RESUMO
Tau aggregation is an appealing target for therapeutic intervention. However, conformational change or aggregation needs to be targeted without inhibiting the normal biology of tau and its role in microtubule stabilization. The number of compound classes being tested at this time are very limited and include Congo red derivatives [2], anthraquinones (Pickhardt et al. 2005 [3], disputed in Crowe et al. 2007 [4]), 2,3-di(furan-2-yl)-quinoxalines , phenylthiazolyl-hydrazide (PTH) [5], polyphenols and porphyrins [6] and cyanine dyes [1, 7, 8]. Herein we have utilized a member of the cyanine dye family (C11) in an organotypic slice culture model of tangle formation. Our results demonstrate that C11 is capable of affecting tau polymerization in a biphasic, dose dependent manner. At submicromolar concentrations (0.001 microM) C11 reduced levels of aggregated tau. However, higher doses resulted in an increase in tau polymerization. These effects can also be seen at the level of individual filaments with changes in filament length and number mirroring the pattern seen via immunoblotting. In addition, this effect is achieved without altering levels of phosphorylation at disease and microtubule binding relevant epitopes.
Assuntos
Carbocianinas/farmacologia , Emaranhados Neurofibrilares/efeitos dos fármacos , Emaranhados Neurofibrilares/metabolismo , Tauopatias/tratamento farmacológico , Tauopatias/metabolismo , Proteínas tau/efeitos dos fármacos , Proteínas tau/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Carbocianinas/uso terapêutico , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Camundongos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Técnicas de Cultura de Órgãos , Polímeros/química , Relação Estrutura-Atividade , Tauopatias/fisiopatologiaRESUMO
We have characterized a novel monoclonal antibody, Tau-66, raised against recombinant human tau. Immunohistochemistry using Tau-66 reveals a somatic-neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau-66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau-66 stains plaques lacking obvious dystrophic neurites (termed herein 'diffuse reticulated plaques') in STG and the hippocampus. Enzyme-linked immunosorbent assay (ELISA) analysis reveals that Tau-66 is specific for tau, as there is no cross-reactivity with MAP2, tubulin, Abeta(1-40), or Abeta(1-42), although Tau-66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau-66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155-244 and 305-314. Tau-66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau-66 epitope is conformation-dependent, perhaps involving a close interaction of the proline-rich and the third microtubule-binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.
Assuntos
Doença de Alzheimer/metabolismo , Anticorpos Monoclonais/química , Proteínas tau/imunologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Western Blotting , Ensaio de Imunoadsorção Enzimática , Epitopos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hibridomas , Imuno-Histoquímica , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Conformação Proteica , Proteínas tau/químicaRESUMO
The structure of TPK1delta, a truncated variant of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae, was determined in an unliganded state at 2.8 A resolution and refined to a crystallographic R-factor of 19.4%. Comparison of this structure to that of its fully liganded mammalian homolog revealed a highly conserved protein fold comprised of two globular lobes. Within each lobe, root mean square deviations in Calpha positions averaged approximately equals 0.9 A. In addition, a phosphothreonine residue was found in the C-terminal domain of each enzyme. Further comparison of the two structures suggests that a trio of conformational changes accompanies ligand-binding. The first consists of a 14.7 degrees rigid-body rotation of one lobe relative to the other and results in closure of the active site cleft. The second affects only the glycine-rich nucleotide binding loop, which moves approximately equals 3 A to further close the active site and traps the nucleotide substrate. The third is localized to a C-terminal segment that makes direct contact with ligands and the ligand-binding cleft. In addition to resolving the conformation of unliganded enzyme, the model shows that the salient features of the cAMP-dependent protein kinase are conserved over long evolutionary distances.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Catálise , Domínio Catalítico , Cristalografia por Raios X , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Evolução Molecular , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos , Conformação Proteica , Subunidades Proteicas , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Alzheimer's disease (AD) is characterized by the presence of amyloid-positive senile plaques and tau-positive neurofibrillary tangles. Aside from these two pathological hallmarks, a growing body of evidence indicates that the amount of oxidative alteration of vulnerable molecules such as proteins, DNA, and fatty acids is elevated in the brains of AD patients. It has been hypothesized that the elevated amounts of protein oxidation could lead directly to the formation of neurofibrillary tangles through a cysteine-dependent mechanism. We have tested this hypothesis in an in vitro system in which tau assembly is induced by fatty acids. Using sulfhydryl protective agents and site-directed mutagenesis, we found that cysteine-dependent oxidation of the tau molecule is not required for its polymerization and may even be inhibitory. However, by adjusting the oxidative environment of the polymerization reaction through the addition of a strong antioxidant or through the addition of an oxidizing system consisting of iron, adenosine diphosphate, and ascorbate, we found that oxidation does play a major role in our in vitro paradigm. The results indicated that fatty acid oxidation, the amount of which is found to be elevated in AD patients, can facilitate the polymerization of tau. However, "overoxidation" of the fatty acids can inhibit the process. Therefore, we postulate that specific fatty acid oxidative products could provide a direct link between oxidative stress mechanisms and the formation of neurofibrillary tangles in AD.
