Identification of presynaptic proteins as neddylation targets

Abstract

Reversible post-translational modifications are essential for the regulation of cell functions. One such modification is the attachment of ubiquitin and ubiquitin-like proteins, including Nedd8 (neural-precursor-cell-expressed developmentally down- regulated 8) (Enchev et al., 2015). It is well established, that neddylation is involved in the post-translational modification of synaptic proteins. As inhibition of neddylation has been demonstrated to decrease the probability of transmitter release (Brockmann, 2019), it can be hypothesised that proteins involved in synaptic transmission are neddylated. However, the target proteins of Nedd8 remain largely unexplored. <br /> The main focus of my work was the identification of presynaptic proteins modified by neddylation. The investigation focused on three key criteria: the covalent binding of Nedd8 to the target protein, the contribution of the neddylation-specific machinery, and the identification of neddylated lysines. For this, an Avi-tagged Nedd8 was used to detect all covalently attached target proteins of Nedd8 with magnetic streptavidin beads. The use of MLN-4924 as a control ensured that the attachment of Nedd8 occurred through the neddylation-specific machinery. <br /> In my thesis, I identified Stx1a and the homologous proteins Stx3 and Stx4 as targets for neddylation. By mutating lysine residues to arginine, I revealed that lysine 117 or 126 play an essential role in the neddylation of Stx1a. In the case of Stx4, lysines 123, 140 and 161 were identified as neddylated residues, while lysines 102, 108, 124 and 151 are suggested to be irrelevant for neddylation. <br /> Additionally, I also identified RIM1&alpha; as a target protein for Nedd8. Experiments using truncated proteins indicate that the neddylated lysine residue is located within the N-terminal region up to PRM 1. <br /> I unexpectedly discovered that the carboxylases are prospective Nedd8 candidates as well. As my work does not provide any additional information, a precise conclusion cannot be drawn at present. <br /> In conclusion, I identified four new neddylation targets: Stx1a, Stx3, Stx4 and RIM1&alpha;. The identification of Stx and RIM1&alpha; as Nedd8 targets will require further investigation of the function of Nedd8 in the synapse. In particular, the discovery of the neddylated lysines of Stx1a and Stx4, and the resulting availability of non-neddylatable variants, increases the potential for investigation of the function of Nedd8.