RESUMO
BACKGROUND: The neuronal adaptor protein X11α is a multidomain protein with a phosphotyrosine binding (PTB) domain, two PDZ (PSD_95, Drosophila disks-large, ZO-1) domains, a Munc Interacting (MI) domain and a CASK interacting region. Amongst its functions is a role in the regulation of the abnormal processing of the amyloid precursor protein (APP). It also regulates the activity of Cu/Zn Superoxide dismutase (SOD1) through binding with its chaperone the copper chaperone for SOD1. How X11α production is controlled has remained unclear. METHODS: Using the neuroblastoma cell line, N2a, and knockdown studies, the effect of the cellular and infectious prion protein, PrP(C) and PrP(Sc), on X11α is examined. RESULTS: We show that X11α expression is directly proportional to the expression of PrP(C), whereas its levels are reduced by PrP(Sc). We also show PrP(Sc) to affect X11α at a functional level. One of the effects of prion infection is lowered cellular SOD1 levels, here by knockdown of X11α we identify that the effect of PrP(Sc) on SOD1 can be reversed indicating that X11α is involved in prion disease pathogenesis. CONCLUSIONS: A role for the cellular and infectious prion protein, PrP(C) and PrP(Sc), respectively, in regulating X11α is identified in this work. GENERAL SIGNIFICANCE: Due to the multiple interacting partners of X11α, dysfunction or alteration in X11α will have a significant cellular effect. This work highlights the role of PrP(C) and PrP(Sc) in the regulation of X11α, and provides a new target pathway to control X11α and its related functions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Príons/fisiologia , Animais , Linhagem Celular Tumoral , Células Cultivadas , Camundongos , Superóxido Dismutase/fisiologia , Superóxido Dismutase-1RESUMO
Western blotting is a significant tool employed for the detection of cell proteins. High-molecular-weight proteins have proven a challenge to detect by western blotting, but proteins even of 100 KDa can still present difficulties in detection. This work reports the development of a heat transfer method that is suitable for both low- and high-molecular-weight proteins. The procedure involves the use of a constant temperature at 78 °C in a dedicated heat transfer module. Through the use of this protocol the neuronal adaptor protein X11α (120 KDa), which prior to this methodology was undetectable endogenously in the neuroblastoma cell line (N2a), was successfully detected in the N2a cell line. The procedure provides a reproducible protocol that can be adapted for other high-molecular-weight proteins, and it provides the advantage that low-molecular-weight proteins are not sacrificed by the methodology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Western Blotting/métodos , Temperatura Alta , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Animais , Camundongos , Peso Molecular , Células Tumorais CultivadasRESUMO
Prion disorders are associated with the accumulation of a misfolded form (PrP(Sc)) of the normal prion protein, PrP(C). Here, we show that estrogen acts as a regulator of the processes of both prion infection and prion maintenance. Estrogen was found to be cell biased in its effect; it protected cells against prion infection in a prevention mode and enabled prion maintenance in a treatment mode. These processes were regulated by the estrogen receptor subtypes Erα and Erß. By using specific receptor agonists, Erα was found to be the main receptor active in slowing prion infection, whereas in chronically infected cells, although Erα allowed partial maintenance of PrP(Sc) levels, Erß was the main receptor involved in maintaining PrP(Sc) in a treatment paradigm. A cell-biased effect of estrogen has been reported for other neurodegenerative disorders, including Alzheimer's disease. Estrogen's effect is dependent on the cell's health status, which impacts the use of estrogen. This work also identified that by targeting the estrogen receptors with the selective estrogen receptor modulators tamoxifen (Tam) and 4-hydroxy-tamoxifen (OHT), PrP(Sc) could be cleared from prion-infected cell culture. Tam and OHT had half-maximal inhibitory concentrations for clearance of PrP(Sc) of 0.47 µM and 0.14 nM, respectively. This work identifies further factors involved in the prion disease process, and through antagonism of the estrogen system, we demonstrate that the estrogen system is a target for controlling PrP(Sc) levels.
Assuntos
Estrogênios/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Animais , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , CamundongosRESUMO
The events leading to the degradation of the endogenous PrP(C) (normal cellular prion protein) have been the subject of numerous studies. Two cleavage processes, α-cleavage and ß-cleavage, are responsible for the main C- and N-terminal fragments produced from PrP(C). Both cleavage processes occur within the N-terminus of PrP(C), a region that is significant in terms of function. α-Cleavage, an enzymatic event that occurs at amino acid residues 110 and 111 on PrP(C), interferes with the conversion of PrP(C) into the prion disease-associated isoform, PrP(Sc) (abnormal disease-specific conformation of prion protein). This processing is seen as a positive event in terms of disease development. The study of ß-cleavage has taken some surprising turns. ß-Cleavage is brought about by ROS (reactive oxygen species). The C-terminal fragment produced, C2, may provide the seed for the abnormal conversion process, as it resembles in size the fragments isolated from prion-infected brains. There is, however, strong evidence that ß-cleavage provides an essential process to reduce oxidative stress. ß-Cleavage may act as a double-edged sword. By ß-cleavage, PrP(C) may try to balance the ROS levels produced during prion infection, but the C2 produced may provide a PrP(Sc) seed that maintains the prion conversion process.
