Abstract
Precise cellular targeting of macromolecular cargos has important biotechnological and medical implications. Using a recently established ‘protein stapling’ method, we linked the proteolytic domain of botulinum neurotoxin type A (BoNT/A) to a selection of ligands to target neuroendocrine tumor cells. The botulinum proteolytic domain was chosen because of its well-known potency to block the release of neurotransmitters and hormones. Among nine tested stapled ligands, the epidermal growth factor was able to deliver the botulinum enzyme into pheochromocytoma PC12 and insulinoma Min6 cells; ciliary neurotrophic factor was effective on neuroblastoma SH-SY5Y and Neuro2A cells, whereas corticotropin-releasing hormone was active on pituitary AtT-20 cells and the two neuroblastoma cell lines. In neuronal cultures, the epidermal growth factor- and ciliary neurotrophic factor-directed botulinum enzyme targeted distinct subsets of neurons whereas the whole native neurotoxin targeted the cortical neurons indiscriminately. At nanomolar concentrations, the retargeted botulinum molecules were able to inhibit stimulated release of hormones from tested cell lines suggesting their application for treatments of neuroendocrine disorders.
Highlights
IntroductionSecretory activity of neuroendocrine cells and neurons relies on soluble N-ethylmaleimide sensitive fusion proteinattachment protein receptors (SNAREs): syntaxin 1, synaptosomal-associated protein 25 (SNAP25), and synaptobrevin (Fasshauer et al 1998; Sutton et al 1998; S€udhof and Rothman 2009; Gao et al 2012)
Our results demonstrate that new ligands can substitute the botulinum receptor-binding domain and allow targeting of distinct neurons and cells of neuroendocrine origin
Increasing efforts have been directed toward modifying several types of botulinum neurotoxins for treatment of diverse hypersecretory disorders including inflammation, asthma, chronic pain, and neuroendocrine tumors (NET), such as acromegaly and Cushing’s disease (Chaddock et al 2004; Foster 2005; Chaddock and Marks 2006; Foster et al 2006; Chen and Barbieri 2009; Foster and Chaddock 2010; Pickett and Perrow 2011; Somm et al 2012)
Summary
Secretory activity of neuroendocrine cells and neurons relies on soluble N-ethylmaleimide sensitive fusion proteinattachment protein receptors (SNAREs): syntaxin 1, synaptosomal-associated protein 25 (SNAP25), and synaptobrevin (Fasshauer et al 1998; Sutton et al 1998; S€udhof and Rothman 2009; Gao et al 2012). These three proteins form a tight complex that allows fusion of secretory vesicles with the plasma membrane in both neurons and endocrine cells. We address the possibility to deliver the botulinum protease to NET cells using a selection of growth factors and neuropeptides by exploiting a novel protein stapling method.
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