RESUMO

It was proposed that insulin-degrading enzyme (IDE) participates in the clearance of amyloid beta (Abeta) in the brain, and its low expression or activity may be relevant for the progression of Alzheimer disease. We performed a longitudinal study of brain level, activity, and distribution of IDE in transgenic mice (Tg2576) expressing the Swedish mutation in human Abeta precursor protein. At 16 months of age, Tg2576 showed a significant 2-fold increment in IDE protein level as compared with 4.5- and 11-month-old animals. The peak of IDE was in synchrony with the sharp accumulation of sodium dodecyl sulfate-soluble Abeta and massive Abeta deposition into plaques. At this stage, IDE appeared surrounding Abeta fibrillar deposits within glial fibrillar acidic protein-positive astrocytes, suggesting that it was locally overexpressed during the Abeta-mediated inflammation process. When primary astrocytes were exposed to fibrillar Abeta in vitro, IDE protein level increased as compared with control, and this effect was reduced by the addition of U0126, a specific inhibitor of the ERK1/2 mitogen-activated protein kinase cascade. We propose that in Tg2576 mice and in contrast to its behavior in Alzheimer brains, active IDE increases with age around plaques as a component of astrocyte activation as a result of Abeta-triggered inflammation.

Assuntos

Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/metabolismo , Insulisina/biossíntese , Placa Amiloide/metabolismo , Fatores Etários , Doença de Alzheimer/patologia , Animais , Astrócitos/metabolismo , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa

RESUMO

Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant disorders characterized by cerebrovascular and parenchymal amyloid deposition and neurofibrillary degeneration. In both conditions, the genetic defects cause the loss of the normal stop codon in the precursor BRI, generating novel 34-residue peptides named ABri and ADan in FBD and FDD, respectively. ABri and ADan show a strong tendency to aggregate into non-fibrillar and fibrillar structures at neutral pH and this property seems to be directly related to neurotoxicity. Here we report that a recombinant insulin-degrading enzyme (rIDE) was capable of degrading monomeric ABri and ADan in vitro more efficiently than oligomeric species. These peptides showed high beta-structure content and were more resistant to proteolysis as compared to the BRI wild-type product of 23 amino acids. Specific sites of cleavage within the C-terminal pathogenic extensions raise the possibility that proteolysis of monomeric soluble precursors by IDE may delay ABri and ADan aggregation in vivo.

Assuntos

Amiloide/genética , Amiloide/metabolismo , Demência/genética , Demência/metabolismo , Insulisina/metabolismo , Mutação , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Amiloide/química , Animais , Sítios de Ligação/genética , Cisteína/química , Dinamarca , Genes Dominantes , Humanos , Técnicas In Vitro , Insulisina/genética , Glicoproteínas de Membrana , Proteínas de Membrana , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Reino Unido

RESUMO

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.

Assuntos

Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Angiopatia Amiloide Cerebral/genética , Insulisina/fisiologia , Adulto , Animais , Western Blotting , Encéfalo/patologia , Angiopatia Amiloide Cerebral/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Imunoprecipitação , Insulina/metabolismo , Insulisina/química , Cinética , Espectrometria de Massas , Microcirculação , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mutação , Peptídeos/química , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Fatores de Tempo

RESUMO

Paramyosin, a vaccine candidate in different helminthiases, was purified from the adult liver fluke Fasciola hepatica using two different procedures. The first started with a crude extraction of paramyosin in high-salt buffer followed by gel filtration chromatography and two precipitation-solubilization cycles; in the second, anion exchange chromatography replaced the gel filtration step. In both cases, the apparent molecular weight of the purified protein determined by sodium dodecyl sulfate gel electrophoresis under reducing and non-reducing conditions was 97 kDa and 200 kDa, respectively. The molecular weights were consistent with the presence of a dimeric protein linked by disulfide bridges. Western blot analysis showed that the dimeric and monomeric forms were both recognized by an antiserum raised against the F. hepatica 97 kDa band (alpha-FhPmy), and by an anti- Schistosoma mansoni paramyosin immune serum. Immunohistochemistry using alpha-FhPmy demonstrated the localization of paramyosin within the subtegumental muscle and in muscle cells surrounding the gut of adult parasites. We also observed labeling of extramuscular structures like testes, surface lamellae of the gut and the tegument of adult flukes.

Assuntos

Fasciola hepatica/metabolismo , Tropomiosina/isolamento & purificação , Tropomiosina/metabolismo , Animais , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Precipitação Fracionada , Proteínas de Helminto/análise , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Proteínas de Helminto/metabolismo , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Masculino , Peso Molecular , Células Musculares/imunologia , Células Musculares/metabolismo , Testículo/imunologia , Testículo/metabolismo , Tropomiosina/análise , Tropomiosina/química

RESUMO

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments.

Assuntos

Cisteína Endopeptidases/metabolismo , Fragmentos de Imunoglobulinas/análise , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Humanos , Immunoblotting , Imunoglobulina G/classificação , Dados de Sequência Molecular , Proteínas de Protozoários

RESUMO

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.

Assuntos

Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Insulisina/metabolismo , Animais , Hemorragia Cerebral/enzimologia , Hemorragia Cerebral/patologia , Demência/genética , Dimerização , Saúde da Família , Variação Genética , Humanos , Fragmentos de Peptídeos/análise , Ratos , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Acidente Vascular Cerebral/genética

RESUMO

Amiloidose é um termo genérico que descreve amplo espectro de doenças caracterizadas pela deposiçao de proteínas fibrilares insolúveis em diversos orgaos. As fibrilas depositadas compoem-se predominantemente de proteínas de baixo peso molecular que sao normalmente solúveis sob condiçoes fisiológicas. Todos os tipos de amilóide compartilham propriedades físicas, estruturais e tintoriais comuns: conformaçao em placa beta, alto grau de insolubilidade e aparência fibrilar à microscopia eletrônica. Uma vez formadas, as fibrilas amilóides sao resistentes à degradaçao proteolítica pelo sistema mononuclear fagocítico. Como resultado, o acúmulo de fibrilas leva a dano celular e disfunçao de órgaos. Embora 18 proteínas diferentes e mais de 60 variantes genéticas já tenham sido identificadas e caracterizadas bioquimicamente como constituintes expressivos dos depósitos amilóides, os mecanismos patogenéticos responsáveis pela formaçao das fibrilas permanecem desconhecidos. Diversos fatores de risco para amiloidogênese têm sido reconhecidos, entre os quais a presença de substituiçoes de aminoácidos nas moléculas precursoras, modificaçoes pós-traduçao relacionadas ao envelhecimento, bem como alteraçoes das vias normais de degradaçao protéica. A colocalizaçao de um grupo de proteínas nao relacionadas entre si (denominadas proteínas amilóide-associadas) com as fibrilas nos depósitos sugere importante papel modulador dessas proteínas na fibrilogênese

Assuntos

Amiloidose , Febre Familiar do Mediterrâneo