RESUMO
Proteolytic processing of the amyloid precursor protein (APP) occurs via two alternative pathways, localized to different subcellular compartments, which result in functionally distinct outcomes. Cleavage by a beta-gamma sequence generates the Abeta peptide that plays a central role in Alzheimer's disease. In the case of alpha-gamma cleavage, a secreted neurotrophic molecule is generated and the Abeta peptide cleaved and destroyed. In both cases, a cytosolic APP intracellular domain (AICD) is generated. We have previously shown that coexpression of APP with the APP-binding protein Fe65 and the histone acetyltransferase Tip60 results in the formation of nuclear complexes (termed AFT complexes), which localize to transcription sites. We now show that blocking endocytosis or the pharmacological or genetic inhibition of the endosomal beta-cleavage pathway reduces translocation of AICD to these nuclear AFT complexes. AICD signaling further depends on active transport along microtubules and can be modulated by interference with both anterograde and retrograde transport systems. Nuclear signaling by endogenous AICD in primary neurons could similarly be blocked by inhibiting beta-cleavage but not by alpha-cleavage inhibition. This suggests that amyloidogenic cleavage, despite representing the minor cleavage pathway of APP, is predominantly responsible for AICD-mediated nuclear signaling.
Assuntos
Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Núcleo Celular/metabolismo , Espaço Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Linhagem Celular , Membrana Celular/metabolismo , Endocitose , Endossomos/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transporte ProteicoRESUMO
The amyloid precursor protein (APP) as well as its homologues, APP-like protein 1 and 2 (APLP1 and APLP2), are cleaved by α-, ß-, and γ-secretases, resulting in the release of their intracellular domains (ICDs). We have shown that the APP intracellular domain (AICD) is transported to the nucleus by Fe65 where they jointly bind the histone acetyltransferase Tip60 and localize to spherical nuclear complexes (AFT complexes), which are thought to be sites of transcription. We have now analyzed the subcellular localization and turnover of the APP family members. Similarly to AICD, the ICD of APLP2 localizes to spherical nuclear complexes together with Fe65 and Tip60. In contrast, the ICD of APLP1, despite binding to Fe65, does not translocate to the nucleus. In addition, APLP1 predominantly localizes to the plasma membrane, whereas APP and APLP2 are detected in vesicular structures. APLP1 also demonstrates a much slower turnover of the full-length protein compared to APP and APLP2. We further show that the ICDs of all APP family members are degraded by the proteasome and that the N-terminal amino acids of ICDs determine ICD degradation rate. Together, our results suggest that different nuclear signaling capabilities of APP family members are due to different rates of full-length protein processing and ICD proteasomal degradation. Our results provide evidence in support of a common nuclear signaling function for APP and APLP2 that is absent in APLP1, but suggest that APLP1 has a regulatory role in the nuclear translocation of APP family ICDs due to the sequestration of Fe65.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Núcleo Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Estrutura Terciária de Proteína/fisiologia , Transdução de Sinais/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Western Blotting , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Histona Acetiltransferases/metabolismo , Humanos , Imuno-Histoquímica , Lisina Acetiltransferase 5 , Microscopia Confocal , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genéticaRESUMO
Bioavailability is a quantitative measure of the capacity of a drug to reach systemic circulation. However, if a drug target is localized in a subcellular organelle, then the drug may not be able to reach it and the effect of the drug will not be attained. Although most drug targets are localized within intracellular compartments, specific targeting of drugs at the subcellular level is not well established. Membrane proteins, lipids, nutrients and some pathogens are internalized into the cell to be targeted to distinct subcellular compartments via membrane trafficking. Recent advances have identified novel methods of subcellular drug targeting, involving the use of conjugation to ligands of cell surface receptors or to lipid anchors. In this review, we focus on the importance of subcellular targeting of drugs, in particular, the mechanism of lipid-anchoring as a novel strategy and its potential application for the treatment of several diseases.
Assuntos
Espaço Intracelular/metabolismo , Lipídeos/administração & dosagem , Proteínas de Membrana Transportadoras/metabolismo , Preparações Farmacêuticas/administração & dosagem , Receptores de Superfície Celular/metabolismo , Animais , Sistemas de Liberação de Medicamentos/métodos , Humanos , Lipídeos/químicaRESUMO
The beta-amyloid precursor protein (APP) plays a major role in Alzheimer's disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes, which may represent sites of transcription. Despite a lack of co-localization with several described nuclear compartments, we have identified a close apposition between AFT complexes and splicing speckles, Cajal bodies and PML bodies. Live imaging revealed that AFT complexes were highly mobile within nuclei and following pharmacological inhibition of transcription fused into larger assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. In support of our earlier findings, transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Neurônios/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Encéfalo/fisiopatologia , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Células Cultivadas , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Humanos , Hibridização in Situ Fluorescente , Lisina Acetiltransferase 5 , Substâncias Macromoleculares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plasmídeos , Regiões Promotoras Genéticas/genética , Estrutura Terciária de Proteína/fisiologia , Receptores Notch/química , Transdução de Sinais/fisiologia , Transativadores , Ativação Transcricional/fisiologiaRESUMO
Endothelial cell apoptosis can be initiated by withdrawing growth factors or serum, and is inhibited by HDL. Our results show that the total lipoprotein population from apolipoprotein E 4/4 (APOE4/4) sera is less anti-apoptotic than total lipoproteins from other APOE genotypes, as measured by caspase 3/7 activity. Moreover, APOE4/4 VLDL antagonizes the antiapoptotic activity of HDL by a mechanism requiring binding of apoE4 on VLDL particles to an LDL family receptor. This ability of APOE4/4 VLDL to inhibit the antiapoptotic effects of HDL presents a potential mechanism by which the expression of several diseases, including atherosclerosis, is enhanced by the APOE4 genotype.