RESUMEN
Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of alpha-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P alpha-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants alpha-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.
Asunto(s)
Encéfalo/metabolismo , Degeneración Nerviosa/metabolismo , Ovillos Neurofibrilares/metabolismo , Neuronas/metabolismo , Proteína 1A de Unión a Tacrolimus/metabolismo , alfa-Sinucleína/metabolismo , Sustitución de Aminoácidos , Encéfalo/patología , Encéfalo/fisiopatología , Dominio Catalítico/genética , Línea Celular Tumoral , Humanos , Inmunosupresores/farmacología , Mutación/genética , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Ovillos Neurofibrilares/efectos de los fármacos , Ovillos Neurofibrilares/genética , Neuronas/efectos de los fármacos , Neuronas/patología , Dinámicas no Lineales , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/genética , Proteína 1A de Unión a Tacrolimus/farmacología , Factores de Tiempo , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/genéticaRESUMEN
A comparative study that deals with the internalisation of different types of magnetoliposomes (MLs) by 3T3 fibroblasts revealed that cationic MLs proved to be superior to neutral and anionic ones. Internalisation was visualised both by optical light and transmission electron microscopy. The latter showed that the cationic MLs ultimately ended up in lysosomal structures. The effect of increasing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) concentrations in the cationic ML coat has been elucidated. High uptake efficiency was only achieved with MLs that carry a high DOTAP payload. However, these structures also demonstrated toxic effects. The use of the saturated distearoyl analogue (DSTAP) at identical concentrations led to improved uptake efficiency and lower toxicity. By using iron-oxide-free vesicles, it was shown that the toxicity was due to lipid bilayer constituents and not the iron oxide. In conclusion, the use of DMPC-DSTAP (96.67:3.33; molar ratio) MLs results in an extremely high labelling of 3T3 fibroblasts with iron oxides (47.66 pg Fe per cell) without evoking any influence on cell viability.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Liposomas/química , Liposomas/farmacología , Magnetismo , Compuestos de Amonio Cuaternario/farmacología , Células 3T3 , Animales , Cationes/química , Supervivencia Celular/efectos de los fármacos , Ácidos Grasos Monoinsaturados/química , Fibroblastos , Liposomas/metabolismo , Ratones , Microscopía Electrónica de Transmisión , Compuestos de Amonio Cuaternario/químicaRESUMEN
Aggregation of alpha-synuclein (alpha-SYN) plays a key role in Parkinson's disease (PD). We have used fluorescence correlation spectroscopy (FCS) to study alpha-SYN aggregation in vitro and discovered that this process is clearly accelerated by addition of FK506 binding proteins (FKBPs). This effect was observed both with E. coli SlyD FKBP and with human FKBP12 and was counteracted by FK506, a specific inhibitor of FKBP. The alpha-SYN aggregates formed in the presence of FKBP12 showed fibrillar morphology. The rotamase activity of FKBP apparently accelerates the folding and subsequent aggregation of alpha-SYN. Since FK506 and other non-immunosuppressive FKBP inhibitors are known to display neuroregenerative and neuroprotective properties in disease models, the observed inhibition of rotamase activity and alpha-SYN aggregation, may explain their mode of action. Our results open perspectives for the treatment of PD with immunophilin ligands that inhibit a specific member of the FKBP family.
Asunto(s)
Proteínas de Escherichia coli/farmacología , Isomerasa de Peptidilprolil/farmacología , Proteína 1A de Unión a Tacrolimus/farmacología , alfa-Sinucleína/química , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/fisiología , Humanos , Microscopía Electrónica , Nefelometría y Turbidimetría , Isomerasa de Peptidilprolil/aislamiento & purificación , Isomerasa de Peptidilprolil/fisiología , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Espectrometría de Fluorescencia , Tacrolimus/farmacología , Proteína 1A de Unión a Tacrolimus/fisiología , alfa-Sinucleína/efectos de los fármacos , alfa-Sinucleína/genética , alfa-Sinucleína/ultraestructuraRESUMEN
For several integrins, the existence of multiple conformational states has been studied intensively. For the integrin alpha2beta1, a major collagen receptor on platelets and other cell types, however, no such experimental data were available thus far. Recently, our group has developed a monoclonal antibody IAC-1 sensitive to the molecular conformation of alpha2beta1 because it only binds to the activated state of alpha2beta1 on platelets, induced upon inside-out signaling. By investigating IAC-1 binding in combination with collagen binding after inside-out stimulation and outside manipulation, we demonstrated the existence of three different conformations of alpha2beta1 on platelets and Chinese hamster ovary cells as follows: (i) a nonactivated, resting state with no collagen nor IAC-1 binding; (ii) an intermediate state, induced by outside manipulation, with collagen but no IAC-1 binding; and (iii) a fully activated state, induced after inside-out stimulation, with both collagen and IAC-1 binding. Moreover, these different conformational states of alpha2beta1 are dependent on the cell type where alpha2beta1 is expressed, as IAC-1 binding to peripheral blood mononuclear cells and Jurkat cells could also be induced by outside manipulation, in contrast to platelets and alpha2beta1-expressing Chinese hamster ovary cells. Finally, we revealed a functional relevance for these different conformational states because the conformation of alpha2beta1, induced after outside manipulation, resulted in significantly more cell spreading on coated collagen compared with nonactivated or inside-out stimulated cells.