Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination

Chueh-Ling Kuo,Charles B Shoemaker,George A Oyler,Nic D Leipzig

doi:10.1371/journal.pone.0020352

Chueh-Ling Kuo, Charles B Shoemaker + Show 2 more

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https://doi.org/10.1371/journal.pone.0020352

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Abstract

Botulinum neurotoxin (BoNT), a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE) proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop ‘targeted F-box’ (TFB) agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only VH (VHH) domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10) led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.

Highlights

Botulism is caused by exposure to Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists to reverse the symptoms of paralysis after onset
The ALcB8 VHH was expressed as a fusion protein with the F-box protein, TrCP, to create a ‘‘targeted F-box’’ (TFB) designed to promote the specific, SCF E3-ligase mediated polyubiquitination of A Lc protease (ALc) and consequent proteasome-mediated degradation [15]
The B8-TrCP TFB fusion protein or ALcB8 alone were expressed within Botulinum neurotoxin (BoNT)/ A intoxicated neuroblastoma Neuro 2A (N2A) cells together with the ALc substrate, SNAP25, expressed as an indicator protein flanked by yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) [26]

Summary

Introduction

Botulism is caused by exposure to Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists to reverse the symptoms of paralysis after onset. The Lc proteases of the seven different BoNT serotypes have distinct active sites that cleave different sites in one or more SNARE proteins [3,4,9,10,11]. To protect against all known forms of BoNT, conventional small molecule drug development would need to be separately performed for each of the seven different drug targets, and perhaps even some of the subtypes. This challenge, together with other extreme hurdles confronting BoNT small molecule drug development, seriously complicates efforts to develop agents to treat botulism. New therapeutic paradigms are urgently needed to counter the enormous risks associated with these easy-to-obtain, easy-to-produce and extremely dangerous bioterror agents

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