title: 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.
authors.affiliation: Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, CA 91343, USA olgav@ucla.edu.
authors.forename: Olga
authors.initials: O
authors.lastname: Vagin
authors.affiliation: Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, CA 91343, USA.
authors.forename: Elmira
authors.initials: E
authors.lastname: Tokhtaeva
authors.affiliation: Department of Biological Sciences, Allergan Inc., Irvine, CA 92612, USA.
authors.forename: Patton E
authors.initials: PE
authors.lastname: Garay
authors.affiliation: The NPI-Semel Institute, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Puneet
authors.initials: P
authors.lastname: Souda
authors.affiliation: Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, CA 91343, USA.
authors.forename: Sara
authors.initials: S
authors.lastname: Bassilian
authors.affiliation: The NPI-Semel Institute, Pasarow Mass Spectrometry Laboratory, UCLA, Los Angeles, CA 90095, USA.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
authors.affiliation: Department of Biological Sciences, Allergan Inc., Irvine, CA 92612, USA.
authors.forename: Ramilla
authors.initials: R
authors.lastname: Lewis
authors.affiliation: Department of Physiology, School of Medicine, UCLA and Veterans Administration Greater Los Angeles Health Care System, Los Angeles, CA 91343, USA.
authors.forename: George
authors.initials: G
authors.lastname: Sachs
authors.affiliation: Department of Biological Sciences, Allergan Inc., Irvine, CA 92612, USA.
authors.forename: Larry
authors.initials: L
authors.lastname: Wheeler
authors.affiliation: Department of Biological Sciences, Allergan Inc., Irvine, CA 92612, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Aoki
authors.affiliation: Department of Biological Sciences, Allergan Inc., Irvine, CA 92612, USA.
authors.forename: Ester
authors.initials: E
authors.lastname: Fernandez-Salas
mesh_terms: D000818:Animals; D019274:Botulinum Toxins, Type A / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000382:microbiology / Q000502:physiology*; D020258:Neurotoxicity Syndromes / Q000378:metabolism* / Q000382:microbiology; D010671:Phenylurea Compounds / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D058112:Septins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D010671:Phenylurea Compounds; D011725:Pyridines; D034741:RNA, Small Interfering; D019274:Botulinum Toxins, Type A; C000602252:SEPTIN2 protein, human; C113158:SEPTIN7 protein, human; D058112:Septins; C116093:N-(2-chloro-4-pyridyl)-N'-phenylurea
keywords: Botulinum toxin A protease; Degradation; Protein stability; Septin; Ubiquitylation