RESUMEN
A hallmark of a group of neurodegenerative diseases such as Alzheimer disease is the formation of neurofibrillary tangles, which are principally composed of bundles of filaments formed by microtubule-associated protein Tau. Clarifying how natively unstructured Tau protein forms abnormal aggregates is of central importance for elucidating the etiology of these diseases. There is considerable evidence showing that zinc, as an essential element that is highly concentrated in brain, is linked to the development or progression of these diseases. Herein, by using recombinant human Tau fragment Tau(244-372) and its mutants, we have investigated the effect of zinc on the aggregation of Tau. Low micromolar concentrations of Zn(2+) dramatically accelerate fibril formation of wild-type Tau(244-372) under reducing conditions, compared with no Zn(2+). Higher concentrations of Zn(2+), however, induce wild-type Tau(244-372) to form granular aggregates in reducing conditions. Moreover, these non-fibrillar aggregates assemble into mature Tau filaments when Zn(2+) has been chelated by EDTA. Unlike wild-type Tau(244-372), low micromolar concentrations of Zn(2+) have no obvious effects on fibrillization kinetics of single mutants C291A and C322A and double mutant C291A/C322A under reducing conditions. The results from isothermal titration calorimetry show that one Zn(2+) binds to one Tau molecule via tetrahedral coordination to Cys-291 and Cys-322 as well as two histidines, with moderate, micromolar affinity. Our data demonstrate that low micromolar zinc accelerates the fibrillization of human Tau protein via bridging Cys-291 and Cys-322 in physiological reducing conditions, providing clues to understanding the relationship between zinc dyshomeostasis and the etiology of neurodegenerative diseases.
Asunto(s)
Cisteína/metabolismo , Ovillos Neurofibrilares , Fragmentos de Péptidos , Zinc/metabolismo , Proteínas tau , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Disulfuros/metabolismo , Humanos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Ovillos Neurofibrilares/química , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Oxidación-Reducción , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.
Asunto(s)
Calcio/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas ras/metabolismo , Proteínas ras/fisiología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/farmacología , Simulación por Computador , Relación Dosis-Respuesta a Droga , Humanos , Iones/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Concentración Osmolar , Unión Proteica/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloride-denatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents.