Assuntos
Curcumina/metabolismo , DNA/metabolismo , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Curcumina/efeitos adversos , Curcumina/uso terapêutico , Eletroforese em Gel de Ágar , Humanos , Substâncias Intercalantes/efeitos adversos , Substâncias Intercalantes/metabolismo , Substâncias Intercalantes/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/patologiaRESUMO
Chronic and complex autoimmune diseases, currently treated palliatively with immunosuppressives, require multi-targeted therapy for greater effectiveness. The naturally occurring polyphenol curcumin has emerged as a powerful "nutraceutical" that interacts with multiple targets to regress diseases safely and inexpensively. Up to 8 g/day of curcumin for 18 months was non-toxic to humans. However, curcumin's utility is limited by its aqueous insolubility. We have demonstrated a heat-mediated 12-fold increase in curcumin's aqueous solubility. Here, we show by SDS-PAGE and surface plasmon resonance that heat-solubilized curcumin binds to proteins. Based on this binding we hypothesized that heat-solubilized curcumin or turmeric would prevent autoantibody targeting of cognate autoantigens. Heat-solubilized curcumin/turmeric significantly decreased binding of autoantibodies from Sjögren's syndrome (up to 43/70%, respectively) and systemic lupus erythematosus (up to 52/70%, respectively) patients as well as an animal model of Sjögren's syndrome (up to 50/60%, respectively) to their cognate antigens. However, inhibition was not specific to autoimmunity. Heat-solubilized curcumin/turmeric also inhibited binding of commercial polyclonal anti-spectrin to spectrin (50/56%, respectively). Thus, we suggest that the multifaceted heat-solubilized curcumin can ameliorate autoimmune disorders. In addition, the non-toxic curcumin could serve as a new protein stain in SDS-PAGE even though it is less sensitive than the Coomassie system which involves toxic chemicals.
Assuntos
Reações Antígeno-Anticorpo/efeitos dos fármacos , Doenças Autoimunes/imunologia , Curcumina/química , Curcumina/farmacologia , Temperatura Alta , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Autoantígenos/imunologia , Autoantígenos/metabolismo , Doenças Autoimunes/dietoterapia , Curcuma/química , Curcuma/metabolismo , Curcumina/metabolismo , Suplementos Nutricionais , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Fatores Imunológicos/metabolismo , Indicadores e Reagentes , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Extratos Vegetais/farmacologia , Síndrome de Sjogren/imunologia , Solubilidade , Espectrina/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.