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1.
Biochem Biophys Res Commun ; 388(2): 434-8, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19682432

RESUMO

Regulation of proteolytic cleavage of the amyloid precursor protein by the aspartic protease BACE may occur by alternative splicing and the generation of enzymatically inactive forms. In fact, the presence of exonic donor and acceptor sites for intron 3 generates the two deficient variants BACE457 and BACE476. In HEK293 cells, when introns are inserted separately in the BACE cDNA, we found that whilst introns 2 and 4 are efficiently spliced out, intron 3 is not removed. On the other hand, splicing to wild-type BACE is restored when intron 3 is flanked by the two other introns. The presence of all three introns also leads to alternative splicing of intron 3 and the generation of BACE476. In contrast, BACE457 expression takes place only after mutating the donor splice site of intron 3, indicating that additional regulatory elements are necessary for the use of the splicing site within exon 4. Overall, our data demonstrate that a complex splicing of intron 3 regulates the maturation of the BACE mRNA. This appears orchestrated by domains present in the exons and introns flanking intron 3. Excessive BACE activity is a risk factor for Alzheimer's disease, therefore this complex regulation might guarantee low neuronal BACE activity and disease prevention.


Assuntos
Processamento Alternativo , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Íntrons/genética , Sequência de Bases , Humanos , RNA Mensageiro/metabolismo
2.
Anal Biochem ; 395(1): 8-15, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19664996

RESUMO

The genetic mutation causing Huntington's disease is a polyglutamine expansion in the huntingtin protein where more than 37 glutamines cause disease by formation of toxic intracellular fragments, aggregates, and cell death. Despite a clear pathogenic role for mutant huntingtin, understanding huntingtin expression during the presymptomatic phase of the disease or during disease progression has remained obscure. Central to clarifying the role in the pathomechanism of disease is the ability to easily and accurately measure mutant huntingtin in accessible human tissue samples as well as cell and animal models. Here we describe a highly sensitive time-resolved Förster resonance energy transfer (FRET) assay for quantification of soluble mutant huntingtin in brain, plasma, and cerebrospinal fluid. Surprisingly, in mice, soluble huntingtin levels decrease during disease progression, inversely correlating with brain aggregate load. Mutant huntingtin is easily detected in human brain and blood-derived fractions, providing a utility to assess mutant huntingtin expression during disease course as well as a pharmacodynamic marker for disease-modifying therapeutics targeting expression, cleavage, or degradation of mutant huntingtin. The design of the homogeneous one-step method for huntingtin detection is such that it can be easily applied to measure other proteins of interest.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Doença de Huntington/diagnóstico , Proteínas Mutantes/isolamento & purificação , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Adulto , Análise de Variância , Animais , Encéfalo/metabolismo , Linhagem Celular , Progressão da Doença , Células-Tronco Embrionárias/metabolismo , Éxons , Feminino , Expressão Gênica , Humanos , Proteína Huntingtina , Doença de Huntington/sangue , Doença de Huntington/líquido cefalorraquidiano , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Proteínas Mutantes/metabolismo , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão , Sensibilidade e Especificidade , Fatores de Tempo , Adulto Jovem
3.
J Cell Biol ; 168(6): 863-8, 2005 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-15767460

RESUMO

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Anticorpos/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Anticorpos/química , Anticorpos Monoclonais/metabolismo , Ligação Competitiva , Western Blotting , Extratos Celulares , Linhagem Celular , Meios de Cultivo Condicionados/análise , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Cinética , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Testes de Precipitina , Especificidade por Substrato , Transfecção
4.
J Neurochem ; 80(5): 799-806, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11948243

RESUMO

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Western Blotting , Endopeptidases , Expressão Gênica , Humanos , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Transgenes
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