Limits...
Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination.

Kuo CL, Oyler GA, Shoemaker CB - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America.

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 V(H) (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.

Show MeSH

Related in: MedlinePlus

TFBs promote target-specific polyubiquitination.M17 cells were co-transfected with HA-ubiquitin, CFP-ALc and expression plasmids for ALc-specific TFB (D5-B8) or BLc-specific TFB (D5-B10) as indicated. 24 hrs post-transfection, cells were treated with 10 µM of MG132 for 6 hrs and cell lysates were prepared. Purified recombinant GST-ALcB8 was added to the cell extracts and CFP-ALc was purified by glutathione affinity. Eluted protein was resolved by SDS-PAGE. CFP-Lc expression levels and HA-ubiquitin modification were detected by Western blotting using sheep anti-ALc antibody or anti-HA antibody and the results shown are representative of four separate experiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3101245&req=5

pone-0020352-g003: TFBs promote target-specific polyubiquitination.M17 cells were co-transfected with HA-ubiquitin, CFP-ALc and expression plasmids for ALc-specific TFB (D5-B8) or BLc-specific TFB (D5-B10) as indicated. 24 hrs post-transfection, cells were treated with 10 µM of MG132 for 6 hrs and cell lysates were prepared. Purified recombinant GST-ALcB8 was added to the cell extracts and CFP-ALc was purified by glutathione affinity. Eluted protein was resolved by SDS-PAGE. CFP-Lc expression levels and HA-ubiquitin modification were detected by Western blotting using sheep anti-ALc antibody or anti-HA antibody and the results shown are representative of four separate experiments.

Mentions: CFP-ALc was co-expressed with the TFBs D5-B8 or D5-B10 in the presence of the proteasome inhibitor, MG132, to permit accumulation of polyubiquitinated proteins. The cells were also co-transfected with an expression plasmid for HA-ubiquitin. The CFP-ALc was purified from cell extracts by affinity to GST-ALcB8. The use of the VHH ALcB8 to purify the ALc should eliminate (by competition) co-purification of any D5-B8 that remained bound to the CFP-ALc in the extract and which could lead to contaminating polyubiquitinated protein. The purified ALc from each extract was analyzed by Western blot (Figure 3) and shown to contain CFP-ALc. The amount of extract loaded was normalized such that the CFP-ALc levels were nearly the same. When an identical Western blot was analyzed for HA, it became clear that the ALc co-expressed with D5-B8 was much more heavily ubiquitinated than ALc co-expressed with D5-B10. These results show that the TFB D5-B8 is promoting ubiquitination of ALc within the transfected M17 cells.


Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination.

Kuo CL, Oyler GA, Shoemaker CB - PLoS ONE (2011)

TFBs promote target-specific polyubiquitination.M17 cells were co-transfected with HA-ubiquitin, CFP-ALc and expression plasmids for ALc-specific TFB (D5-B8) or BLc-specific TFB (D5-B10) as indicated. 24 hrs post-transfection, cells were treated with 10 µM of MG132 for 6 hrs and cell lysates were prepared. Purified recombinant GST-ALcB8 was added to the cell extracts and CFP-ALc was purified by glutathione affinity. Eluted protein was resolved by SDS-PAGE. CFP-Lc expression levels and HA-ubiquitin modification were detected by Western blotting using sheep anti-ALc antibody or anti-HA antibody and the results shown are representative of four separate experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101245&req=5

pone-0020352-g003: TFBs promote target-specific polyubiquitination.M17 cells were co-transfected with HA-ubiquitin, CFP-ALc and expression plasmids for ALc-specific TFB (D5-B8) or BLc-specific TFB (D5-B10) as indicated. 24 hrs post-transfection, cells were treated with 10 µM of MG132 for 6 hrs and cell lysates were prepared. Purified recombinant GST-ALcB8 was added to the cell extracts and CFP-ALc was purified by glutathione affinity. Eluted protein was resolved by SDS-PAGE. CFP-Lc expression levels and HA-ubiquitin modification were detected by Western blotting using sheep anti-ALc antibody or anti-HA antibody and the results shown are representative of four separate experiments.
Mentions: CFP-ALc was co-expressed with the TFBs D5-B8 or D5-B10 in the presence of the proteasome inhibitor, MG132, to permit accumulation of polyubiquitinated proteins. The cells were also co-transfected with an expression plasmid for HA-ubiquitin. The CFP-ALc was purified from cell extracts by affinity to GST-ALcB8. The use of the VHH ALcB8 to purify the ALc should eliminate (by competition) co-purification of any D5-B8 that remained bound to the CFP-ALc in the extract and which could lead to contaminating polyubiquitinated protein. The purified ALc from each extract was analyzed by Western blot (Figure 3) and shown to contain CFP-ALc. The amount of extract loaded was normalized such that the CFP-ALc levels were nearly the same. When an identical Western blot was analyzed for HA, it became clear that the ALc co-expressed with D5-B8 was much more heavily ubiquitinated than ALc co-expressed with D5-B10. These results show that the TFB D5-B8 is promoting ubiquitination of ALc within the transfected M17 cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, United States of America.

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 V(H) (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.

Show MeSH
Related in: MedlinePlus