Tübingen 2018

Development of a shuttle mechanism using detoxified botulium toxin

Inspiration

It has been demonstrated, that some botulinum neurotoxins (BoNTs) can be used for delivery of enzymatically active proteins.¹ However, the remarkable toxicity of BoNTs has stopped this substance class from being employed for this feature. In 2017 Vazquez-Cintron et al. has shown a series of point mutations in botulinum toxin C that reduce its toxicity by a factor of approximately 5 x 10⁶, while still being able to transport the light chain into the target cell cytosol.² Combining these approaches should enable for constructs trafficking a wide range of molecules into neurons with a BoNTs intrinsic, excellent specificity and activity.

Project idea

Our project tries to explore the range of capabilities for internalization of the nontoxic botulinus toxin C, by fusing various proteins and small molecular compounds to it. By trying to use cleavable linkers, we will attempt to release our fused molecules from the botulinum toxin light chain once it has invaded the target cell cytoplasm. This should increase the activity of especially the small molecular compounds. Finally we wish to make our delivery system as modular as possible.

Coupling and assays

We will develop a library of different detoxified botulinum toxin derivatives which can carry other proteins, small molecules, and fluorochromes by specific linkers.

• SNAP-25 cleavage assay for proof of the detoxification

Modelling and VR

We try to predict changes of the tertiary structure of botulinum toxin C1 caused by the detoxifying point mutations introduced in the toxin. Furthermore we are developing a virtual reality (VR) protein viewer for smartphones, that is easily and cheaply accessible for anyone, giving scientists an immersive experience while working with proteins.

We want you - for cooperations

1. Bade, S. , Rummel, A. , Reisinger, C. , Karnath, T. , Ahnert‐Hilger, G. , Bigalke, H. and Binz, T. (2004), Botulinum neurotoxin type D enables cytosolic delivery of enzymatically active cargo proteins to neurones via unfolded translocation intermediates. Journal of Neurochemistry, 91: 1461-1472. http://doi.org/10.1111/j.1471-4159.2004.02844.x
2. Vazquez-Cintron, E. J., Beske, P. H., Tenezaca, L., Tran, B. Q., Oyler, J. M., Glotfelty, E. J., … Ichtchenko, K. (2017). Engineering Botulinum Neurotoxin C1 as a Molecular Vehicle for Intra-Neuronal Drug Delivery. Scientific Reports, 7, 42923. http://doi.org/10.1038/srep42923