Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/metabolismo , Enfermedad de Huntington/metabolismo , Microscopía de Fuerza Atómica , Agregación Celular , Proteínas de Unión al ADN/fisiología , Humanos , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/fisiopatología , Proteínas de Microfilamentos/fisiología , Unión Proteica/fisiologíaRESUMEN
BACKGROUND: Huntington's disease is an autosomal dominant progressive neurodegenerative disease associated with dramatic expansion of a polyglutamine sequence in exon 1 of the huntingtin protein htt that leads to cytoplasmic, and even nuclear aggregation of fibrils. METHODS: We have studied the in vitro fibril formation of mutant exon 1, and the shorter wild-type exon 1, with use of atomic force microscopy (AFM). RESULTS: Large aggregates are formed spontaneously after cleavage of the glutathione-S-transferase fusion protein of the mutant exon 1 protein. The AFM data showed that, unlike fibrils assembled by such proteins as amyloid beta-peptide and alpha-synuclein, htt forms fibrils with extensive branched morphologic features. Branching can be observed even at earlier stages of the htt self-assembly, but the effect is much more pronounced at late stages of aggregation. We also found that fusing of htt with green fluorescent protein does not change the branched-type morphologic features of the aggregates. CONCLUSIONS: On the basis of the results obtained, we propose a model for htt fibrillization that explains branched morphologic features of the aggregates.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Modelos Químicos , Modelos Moleculares , Nanoestructuras/química , Nanoestructuras/ultraestructura , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/ultraestructura , Proteínas Nucleares/química , Proteínas Nucleares/ultraestructura , Simulación por Computador , Cristalización/métodos , Dimerización , Proteína Huntingtina , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Unión Proteica , Conformación ProteicaRESUMEN
Atomic force microscopy (AFM) was applied to directly visualize the end-to-end DNA interaction mediated by magnesium cations. We took advantage of the APS-mica, allowing the preparation of samples in a broad range of monovalent and divalent cations to separate the effects of Mg(2+) and Na(+) cations on the interaction of restriction DNA fragments with cohesive end. The AFM data clearly show that DNA restriction fragments with cohesive ends form substantial amount of circles in the presence of Mg(2+) cations, suggesting that Mg(2+) cations stabilize the interaction of cohesive ends. This effect depends on the MgCl(2) concentration, so that the yield of circles approaches 18% in the presence of 50 mM MgCl(2). Furthermore, we demonstrate that this conferred cohesive end stability is specific for divalent cations, as substitution of MgCl(2) with NaCl leads to a near complete loss of cohesive end stability. We further demonstrate that cohesive end stabilization is achieved by substituting Mg(2+) with Ca(2+), Mn(2+), or Zn(2+). The data obtained suggest that the end stabilization mediated by divalent cations is primarily the result of inter-base interactions rather than bridging of phosphate moieties.