RESUMO
G-protein coupled receptor kinases (GRKs) constitute a serine/threonine kinase family playing a major role in agonist-induced phosphorylation and desensitization of G-protein coupled receptors. Recently, GRK2 and GRK5 have been demonstrated to phosphorylate alpha-synuclein (Ser129) and other synuclein isoforms. We studied colocalization of GRK2, GRK5, alpha-synuclein, and tau in neurodegenerative disorders characterized by fibrillary tau inclusions and/or alpha-synuclein-enriched Lewy bodies. We found that Lewy bodies were negative for both GRK2 and GRK5 in Lewy body disease (LBD) and LBD mixed with Alzheimer disease (AD + LBD). Instead, GRK2 but not GRK5 colocalized with 40% to 50% of neurofibrillary tangles in AD + LBD and AD brains. In disorders with less prominent alpha-synucleinopathy, neuronal and glial fibrillary tau deposits known to contain distinct subsets of tau isoforms were also positive for GRK2. These deposits included tufted astrocytes and coiled bodies in progressive supranuclear palsy, astrocytic plaques in corticobasal degeneration, and Pick bodies in Pick disease. In addition, paired helical filaments isolated from AD and AD + LBD brains were found to immunogold-label for GRK2, suggesting that GRK2 could be a potential tau kinase associated with fibrillary tau. Our studies indicate that GRK2 is a novel component of neuronal and glial fibrillary tau deposits with no preference in tau isoform binding. GRK2 may play a role in hyperphosphorylation of tau in tauopathies.
Assuntos
Encéfalo/enzimologia , Doenças Neurodegenerativas/enzimologia , Emaranhados Neurofibrilares/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinases de Receptores Adrenérgicos beta/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Doença de Alzheimer/fisiopatologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Feminino , Quinase 2 de Receptor Acoplado a Proteína G , Quinase 5 de Receptor Acoplado a Proteína G , Humanos , Corpos de Lewy/enzimologia , Corpos de Lewy/genética , Corpos de Lewy/patologia , Doença por Corpos de Lewy/embriologia , Doença por Corpos de Lewy/patologia , Doença por Corpos de Lewy/fisiopatologia , Masculino , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Emaranhados Neurofibrilares/genética , Emaranhados Neurofibrilares/patologia , Neuroglia/enzimologia , Neuroglia/patologia , Neurônios/enzimologia , Neurônios/patologia , Fosforilação , Doença de Pick/enzimologia , Doença de Pick/patologia , Doença de Pick/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Paralisia Supranuclear Progressiva/enzimologia , Paralisia Supranuclear Progressiva/patologia , Paralisia Supranuclear Progressiva/fisiopatologia , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Quinases de Receptores Adrenérgicos beta/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Four (CYP195A2, CYP199A2, CYP203A1, and CYP153A5) of the seven P450 enzymes, and palustrisredoxin A, a ferredoxin associated with CYP199A2, from the metabolically diverse bacterium Rhodopseudomonas palustris have been expressed and purified. A range of substituted benzenes, phenols, benzaldehydes, and benzoic acids was shown to bind to the four P450 enzymes. Monooxygenase activity of CYP199A2 was reconstituted with palustrisredoxin A and putidaredoxin reductase of the P450cam system from Pseudomonas putida. We found that 4-ethylbenzoate and 4-methoxybenzoate were oxidized to single products, and 4-methoxybenzoate was demethylated to form 4-hydroxybenzoate. Crystals of substrate-free CYP199A2 which diffracted to approximately 2.0A have been obtained.