusion.


Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular , Cristalografia por Raios X , Dimerização , Humanos , Células Híbridas , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Fusão de Membrana , Camundongos , Mitocôndrias/ultraestrutura , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
7.
J Cell Biol ; 160(2): 189-200, 2003 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-12527753

RESUMO

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.


Assuntos
Embrião de Mamíferos/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Membrana/deficiência , Mitocôndrias/metabolismo , Proteínas Mitocondriais/deficiência , Trofoblastos/metabolismo , Animais , Movimento Celular/genética , Células Cultivadas , Proteínas do Citoesqueleto/genética , Perda do Embrião/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/genética , Feminino , Feto , GTP Fosfo-Hidrolases , Genes Letais/genética , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Substâncias Macromoleculares , Masculino , Potenciais da Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Placenta/anormalidades , Placenta/metabolismo , Placenta/fisiopatologia , Trofoblastos/patologia , Utrofina
9.
EMBO J ; 21(4): 590-601, 2002 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-11847107

RESUMO

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.


Assuntos
Endocitose , Imunoglobulina G/metabolismo , Receptores Fc/metabolismo , Androstadienos/farmacologia , Animais , Compartimento Celular , Linhagem Celular , Cães , Proteínas de Fluorescência Verde , Antígenos de Histocompatibilidade Classe I , Ligantes , Proteínas Luminescentes/metabolismo , Nocodazol/farmacologia , Wortmanina
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