RESUMO
When overexpressed, a short cytoplasmic domain of the amyloid precursor protein (APP), normally unmasked in the brain of Alzheimer's disease patients, activates caspase-3 and induces neuronal death. Death induction by this "Jcasp" domain is lost when tyrosine 653 is changed into an aspartate, suggesting specific interactions with unknown partners. To identify these putative partners and start to elucidate the mechanisms involved in Jcasp-induced cell death, we internalized a biotinylated version of the peptide into primary neurons and analyzed intracellular interacting proteins by pull-down and mass spectrometry. We find that SET protein, also called template-activating factor (TAF1beta) or phosphatase 2A inhibitor 2 (I2(PP2A)), specifically binds Jcasp early after internalization and that SET and Jcasp interact directly in vitro. Down-regulation of SET reduces Jcasp-induced cell death, confirming a role of this protein in Jcasp-induced apoptosis. Conversely, SET gain of function increases cell death, which suggests that SET level is crucial for neuronal survival/death. Taken together, these results suggest that SET is part of a neuronal apoptotic pathway related to Alzheimer's disease and provides a new entry in the analysis of this pathology.
Assuntos
Precursor de Proteína beta-Amiloide/química , Apoptose , Proteínas Cromossômicas não Histona/fisiologia , Citoplasma/metabolismo , Neurônios/patologia , Fatores de Transcrição/fisiologia , Doença de Alzheimer/metabolismo , Animais , Ácido Aspártico/química , Bioensaio , Encéfalo/embriologia , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Proteínas de Ligação a DNA , Regulação para Baixo , Chaperonas de Histonas , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Modelos Biológicos , Neurônios/metabolismo , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes/química , Coloração pela Prata , Tirosina/químicaRESUMO
This report presents the first hsp90 complete cDNA sequences from two Lepidoptera. The Bombyx mori full sequence was reconstituted from 15 partial cDNA clones belonging to expressed sequence tag libraries obtained from different tissues or cultured cells, thus showing the ubiquitous expression of the hsp90 gene. The Spodoptera frugiperda cDNA was isolated as a full-length clone from a cDNA library established from the Sf9 cell line. Both cDNAs are highly homologous and display the classical amino acid (aa) stretches representing the HSP90 signature. They potentially encode a 716 aa (B. mori) and a 717 aa (S. frugiperda) protein, with a calculated molecular mass of 83 kDa similar to the Drosophila homologous protein. We show that, unlike the vertebrates, hsp90 is a unique gene in both S. frupiperda and B. mori genomes. Sequencing of the corresponding genomic region shows that, contrary to the dipteran homologous gene, the lepidopteran hsp90 gene does not display any intron. Phylogenetic analysis based on the two lepidopteran and 23 other HSP90 aa sequences displays a high consistency with known phylogeny at both high and low taxonomic levels. Transcriptional analysis performed in S. frugiperda shows that the induction of the hsp90 gene only occurs 14 degrees C above physiological growth conditions (42 degrees C).