RESUMEN
Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP7B-/-) showed autophagy deficiency at elevated copper condition. This phenotype was complemented by heterologous ATP7B expression. These findings suggest a cooperative role of ATP7B and LC3B in autophagy-mediated copper clearance.
Asunto(s)
ATPasas Transportadoras de Cobre/metabolismo , Cobre/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Secuencia de Aminoácidos , Transporte Biológico/efectos de los fármacos , Cobre/farmacología , ATPasas Transportadoras de Cobre/química , Células Hep G2 , Humanos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
The synthesis of a sulfate-modified dendritic peptide amphiphile and its self-assembly into one-dimensional rod-like architectures in aqueous medium is reported. The influence of the ionic strength on the supramolecular polymerization was probed via circular dichroism spectroscopy and cryogenic transmission electron microscopy. Physiological salt concentrations efficiently screen the charges of the dendritic building block equipped with eight sulfate groups and trigger the formation of rigid supramolecular polymers. Since multivalent sulfated supramolecular structures mimic naturally occurring L-selectin ligands, the corresponding affinity was evaluated using a competitive SPR binding assay and benchmarked to an ethylene glycol-decorated supramolecular polymer.
RESUMEN
Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer's disease and Parkinson's disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Enfermedad de Niemann-Pick Tipo C/metabolismo , Estrés Oxidativo , Catalasa/genética , Catalasa/metabolismo , Diferenciación Celular , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/citología , Péptidos y Proteínas de Señalización Intracelular/genética , Neuronas/citología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Niemann-Pick disease type C1 (NPC1) is a rare inherited lipid storage disorder caused by mutations in the NPC1 gene. Mutations lead to impaired lipid trafficking and subsequently to accumulation of cholesterol and sphingolipids. NPC1-patients present variable multisystemic symptoms, including neurological deficits. Here, we describe the generation of human iPSC lines obtained from fibroblasts of a male individual, carrying the homozygous mutation p.I1061T, and an unrelated and healthy male individual. A non-integrating Sendai virus system, containing KLF4, OCT3/4, SOX2 and C-MYC, was used for reprogramming. These cell lines provide a valuable resource for studying the pathophysiology of multisystemic NPC1-disease.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Niemann-Pick Tipo C , Fibroblastos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Factor 4 Similar a Kruppel , Masculino , Mutación/genética , Proteína Niemann-Pick C1 , Virus Sendai/genéticaRESUMEN
Fluorescent nanomaterials are expected to revolutionize medical diagnostic, imaging, and therapeutic tools due to their superior optical and structural properties. Their inefficient water solubility, cell permeability, biodistribution, and high toxicity, however, limit the full potential of their application. To overcome these obstacles, a water-soluble, fluorescent, cytocompatible polymer-single-walled carbon nanotube (SWNT) complex is introduced for bioimaging applications. The supramolecular complex consists of an alkylated polymer conjugated with neutral hydroxylated or charged sulfated dendronized perylene bisimides (PBIs) and SWNTs as a general immobilization platform. The polymer backbone solubilizes the SWNTs, decorates them with fluorescent PBIs, and strongly improves their cytocompatibility by wrapping around the SWNT scaffold. In photophysical measurements and biological in vitro studies, sulfated complexes exhibit superior optical properties, cellular uptake, and intracellular staining over their hydroxylated analogs. A toxicity assay confirms the highly improved cytocompatibility of the polymer-wrapped SWNTs toward surfactant-solubilized SWNTs. In microscopy studies the complexes allow for the direct imaging of the SWNTs' cellular uptake via the PBI and SWNT emission using the 1st and 2nd optical window for bioimaging. These findings render the polymer-SWNT complexes with nanometer size, dual fluorescence, multiple charges, and high cytocompatibility as valuable systems for a broad range of fluorescence bioimaging studies.
Asunto(s)
Dendrímeros/química , Diagnóstico por Imagen/métodos , Electricidad , Nanotubos de Carbono/química , Muerte Celular/efectos de los fármacos , Fluorescencia , Células HeLa , Humanos , Nanotubos de Carbono/toxicidad , Imagen ÓpticaRESUMEN
A series of water-soluble, hydroxylated and sulphated, polyglycerol (PG) dendronised, monofunctional perylene bisimides (PBIs) were synthesised in three generations. Their photophysical properties were determined by absorption and emission spectroscopy and their suitability as potential biolabels examined by biological in vitro studies after bioconjugation. It could be shown that the photophysical properties of the PBI labels can be improved by increasing the sterical demand and ionic charge of the attached dendron. Thereby, charged labels show superior suppression of aggregation over charge neutral labels owing to electrostatic repulsion forces on the PG-dendron. The ionic charges also enabled a reduction in dendron generation while retaining the labels' outstanding fluorescence quantum yields (FQYs) up to 100 %. These core-unsubstituted perylene derivatives were successfully applied as fluorescent labels upon bioconjugation to the therapeutic antibody cetuximab. The dye-antibody conjugates showed a strongly enhanced aggregation tendency compared to the corresponding free dyes. Biological evaluation by receptor-binding, cellular uptake, and cytotoxicity studies revealed that labelling did not affect the antibody's function, which renders the noncharged and charged dendronised PBIs suitable candidates as fluorescent labels in biological imaging.
RESUMEN
Near-infrared (NIR) fluorescent dyes are gaining increased attention due to their potential to serve as molecular probes for in vivo imaging. Here, we demonstrate that oligoglycerol dendrons effectively enhance the fluorescence properties of an NIR dye by increasing the solubility in water and the prevention of aggregate formation. First- and second-generation oligoglycerol dendrons were conjugated to an NIR dye via a dipolar-cycloaddition (click) reaction. The two new dye conjugates exhibited enhanced NIR fluorescent emission and considerably higher fluorescent quantum yields than the dye alone. The high photostability measured for one of the oligoglycerol-linked dyes, in comparison to commonly used fluorogenic dyes such as Cy5 and Cy7, was validated using fluorescence microscopy of macrophages.