Assuntos
Ácidos Graxos não Esterificados/farmacologia , Polímeros/metabolismo , Proteínas tau/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Antioxidantes/farmacologia , Soluções Tampão , Hidroxitolueno Butilado/farmacologia , Cisteína/genética , Cisteína/metabolismo , Ditiotreitol/farmacologia , Ácidos Graxos não Esterificados/antagonistas & inibidores , Humanos , Microscopia Eletrônica , Mutagênese Sítio-Dirigida , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas tau/antagonistas & inibidores , Proteínas tau/genética , Proteínas tau/ultraestruturaRESUMO
Tissue transglutaminase (tTG) is up-regulated in Alzheimer's disease brain and localizes to neurofibrillary tangles with the tau protein. Tau is an in vitro tTG substrate, being cross-linked and/or polyaminated. Further, the Gln and Lys residues in tau that are modified by tTG in vitro are located primarily within or adjacent to the microtubule-binding domains. Considering these and other previous findings, this study was carried out to determine if tau is modified in situ by tTG in human neuroblastoma SH-SY5Y cells, and whether tTG-catalyzed tau polyamination modulates the function and/or metabolism of tau in vitro. For these studies, SH-SY5Y cells stably overexpressing tTG were used. tTG coimmunoprecipitated with tau, and elevating intracellular calcium levels with maitotoxin resulted in a 52 +/- 4% increase in the amount of tTG that coimmunoprecipitated with tau. The increase in association of tTG with tau after treatment with maitotoxin corresponded to a coimmunolocalization of tTG, tTG activity, and tau in the cells. Further, tau was modified by tTG in situ in response to maitotoxin treatment. In vitro polyaminated tau was significantly less susceptible to micro-calpain proteolysis; however, tTG-mediated polyamination of tau did not significantly alter the microtubule-binding capacity of tau. Thus, tau interacts with and is modified by tTG in situ, and modification of tau by tTG alters its metabolism. These data indicate that tau is likely to be modified physiologically and pathophysiologically by tTG, and tTG may play a role in Alzheimer's disease.
Assuntos
Oxocinas , Transglutaminases/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/enzimologia , Animais , Cálcio/metabolismo , Calpaína/metabolismo , Cobaias , Humanos , Técnicas de Imunoadsorção , Toxinas Marinhas/farmacologia , Microtúbulos/metabolismo , Neuroblastoma , Emaranhados Neurofibrilares/enzimologia , Poliaminas/metabolismo , Proteínas Recombinantes/metabolismo , Células Tumorais CultivadasRESUMO
A large family of isoquinoline sulfonamide compounds inhibits protein kinases by competing with adenosine triphosphates(ATP), yet interferes little with the activity of other ATP-using enzymes such as ATPases and adenylate cyclases. One such compound, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CK17), is selective for casein kinase-1 isolated from a variety of sources. Here we report the crystal structure of the catalytic domain of Schizosaccharomyces pombe casein kinase-1 complexed with CK17, refined to a crystallographic R-factor of 17.8% at 2.5 angstrom resolution. The structure provides new insights into the mechanism of the ATP-competing inhibition and the origin of their selectivity toward different protein kinases. Selectivity for protein kinases versus other enzymes is achieved by hydrophobic contacts and the hydrogen bond with isoquinoline ring. We propose that the hydrogen bond involving the ring nitrogen-2 atom of the isoquinoline must be preserved, but that the ring can flip depending on the chemical substituents at ring positions 5 and 8. Selectivity for individual members of the protein kinase family is achieved primarily by interactions with these substituents.
Assuntos
Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases , Sulfonamidas , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Inibidores Enzimáticos/química , Ligação de Hidrogênio , Isoquinolinas/química , Isoquinolinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate.
