RESUMO
Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild-type LCA was concentrated in plasma-membrane-associated clusters, colocalizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Neurônios/fisiologia , Síndromes Neurotóxicas/metabolismo , Septinas/metabolismo , Animais , Toxinas Botulínicas Tipo A/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação/genética , Neurônios/microbiologia , Síndromes Neurotóxicas/microbiologia , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Septinas/genéticaRESUMO
Botulinum neurotoxin serotype A (BoNT/A) causes transient muscle paralysis by entering motor nerve terminals (MNTs) where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206) to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs), making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs). Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3) as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.
Assuntos
Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Animais , Toxinas Botulínicas Tipo A/genética , Membrana Celular/genética , Células HEK293 , Humanos , Camundongos , Células PC12 , Transporte Proteico/genética , Ratos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismoRESUMO
Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT/A last from 3 to 12 months, whereas the effects of BoNT/E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT/A light chain (BoNT/A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25(197). In contrast, the short-duration BoNT/E serotype is cytoplasmic. Mutations in the BoNT/A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.
