Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. BoNTs consist of three essential domains for toxicity: the cell binding domain (Hc), the translocation domain (Hn) and the catalytic domain (LC). The binding function of the Hc domain is essential for BoNTs to bind the neuronal cell membrane, therefore removal of the Hc domain results in a product that retains the endopeptidase activity of the LC but is non-toxic. Functional derivatives (LHn) of the parent neurotoxin composed of Hn and LC domains have been recombinantly produced and characterised. The crystallographic structures of LHn from serotypes A and B are reported here and demonstrate the stability of the LHn fragment in comparison to the full length toxins. The activity of LHn has been assessed on recombinant substrates and on cultured neuronal cells. LHn retains the capacity to internalise and cleave its intracellular SNARE substrate when applied to the cells at high concentration. These activities demonstrate the utility of engineered botulinum neurotoxin fragments as analytical tools to study the mechanisms of action of BoNT neurotoxins and of SNARE proteins. Targeted secretion inhibitors (TSI) are a new class of engineered biopharmaceutical molecules derived from the botulinum neurotoxins. These functional derivatives are expressed as single-chain proteins and require post-translational activation into di-chain molecules for function. A range of BoNT derivatives are presented and demonstrate the successful use of engineered SNARE substrate peptides at the LC-Hn interface to give these molecules self-activating capabilities while retaining the functions of LHn. Several novel molecules with therapeutic potential have been produced and their crystallisation for structural investigation is reported. These results provide an understanding of the structural implications and challenges of engineering therapeutic molecules that combine functional properties of the LHn fragment from BoNTs with specific ligand partners to target different cell types.
|Date of Award||25 Jan 2012|
|Supervisor||Ravi Acharya (Supervisor)|