Assuntos
Príons/metabolismo , Animais , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Polineuropatias/metabolismo , Príons/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Prion disorders are characterised by the accumulation of a misfolded isoform (PrPSc) of the host encoded prion protein (PrPC). This paper examines the antiprion potential of cyclodextrin (CD) analogues and it identifies sulphated-beta-cyclodextrin, with a half-maximal inhibitory concentration (IC50) of 2.4 microM, as having 31-fold greater antiprion activity than that previously reported for beta-cyclodextrin (betaCD). METHODS: Scrapie infected cells were treated with a range of betaCD analogues. This enabled a CD structure to antiprion activity analysis to be carried out. The metachromatic activity of each of the cyclodextrins was determined, this test is employed to mimic complexation of glycosaminogylcans to a cell membrane. RESULTS: Sulphated-betaCD had an IC50 of 2.4 microM and it was the only CD found to have metachromatic activity. Its activity was equivalent to that of heparin and heparin sulphate, this may account for sulphated-betaCD's superior antiprion action. GENERAL SIGNIFICANCE: In solution heparin can form a helical structure with a hydrophobic interior, the hydrophobic interior of cyclic CDs is vital for CD molecule encapsulation. The controlled CD structure, however, restricts degradation by human enzymes; consequently sulphated-CDs could be ideal candidates in the search for prion therapeutics. Sulphated-CDs may open up avenues for the treatment of TSEs.
Assuntos
Ciclodextrinas/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Western Blotting , Linhagem Celular Tumoral , Ciclodextrinas/química , Relação Dose-Resposta a Droga , Heparina/química , Heparina/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Proteínas PrPSc/biossíntese , Relação Estrutura-Atividade , Sulfatos/química , beta-Ciclodextrinas/química , beta-Ciclodextrinas/farmacologiaRESUMO
Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders associated with the conversion of a normal host prion protein (PrP(C)) into a pathogenic isoform (PrP(Sc)). Despite years of research, there is still no known cure for TSEs. Amphotericin B (AmB), an anti-fungal antibiotic, has antiprion activity but its usage is limited by its toxicity. This study assessed the antiprion properties of new amphotericin analogues in which the exocyclic carboxyl groups were replaced by methyl groups. These analogues reduced levels of the abnormal PrP(Sc) isoform of the mouse prion protein in cultured cells. 16-descarboxyl-16-methyl-amphotericin B (16B) had antiprion activity equivalent to that of amphotericin B and was significantly less toxic to cells as determined by a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide dye reduction assay. A non-anti-fungal analogue, 16-descarboxyl-16-methyl-19-O-(6-deoxyhexosyl)-19-O-desmycosaminyl-amphotericin (16-19B) had higher antiprion activity and significantly lower toxicity than AmB. Some of the new amphotericin analogues may have potential as antiprion drugs.
Assuntos
Anfotericina B/análogos & derivados , Anfotericina B/farmacologia , Proteínas PrPSc/metabolismo , Anfotericina B/química , Animais , Antioxidantes/metabolismo , Ácido Ascórbico/farmacologia , Catalase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Polienos/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismoRESUMO
Central to Prion diseases is the normal endogenous Prion protein, PrPC. In spite of years of research the exact function of this protein remains enigmatic. Numerous binding partners have been identified for PrPC and due to the presence of a repeated sequence of PHGGGWGQ in the proteins amino-terminus it can bind metal ions. The protein is a complex molecule and each portion of PrPC possesses different roles for function and/ or trafficking. As understanding the role of PrPC is central to these disorders the structure/function relationship will be reviewed here.
Assuntos
Doenças Priônicas/metabolismo , Príons/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Cobre/metabolismo , Glicosilação , Proteínas de Choque Térmico/metabolismo , Humanos , Laminina/metabolismo , Doenças Priônicas/fisiopatologia , Transdução de Sinais/fisiologiaRESUMO
Prion diseases are fatal neurodegenerative disorders that are caused by the conversion of a normal host-encoded protein, PrP(C), to an abnormal, disease-causing form, PrP(Sc). This paper reports that cyclodextrins have the ability to reduce the pathogenic isoform of the prion protein PrP(Sc) to undetectable levels in scrapie-infected neuroblastoma cells. Beta-cyclodextrin removed PrP(Sc) from the cells at a concentration of 500 microM following 2 weeks of treatment. Structure activity studies revealed that antiprion activity was dependent on the size of the cyclodextrin. The half-maximal inhibitory concentration (IC(50)) for beta-cyclodextrin was 75 microM, whereas alpha-cyclodextrin, which possessed less antiprion activity, had an IC(50) of 750 microM. This report presents cyclodextrins as a new class of antiprion compound. For decades, the pharmaceutical industry has successfully used cyclodextrins for their complex-forming ability; this ability is due to the structural orientation of the glucopyranose units, which generate a hydrophobic cavity that can facilitate the encapsulation of hydrophobic moieties. Consequently, cyclodextrins could be ideal candidates for the treatment of prion diseases.