RESUMO
Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.
Assuntos
Precursor de Proteína beta-Amiloide , Terapêutica com RNAi , Animais , Camundongos , Primatas/genética , Primatas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
The aryl hydrocarbon receptor (AhR) exists in the absence of a ligand as a tetrameric complex composed of a 95-105 kDa ligand binding subunit, a dimer of hsp90, and the immunophilin-like X-associated protein 2 (XAP2). XAP2 has a highly conserved carboxy terminal tetratricopeptide repeat domain that is required for both hsp90 and AhR binding. Hsp 90 appears to be involved in the initial folding of newly synthesized AhR, stabilization of ligand binding conformation of the receptor, and inhibition of constitutive dimerization with ARNT. XAP2 is capable of stabilizing the AhR, as well as enhancing cytoplasmic localization of the receptor. XAP2 binds to both the AhR and hsp90 in the receptor complex, and is capable of independently binding to both hsp90 and the AhR. However, the exact functional role for XAP2 in the AhR complex remains to be fully established.
Assuntos
Proteínas/metabolismo , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Sequência de Aminoácidos , Animais , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Subunidades ProteicasRESUMO
To further understand the role that the hepatitis B virus X-associated protein 2 (XAP2) plays in regulating aryl hydrocarbon receptor (AhR) function, a point mutation was introduced at tyrosine 408 of the AhR, changing the residue to an alanine or lysine. These mutations resulted in the loss of AhR binding to endogenous XAP2 in COS-1 cells and reduced binding of exogenously expressed XAP2. Cellular localization of the mutant AhR-yellow fluorescent protein fusion proteins remained nuclear when XAP2 was co-expressed, while the non-mutant receptor was redistributed to the cytoplasm. XAP2 expression caused an overall repression of constitutive and ligand-induced AhR transcriptional activity. However, increased expression of XAP2 had no effect on the AhRY408A mutant transcriptional activity. Additionally the XAP2 binding-deficient AhR mutants showed overall higher transcriptional activity when compared with the non-mutant receptor. Interestingly reduced incorporation of the Hsp90 associated co-chaperone p23 in the unliganded AhR complex was observed with increasing XAP2 expression. The displacement of p23 from Hsp90 did not occur when increasing levels of XAP2 were introduced in COS-1 cells in the absence of the AhR; thus this displacement event occurs specifically within an AhR complex. Finally XAP2 itself was capable of existing in multimeric complexes, and these complexes did not require Hsp90 or AhR to form. However, it is not yet clear whether XAP2 can exist within the AhR complex in more than one copy.
Assuntos
Proteínas de Choque Térmico HSP90/fisiologia , Chaperonas Moleculares/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Proteínas/fisiologia , Receptores de Hidrocarboneto Arílico/fisiologia , Transcrição Gênica , Animais , Células COS , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Chaperonas Moleculares/metabolismo , Fosfoproteínas/metabolismo , Dibenzodioxinas Policloradas/farmacologiaRESUMO
We have synthesized [(125)I]4'-iodoflavone to study Ah receptor (AhR)-ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). This radioligand allows the comparison of AhR-ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin. Specific binding of [(125)I]4'-iodoflavone with the AhR was detected as a single radioactive peak ( approximately 9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A approximately 6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [(125)I]4'-iodoflavone-AhR 6 S complex, compared with [(125)I]2-iodo-7,8-dibromodibenzo-p-dioxin, was recovered in nuclear extracts. A comparison of the ability of 4'-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4'-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [(125)I]4'-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR.
