RESUMEN
The neuronal ceroid lipofuscinoses (NCLs) are inherited neurodegenerative diseases characterized by massive accumulation of autofluorescent storage bodies in neurons and other cells. A late-onset form of NCL occurs in Tibetan terrier dogs. Gel electrophoretic analyses of isolated storage body proteins from brains of affected dogs indicated that a protein of approximately 50 kDa was consistently prominent and a 16 kDa component was present in some brain storage body preparations. Mass spectral analysis identified the 50 kDa protein as glial fibrillary acidic protein (GFAP), isoform 2. GFAP identification was supported by immunoblot and immunohistochemical analyses. Histone H4 was the major protein in the 16 kDa component. Specific accumulation of GFAP and histone H4 in storage bodies has not been previously reported for any of the NCLs. Tibetan terrier NCL may be the canine correlate of one of the human adult-onset NCLs for which the genetic bases and storage body compositions have not yet been determined.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Proteína Ácida Fibrilar de la Glía/biosíntesis , Histonas/biosíntesis , Lipofuscinosis Ceroideas Neuronales/metabolismo , Secuencia de Aminoácidos , Animales , Modelos Animales de Enfermedad , Perros , Humanos , Inmunohistoquímica , Espectrometría de Masas/métodos , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Isoformas de ProteínasRESUMEN
Little is known of the plant branched-chain alpha-ketoacid dehydrogenase complex. We have undertaken a detailed study of the structure of the dihydrolipoyl acyltransferase (BCE2) subunit that forms the core of the complex, to which two other enzymes attach. Mature Arabidopsis thaliana BCE2 was expressed in Escherichia coli. The soluble recombinant protein was purified using a Superose 6 size-exclusion column to >90% homogeneity and was catalytically active. The recombinant protein formed a stable complex with a native molecular mass of 0.95 MDa and an S coefficient of 19.4, consistent with formation of a 24-mer. Negative-staining transmission electron microscopy of the recombinant protein confirmed that BCE2 forms a core with octagonal symmetry. Despite divergence of mammalian and plant BCE2s, there is clearly conservation of structure that is independent of primary sequence.
Asunto(s)
Aciltransferasas/química , Proteínas de Plantas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Microscopía Electrónica , Complejos Multienzimáticos/química , Proteínas de Plantas/metabolismo , Conformación Proteica , Proteínas Quinasas/química , Subunidades de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Ultracentrifugación/métodosRESUMEN
Pyruvate dehydrogenase kinase (PDK) specifically phosphorylates the E1alpha subunit of the pyruvate dehydrogenase complex (PDC). Sequence analysis of cloned PDKs led to the proposal that they are mechanistically related to prokaryotic 2-component His-kinases. The reaction mechanism of protein His-kinases involves autophosphorylation of a specific His residue followed by phosphotransfer to an Asp residue. Treatment of recombinant Arabidopsis thaliana PDK with the His-directed reagents diethyl pyrocarbonate (DEPC) and dichloro-(2,2':6', 2"-terpyridine)-platinum(II) dihydrate led to a marked inhibition of autophosphorylation. In addition, DEPC treatment abolished the ability of PDK to trans-phosphorylate and inactivate PDC. These results validate the prediction that PDKs require His residues for activity.
Asunto(s)
Histidina/metabolismo , Inhibidores de Proteínas Quinasas , Piruvato Deshidrogenasa (Lipoamida) , Arabidopsis/enzimología , Arabidopsis/genética , Dietil Pirocarbonato/farmacología , Histidina Quinasa , Compuestos Organoplatinos/farmacología , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
An Arabidopsis cDNA encoding the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) was isolated from a lambdaPRL2 library. The cDNA is 1709 bp in length, with a continuous open reading frame of 1440 bp encoding a protein of 480 amino acids with a calculated molecular mass of 50,079 D. Southern analysis suggests that a single gene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit-binding, and catalytic domains, although it is unusual in having only a single lipoyl domain. The in vitro synthesized plastid E2 precursor protein has a relative molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon incubation of the precursor with pea (Pisum sativum) chloroplasts, it was imported and processed to a mature-sized relative molecular weight of 60,000. The imported protein was located in the chloroplast stroma, associated with the endogenous pyruvate dehydrogenase. Catalytically active recombinant plastid E2 was purified as a glutathione S-transferase fusion protein. Analysis of plastid E2 mRNA by reverse transcriptase-polymerase chain reaction showed highest expression in flowers, followed by leaves, siliques, and roots. The results of immunoblot analysis indicate that protein expression was similar in roots and flowers, less similar in leaves, and even less similar in siliques. This is the first report, to our knowledge, describing a plastid E2.