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James, N. G.

Publications and source records attributed to James, N. G..

2 recordsLinked to original sources

A mutation associated with Charcot-Marie-Tooth disease enhances the formation of stable dynamin 2 complexes in cells

Mutations in dynamin 2 (DNM2) have been associated with two distinct motor disorders, Charcot-Marie-Tooth neuropathies (CMT) and centronuclear myopathy (CNM). The majority of these mutations are clustered in the pleckstrin homology domain (PHD) which engage in intramolecular interactions that suppress dynamin self-assembly and GTPase activation. CNM mutations in the PHD interferes with these intramolecular interactions, thereby blocking the formation of the auto-inhibited state. CMT mutations are located primarily on the opposite surface of the PHD, which is specialized for lipid PIP2 binding. It has been speculated that the distinct locations and interactions of residues mutated in CMT and CNM explain why each set of mutations cause either one disease or the other, despite their close proximity within the PHD sequence. We show that at least one CMT-causing mutant, lacking residues 555DEE557 ({Delta}DEE), displays this inability to undergo auto-inhibition as observed in CNM-linked mutants. This {Delta}DEE deletion mutant induces the formation of abnormally large cytoplasmic inclusions similar to those observed for CNM-linked mutant R369W. We also found substantially reduced migration from the membrane of the {Delta}DEE deletion mutant. These findings call into question the molecular mechanism currently believed to underlie the absence of pathogenic overlap between DNM2-dependent CMT and CNM.

cell biology↗

Characterization of clostridium botulinum neurotoxin serotype A (BoNT/A) and fibroblast growth factor receptor interactions using a novel receptor dimerization assay

Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that also serves as an effective therapeutic for a variety of neuromuscular and glandular diseases and disorders. The observed pharmacological effect of BoNT/A is due to specific targeting and entry into motor nerve terminals within muscles followed by cleavage of the SNARE protein, SNAP-25, inducing a block in neurotransmission and temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells are mediated by its binding domain (HC/A), which binds to GT1b ganglioside and protein cell surface receptors. Previously, fibroblast growth factor receptor 3 (FGFR3) was identified as a BoNT/A receptor, in addition to synaptic vesicle protein (SV2). To further study BoNT/A interactions with FGFRs, an FCS & TIRF receptor dimerization assay was developed to measure dimerization of FGFRs in live cells, as FGFR dimerization can be considered an indirect measure for receptor-ligand binding interaction and downstream signaling. The ability of HC/A to facilitate dimerization of three FGFR subtypes (FGFR1-3) was assessed. Recombinant HC/A (rHC/A) was shown to dimerize FGFR subtypes in the rank order FGFR3c > FGFR2b > FGFR1c. With potencies (EC50 values) defined as the concentration of ligand required to dimerize 50% of the receptors, wild type rHC/A dimerized FGFR3c with an EC50 of 24 nM, similar to FGF9, a native FGFR3c ligand, which had an EC50 of 15 nM, while FGFR1c and FGFR2b required higher rHC/A concentrations ([≥]100 nM and 68 nM, respectively). Furthermore, addition of the GT1b ganglioside to the culture media resulted in increased dimerization, whereas a ganglioside mutant variant of HC/A (rHC/A W1266L;Y1267S) showed decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of HC/A (rHC/A T1145A;T1146A). These results support a model wherein BoNT/A interacts with FGFRs, gangliosides, and SV2 on the cell surface to facilitate cell uptake.

cell biology↗