Assuntos
Flavonoides/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ligação Competitiva , Citosol/metabolismo , Flavonoides/química , Células HeLa , Humanos , Radioisótopos do Iodo , Ligantes , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Dibenzodioxinas Policloradas/química , Dibenzodioxinas Policloradas/metabolismo , Transporte Proteico , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/química , Espectrometria de Fluorescência , Células Tumorais CultivadasRESUMO
The mouse aryl hydrocarbon receptor (mAhR) is a ligand-activated transcription factor that exists in a tetrameric, core complex with a dimer of the 90-kDa heat shock protein, and the hepatitis B virus X-associated protein 2 (XAP2). Transiently expressed mAhR-YFP (yellow fluorescent protein fused with the mAhR) localizes throughout cells, with a majority occupying nuclei. Co-expression of XAP2 with mAhR-YFP results in a distinct redistribution to the cytoplasm. We have utilized several approaches to attempt to identify the mechanism by which XAP2 modulates the sub-cellular localization of the mAhR. The nuclear export inhibitor, leptomycin B, was used to demonstrate that XAP2 inhibits ligand-independent nucleocytoplasmic shuttling of the receptor. Results from cytoskeletal disruption and the addition of an alternate nuclear localization sequence (NLS) to mAhR-YFP suggest that XAP2 does not physically tether the complex in the cytoplasm. The use of a rabbit polyclonal antibody raised against a portion of the bipartite NLS of the mAhR revealed that XAP2 does not appear to block access to the NLS. However, XAP2 hinders importin beta binding to the mAhR complex, suggesting that XAP2 alters the conformation of the bipartite NLS of mAhR. XAP2 also represses the transactivation potential of the AhR, in contrast to previously published reports, perhaps by stabilizing the receptor complex and/or blocking nucleocytoplasmic shuttling of the AhR complex.
Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas/química , Transcrição Gênica , beta Carioferinas/metabolismo , Actinas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Células COS , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citosol/metabolismo , Deleção de Genes , Vetores Genéticos , Proteínas de Choque Térmico HSP90/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Camundongos , Microscopia de Fluorescência , Sinais de Localização Nuclear , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Transfecção , Tubulina (Proteína)/metabolismoRESUMO
The cytosolic Ah receptor (AhR) heterocomplex consists of one molecule of the AhR, a 90-kDa heat shock protein (Hsp90) dimer, and one molecule of the hepatitis B virus X-associated protein 2 (XAP2). Serine residues 43,53,131-2, and 329 on XAP2-FLAG were identified as putative phosphorylation sites using site-directed mutagenesis followed by two-dimensional phosphopeptide mapping analysis. Protein kinase CK2 (CK2) was identified as the 45-kDa kinase from COS 1 cell or liver extracts that was responsible for phosphorylation of serine 43 in the XAP2 peptide 39-57. Loss of phosphorylation at any or all of the serine residues did not significantly affect the ability of XAP2-FLAG to bind to the murine AhR in rabbit reticulocyte lysate or Hsp90 in COS-1 cells. Furthermore, all of these serine mutants were able to sequester murine AhR-YFP into the cytoplasm as well as wild-type XAP2. YFP-XAP2 S53A was unable to enter the nucleus, indicating a potential role of phosphorylation in nuclear translocation of XAP2.
Assuntos
Vírus da Hepatite B/metabolismo , Proteínas/metabolismo , Serina , Alanina , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Mapeamento de Peptídeos , Fosfopeptídeos/química , Fosforilação , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
The aryl hydrocarbon receptor (AhR) mediates the toxicologic and carcinogenic properties of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In the cytoplasm, the AhR is complexed with a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2). Most studies that have examined the ability of XAP2 to modulate the AhR have characterized the mouse receptor (mAhR). However, the amino acid sequence of mAhR is significantly different from human AhR (hAhR) in the carboxy terminal half of the protein, and this could lead to differences in the behavior of the two receptors. mAhR-yellow fluorescent protein (YFP) and hAhR-YFP were used to compare nucleocytoplasmic shuttling properties and the ability of XAP2 to modulate their activity. As reported previously, mAhR localized predominantly in the nucleus and was redistributed to the cytoplasm by coexpression of XAP2 in COS-1 cells. Leptomycin B treatment revealed that XAP2 blocked mAhR-YFP translocation to the nucleus in the absence of ligand. In contrast, hAhR-YFP localized predominantly in the cytoplasm, and coexpression of XAP2 did not affect this localization, and did not block nuclear accumulation in the presence of leptomycin B. An XAP2 fusion protein with a nuclear localization signal fused to the carboxy terminus (XAP2-NLS) was utilized to test whether this protein could drag the AhR into the nucleus. Coexpression of mAhR-YFP and XAP2-NLS caused cytoplasmic localization of the mAhR, while hAhR-YFP was partially localized in the nucleus, suggesting that XAP2 remains bound to the hAhR during nucleocytoplasmic shuttling. The presence of XAP2 in the ligand-bound hAhR complex enhanced the rate of nuclear translocation but repressed transcriptional activity. Together, these results suggest that the hAhR differs biochemically from the mAhR.