Assuntos
Modelos Moleculares , Conformação Proteica , Proteínas Quinases/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Caseína Quinases , Cristalografia por Raios X , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Schizosaccharomyces/enzimologia , Alinhamento de Sequência , Deleção de Sequência , Especificidade por SubstratoRESUMO
We have examined the activity and substrate specificity of the Saccharomyces cerevisiae Hrr25p and the Schizosaccharomyces pombe Hhp1, Hhp2, and Cki1 protein kinase isoforms. These four gene products are isotypes of casein kinase I (CKI), and the sequence of these protein kinases predicts that they are protein serine/threonine kinases. However, each of these four protein kinases, when expressed in Escherichia coli in an active form, was recognized by anti-phosphotyrosine antibodies. Phosphoamino acid analysis of 32P-labeled proteins showed phosphorylation on serine, threonine, and tyrosine residues. The E. coli produced forms of Hhp1, Hhp2, and Cki1 were autophosphorylated on tyrosine, and both Hhp1 and Hhp2 were capable of phosphorylating the tyrosine-protein kinase synthetic peptide substrate polymer poly-E4Y1. Immune complex protein kinases assays from S. pombe cells showed that Hhp1-containing precipitates were associated with a protein-tyrosine kinase activity, and the Hhp1 present in these immunoprecipitates was phosphorylated on tyrosine residues. Although dephosphorylation of Hhp1 and Hhp2 by Ser/Thr phosphatase had little effect on the specific activity, tyrosine dephosphorylation of Hhp1 and Hhp2 caused a 1.8-to 3.1-fold increase in the Km for poly-E4Y1 and casein. These data demonstrate that four different CKI isoforms from two different yeasts are capable of protein-tyrosine kinase activity and encode dual-specificity protein kinases.
Assuntos
Isoenzimas/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Caseína Quinases , Genes Fúngicos , Isoenzimas/genética , Dados de Sequência Molecular , Peptídeos/química , Fosforilação , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Especificidade por Substrato , TirosinaRESUMO
Two cDNAs encoding casein kinase-1 have been isolated from a yeast cDNA library and termed CKI1 and CKI2. Each clone encodes a protein of approximately 62,000 Da containing a highly conserved protein kinase domain surrounded by variable amino- and carboxy-terminal domains. The proteins also contain two conserved carboxy-terminal cysteine residues that comprise a consensus sequence for prenylation. Consistent with this posttranslational modification, cell fractionation experiments demonstrate that intact CKI1 is found exclusively in yeast cell membranes. Gene disruption experiments reveal that, although neither of the two CKI genes is essential by itself, at least one CKI gene is required for yeast cell viability. Spores deficient in both CKI1 and CKI2 fail to grow and, therefore, either fail to germinate or arrest as small cells before bud emergence. These results suggest that casein kinase-1, which is distributed widely in nature, plays a pivotal role in eukaryotic cell regulation.
Assuntos
Genes Fúngicos/genética , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Caseína Quinases , Membrana Celular/enzimologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Dados de Sequência Molecular , Fenótipo , Proteínas Quinases/química , Proteínas Quinases/isolamento & purificação , Saccharomyces cerevisiae/enzimologiaRESUMO
A mutant catalytic subunit of adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase has been isolated from Saccharomyces cerevisiae that is no longer subject to regulation yet retains its catalytic activity. Biochemical analysis of the mutant subunit indicates a 100-fold decreased affinity for the regulatory subunit. The mutant catalytic subunit exhibits approximately a threefold increase in Michaelis constant for adenosine triphosphate and peptide cosubstrates, and is essentially unchanged in its catalytic rate. The nucleotide sequence of the mutant gene contains a single nucleotide change resulting in a threonine-to-alanine substitution at amino acid 241. This residue is conserved in other serine-threonine protein kinases. These results identify this threonine as an important contact between catalytic and regulatory subunits but only a minor contact in substrate recognition.
Assuntos
AMP Cíclico/farmacologia , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Catálise , Genes Fúngicos , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Fosforilação , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Relação Estrutura-Atividade , TreoninaRESUMO
Rat liver nuclei contain a protein phosphatase that is indistinguishable from the catalytic subunit of protein phosphatase-1 in its molecular mass, sensitivity to inhibitor-1 and inhibitor-2 and specificity for the beta-subunit of phosphorylase kinase. This activity is not bound to the outer nuclear membrane, but located within the nucleus. The average level of protein phosphatase-1 activity in nuclei is at least 5-fold higher than its average extranuclear concentration.
Assuntos
Fígado/enzimologia , Fosfoproteínas Fosfatases/análise , Animais , Núcleo Celular/enzimologia , AMP Cíclico/fisiologia , Feminino , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilase Fosfatase/análise , Proteínas Quinases/análise , Proteína Fosfatase 1 , Ratos , Ratos EndogâmicosRESUMO
A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.