Recruitment of septin cytoskeletal proteins by Botulinum toxin A protease determines its remarkable stability

Abstract

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.

Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells

“…For experiments with cycloheximide, this was added 2 hours before other inhibitors. As a wide range of concentrations for lactacystin are reported in the literature, effectiveness of the 2.5 nM concentration, as demonstrated previously in other cell lines [11], was confirmed in HGE-20 cells by incubation with lactacystin, lysis, and western blot with ubiquitin antibodies (data not shown). After incubation with inhibitors, cells were washed twice with ice cold phosphate buffered saline containing 1mM MgCl 2 and 1 mM CaCl 2 (PBS ++ ) on ice, then lysed with 50 mM Tris pH 7.5 containing 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate and Complete Protease Inhibitor Cocktail, 1 tablet/50 ml, (Roche Diagnostics, Indianapolis, IN, USA) at 4°C for 30 min.…”

“…Previous data showing similar effects of FCF and septin silencing on various cellular processes, including mitosis and cell migration [64], protein stability [11], glucose transport [65], store-operated Ca 2+ influx [66], ciliogenesis [63], epithelial junctions [67], gastrulation during embryogenesis [68], and bacterial invasion [69], demonstrate that FCF is a valuable tool to interfere with septin dynamics. Exposure of AGS and HGE-20 cells to FCF for 12 hours resulted in about a 2-fold decrease in ErbB2 protein levels in both cell lines (Fig.…”

High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

“…Contrast and brightness settings were adjusted so that all pixels were in the linear range. The coefficient of colocalization of Na,K-ATPase ␣ 1 subunit with LMO7b (means Ϯ standard errors of the means [SEM]) was calculated by using ZEN 2009 software (Carl Zeiss MicroImaging GmbH, Germany) as previously described (33). Imaging work was performed at the Northwestern University Center for Advanced Microscopy (Cell Imaging Facility and Nikon Imaging Center), which was generously supported by NCI CCSG P30 CA060553, awarded to the Robert H. Lurie Comprehensive Cancer Center.…”

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

“…In these cell types, FCF may be a useful tool to analyze higher-order septin dynamics, but conclusions about septin functions in various cellular processes based on FCF-induced effects should be made with the utmost caution. In systems in which septin genes are not easily mutated, numerous studies have attempted to address this issue using RNA interference-mediated depletion of septins, and in most cases this mimics the FCF effect of interest (30)(31)(32)52). However, it has been well established that experimental activation of the RNA interference system via the introduction of synthetic small RNA molecules is associated with cytotoxicity and various indicators of general cellular stress (53)(54)(55)(56)(57), not unlike what we have documented for FCF in multiple cell types.…”