Research

C3 project

Uptake and binding of C3

C3(bot) is produced as a single-chain exoenzyme (~25 kDa) by the gram-positive bacterium Clostridium botulinum. It selectively ADP-ribosylates the small GTPases RhoA, B, C and thereby inhibits their downstream signaling pathways. Due to its high selectivity (only 3 GTPases from the Ras superfamily of 150 GTPases) it is used as a pharmacological tool to study cellular Rho functions. Unlike classical clostridial toxins, C3 does not possess a receptor binding and translocation domain to enter the cell autonomously. We were able to show that the intermediate filament vimentin is essential as a receptor for C3 binding(Rohrbeck et al., 2014). Furthermore, involvement of clathrin- and calveolin-dependent endocytosis could be excluded. Instead, a dynamin-dependent endocytosis mechanism was identified in the HT22 and J774A.1 cells(Rohrbeck et al., 2015). In addition, there is evidence for another interaction partner in C3 uptake. The involvement of an Arg-Gly-Asp (RGD) motif within C3 and the inhibitory effect of β1-integrin antibodies suggest the involvement of integrins.

The aim of the research is to understand the basic mechanism of C3 uptake into the cell. New insights into the uptake mechanism of C3 may usefully change the therapy of acute neuronal injuries such as spinal cord injuries, peripheral nerve lesions or neurodegenerative diseases.

Intracellular distribution of C3

Findings with selective inhibitors of lysosome and proteosome functions show that both lysosomal and proteasomal degradation is the main inactivation pathway of C3(Naunyn-Schmiedeberg's Arch Pharmacol (2019) 392, P177). Cell fractionation and immunocytochemical experiments show that C3 is enriched in membranous compartments.

C3-mediated effects

C3 exoenzyme increases both the growth and the degree of branching of axons and dendrites in primary hippocampal neurons. In contrast, the enzyme-deficient mutant C3-E174Q only has an axonotrophic effect(Ahnert-Hilger et al., 2004). The exact molecular mechanism of action of the neurotrophic effect of C3 is still unknown(Just et al., 2011). Therefore, our research group investigated effects of C3 on different signaling pathways.

In immortalized murine hippocampal neurons, C3 causes strong growth inhibition via reduced cyclin D1 expression. The anti-apoptotic effect of C3 on this cell line is modulated via Rho-dependent transcription factors; both the expression of procaspase-3 and other pro-apoptotic proteins is reduced(Rohrbeck et al., 2012). Further analyses showed that C3 leads to an altered abundance of target proteins involved in the regulation of the cell cycle and cell death(von Elsner et al., 2016). Analysis of different mitogen-activated protein kinase (MAPK) signaling pathways identified a C3-mediated Rho-dependent reduced activity of the MKK3/6-p38 and JNK-c-Jun pathway associated with the observed growth inhibition(von Elsner et al., 2017).

Inhibition of cell cycle progression is associated with increased neuroprotection and neuroregeneration after spinal cord injury, so the results provide initial evidence for the signaling pathways of C3-mediated axono- and dendritotrophic effects.