RESUMO
alpha-Synuclein is a highly conserved presynaptic protein with probable roles in normal synaptic development and plasticity as well as neurodegenerative disease, although its molecular function is not yet clear. To identify potential protein binding partners of alpha-synuclein, we performed co-immunoprecipitations using a monoclonal antibody (H3C) against its C-terminus. More than 20 detectable proteins were specifically co-immunoprecipitated from zebra finch and mouse forebrain extracts. One of these, with relative mobility of 55 kDa, was identified through microsequencing as a mixture of alpha- and beta-tubulin. Tubulin was specifically recovered from a mouse forebrain cytosolic extract by a GST/alpha-synuclein fusion protein immobilized on glutathione-Sepharose beads. In the converse experiment, alpha-synuclein bound to a column prepared from purified bovine brain tubulin immobilized upon CNBr-Sepharose. alpha-Synuclein does not appear to bind assembled microtubules, however, as alpha-synuclein did not pellet with polymerized microtubules in a standard assay for microtubule-associated proteins. Likewise, when a fusion construct of alpha-synuclein and green fluorescent protein (GFP) was expressed in African green monkey kidney epithelial (CV-1) cells, the fusion protein did not colocalize with endogenous microtubules. We conclude that alpha-synuclein may interact specifically with heterodimeric tubulin, but not microtubules, in the neuronal cytosol.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/metabolismo , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Linhagem Celular , Chlorocebus aethiops , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , Aves Canoras , Especificidade da Espécie , Sinucleínas , Extratos de Tecidos , Transfecção , Tubulina (Proteína)/metabolismo , alfa-SinucleínaRESUMO
While alpha- and beta-synuclein largely overlap in their expression in the vertebrate brain, only alpha-synuclein accumulates in the fibrillar aggregates typical of Parkinson's disease. It is thus critical to have immunological reagents that distinguish between these two protein isoforms. The monoclonal antibody Syn-1 (Transduction Labs) has been frequently used for the specific detection of alpha-synuclein. In this report, the epitope for Syn-1 is localized within residues 91-99 of human alpha-synuclein. Sequence differences exist in this domain that account for the specificity of Syn-1 for alpha- versus beta-synuclein. However, Syn-1 also displays reactivity with additional species (approximately 45 kDa) in brain homogenates from both wild-type and alpha-synuclein null mice, indicating a potential for cross-reactivity with a protein species that is unrelated to alpha-synuclein in brain tissue or extracts.
Assuntos
Anticorpos Monoclonais , Especificidade de Anticorpos , Encéfalo/metabolismo , Mapeamento de Epitopos , Proteínas do Tecido Nervoso/imunologia , Animais , Encéfalo/imunologia , Células Cultivadas , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Mutação , Proteínas do Tecido Nervoso/genética , Ratos , Proteínas Recombinantes , Especificidade da Espécie , Sinucleínas , alfa-Sinucleína , beta-SinucleínaRESUMO
The t(8;21)(q22;q22) translocation, present in approximately 5% of adult acute myeloid leukemia (AML) cases, produces the AML1/ETO (AE) fusion protein. Dysregulation of the Pit/Oct/Unc (POU) domain-containing transcription factor POU4F1 is a recurring abnormality in t(8;21) AML. In this study, we showed that POU4F1 overexpression is highly correlated with, but not caused by, AE. We observed that AE markedly increases the self-renewal capacity of myeloid progenitors from murine bone marrow or fetal liver and drives the expansion of these cells in liquid culture. POU4F1 is neither necessary nor sufficient for these AE-dependent properties, suggesting that it contributes to leukemia through novel mechanisms. To identify targets of POU4F1, we performed gene expression profiling in primary mouse cells with genetically defined levels of POU4F1 and identified 140 differentially expressed genes. This expression signature was significantly enriched in human t(8;21) AML samples and was sufficient to cluster t(8;21) AML samples in an unsupervised hierarchical analysis. Among the most highly differentially expressed genes, half are known AML1/ETO targets, implying that the unique transcriptional signature of t(8;21) AML is, in part, attributable to POU4F1 and not AML1/ETO itself. These genes provide novel candidates for understanding the biology and developing therapeutic approaches for t(8;21) AML.
Assuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Perfilação da Expressão Gênica , Leucemia Mieloide Aguda/genética , Fator de Transcrição Brn-3A/genética , Fator de Transcrição Brn-3A/fisiologia , Translocação Genética/genética , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Feto/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Brn-3A/metabolismoRESUMO
A syndrome of headache, chills, sweating, nausea, and exhaustion during and after haemodialysis is described and likened to metal fume fever. A patient has been cured of this syndrome following removal of copper-containing parts from the water-path of her home dialysis system.