Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system

Abstract

Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.botulinum toxin | HECT domain | synaptic transmission | Clostridium | SNAP25

Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication

“…Cells normally degrade foreign proteins such as bacterial or viral proteins through the ubiquitin-proteasome system (UPS). In this regard, the catalytic LC of BoNT/E (LCE), which also cleaves SNAP25 but causes only transient paralysis, is degraded relatively rapidly as a consequence of ubiquitination by the E3 ubiquitin ligase TRAF2 (12). In contrast, the LC of BoNT/A (LCA) is quite stable (12), which is consistent with the proteolytic activity of LCA remaining detectable in neuronal cultures for months (13,14).…”

“…1A). In a previous study, we found that LCA shows detectable, albeit minimal, ubiquitination in the presence of proteasome inhibitor (12). However, given its already long half-life, no significance could be attributed to this finding.…”

“…We have described a tractable cell culture model for studying the persistence of LCA (12). In this model, HEK293 cells are induced with doxycycline to transiently express codon-optimized GFP-tagged BoNT LCs.…”

“…This has necessitated the transfection of plasmids encoding BoNT LCs that have been codon-optimized for mammalian expression to study BoNT persistence (12,(40)(41)(42). It is natural to ask whether discoveries based on transfection of BoNT LCs can be extended to intoxication with the holotoxin.…”

“…In this regard, the catalytic LC of BoNT/E (LCE), which also cleaves SNAP25 but causes only transient paralysis, is degraded relatively rapidly as a consequence of ubiquitination by the E3 ubiquitin ligase TRAF2 (12). In contrast, the LC of BoNT/A (LCA) is quite stable (12), which is consistent with the proteolytic activity of LCA remaining detectable in neuronal cultures for months (13,14). The activity of ubiquitin ligases toward proteins is highly specific; accordingly, despite 35% sequence identity between LCE and LCA, there is no evidence that TRAF2 ubiquitinates the highly stable LCA (12,15).…”