RESUMEN
Autosomal dominant early-onset Alzheimer's disease results mainly from mutations of the presenilin 1 (PSEN1) gene, which codes for an integral membrane protein of 467 amino acids. The hydrophilic loop (amino acids 263-407) of PSEN1, in which many pathogenic mutations have been localized, appears to be crucial for the protein function since it includes the binding domains to different PSEN1 partners. Using circular dichroism (CD) we analyzed the structural effects of the pathogenic L392V mutation and compared them with those of the E318G substitution. This study revealed that, the L392V mutation, in a phospholipidic medium which mimics the in vivo membrane environment, reduces the alpha helix content of the PSEN1 loop, whereas the E318G substitution, considered as a polymorphism, does not. These results suggest that the pathogenic effect of some PSEN1 mutations within the hydrophilic loop could be the alteration of the interaction to the different binding proteins through a disruption of the secondary structure.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Mutación/genética , Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/genética , Dicroismo Circular , Escherichia coli/metabolismo , Humanos , Linaje , Plásmidos , Presenilina-1 , Estructura Secundaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/químicaRESUMEN
The process of the detoxification of A-destruxin into a polar linear peptide in vivo in Locusta migratoria has been demonstrated using both high performance liquid chromatography and negative-ion fast-atom bombardment mass spectrometry. The final structure of the metabolite, after synthesis and comparison of retention time with the component in the crude biological mixture, was elucidated by positive-ion fast-atom bombardment linked-scan mass spectrometry. Diagnostic fragment ions produced by collision-induced dissociation are discussed.
Asunto(s)
Depsipéptidos , Saltamontes/metabolismo , Micotoxinas/análisis , Péptidos Cíclicos/análisis , Animales , Cromatografía Líquida de Alta Presión , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
A cyclodepsipeptide-glutathionyl conjugate has been analysed for the first time using negative-ion fast-atom bombardment linked-scan mass spectroscopy. Fragment anions produced by collision-induced dissociation are discussed.
Asunto(s)
Depsipéptidos , Proteínas Fúngicas , Glutatión/química , Micotoxinas/química , Péptidos Cíclicos/química , Hongos Mitospóricos/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
A new strategy, based on sequential product-ion spectra (MS2 and MS3) and reaction-intermediate scanning (MS3), provides a rapid and unambiguous sequence determination of E-destruxin, a cyclodepsipeptidic toxin from Metarrhizium anisopliae fungus.
Asunto(s)
Depsipéptidos , Proteínas Fúngicas , Hongos Mitospóricos/química , Micotoxinas/análisis , Péptidos Cíclicos/análisis , Secuencia de Aminoácidos , Espectrometría de Masas , Datos de Secuencia MolecularRESUMEN
The conjugation products of E-destruxin (cyclodepsipeptide) with glutathione have been identified by fast-atom bombardment mass spectrometry. Appropriate conditions were developed for cyclodepsipeptide substrate, which enabled the direct, dynamic mass spectra analysis of spontaneous as well as glutathione-s-transferase catalyzed conjugation reactions. Application to the most active cyclopeptide in the series of destruxins yielded glutathionyl adduct.
Asunto(s)
Depsipéptidos , Proteínas Fúngicas , Glutatión/química , Micotoxinas/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Glutatión Transferasa/metabolismo , Hongos Mitospóricos/química , Datos de Secuencia Molecular , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
To study the behavior of the toxins E and A destruxin in biological media, a method was developed with direct injection on to a liquid chromatographic (LC) column. Either slightly lipophilic C1 and C4 "wide-pore" packings or the column-switching approach with a guard column were used. To confirm the results of the direct LC analysis, a "classical approach" with pretreatment prior to injection on to a C8 packed column was also developed. Further, parallel fast atom bombardment MS monitoring in the negative-ion mode was carried out on the same biological samples, to obtain complementary information on the destruxin metabolism in locusts. Thus, the behaviours of E and A destruxin were examined in vivo in different organs of locusts. For E destruxin, several detoxication processes could be observed, such as hydrolysis and conjugation with glutathione.
Asunto(s)
Depsipéptidos , Proteínas Fúngicas , Saltamontes/metabolismo , Micotoxinas/farmacocinética , Péptidos Cíclicos/farmacocinética , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Cuerpo Adiposo/química , Hemolinfa/química , Indicadores y Reactivos , Masculino , Túbulos de Malpighi/química , Datos de Secuencia Molecular , Micotoxinas/análisis , Péptidos Cíclicos/análisis , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría UltravioletaRESUMEN
Multidimensional chromatographies (MDC) implying either a single column with an unique packing designed for providing several separation modes such as internal surface reversed-phase (ISRP), or C1 large pores particles, or multi-column switching technique using the previous supports as guard columns, allow fast and easy direct monitoring of pesticides into plant or animal biological media. In such a way, the comparative study of the agricultural fungicide cymoxanil in two strains of Botrytis cinerea of different sensitivities gave concordant conclusions with the previous work using radiolabelled cymoxanil and TLC. Moreover, as a comparison, a study of the metabolism of this xenobiotic was also followed in animal biological fluids likely to contain peptidases. In locust or lobster haemolymph, an unusual isomerization into a cyclic compound was thus demonstrated accompanying the classical peptidase-depending metabolism generally observed in plants. The same chromatographic approaches were tested and compared for monitoring the metabolism of the natural insecticide toxin E-destruxin in insects. A detoxication process leading to the less toxic E-diol destruxin was evidenced in the organs or haemolymph of the locust Locusta migratoria chosen as a model of non-target insects.