Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation

Abstract

While the role of ubiquitin in protein degradation is well established, the role of other ubiquitin-like proteins (UBLs) in protein degradation is less clear. Neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the UBL with the highest level of amino acids identified when compared to ubiquitin. Here we tested if the N-terminal addition of NEDD8 to a protein of interest could lead to degradation. Mutation of critical glycine residues required for normal NEDD8 processing resulted in a non-cleavable fusion protein that was rapidly degraded within the cells by both the proteasome and autophagy. Both degradation pathways were dependent on a functional ubiquitin-conjugation system as treatment with MLN7243 increased levels of non-cleavable NEDD8-GFP. The degradation of non-cleavable, N-terminal NEDD8-GFP was not due to a failure of GFP folding as different NEDD8-GFP constructs with differing abilities to fold and fluoresce were similarly degraded. Though the fusion of NEDD8 to a protein resulted in degradation, treatment of cells with MLN4924, an inhibitor of the E1 activating enzyme for NEDD8, failed to prevent degradation of other destabilized substrates. Taken together these data suggest that under certain conditions, such as the model system described here, the covalent linkage of NEDD8 to a protein substrate may result in the target proteins degradation.

NEDD8 activity is important for direct antigen MHC class I antigen presentation

“…In other instances, such as N-terminal fusion of SUMO, there is little to no impact either protein stability or antigen presentation (22). We have recently reported the N-terminal fusion of NEDD8 to a protein induces rapid degradation (25) and in this report sought to determine if the induction of protein degradation by NEDD8 fusion would enhance antigen presentation. By comparing cells infected with rVV expressing the different constructs, we were able to directly compare the effect of NEDD8-fusion with ubiquitin fusion and found that while NEDDylation of the cytosolic form of ovalbumin increased the rate of antigen presentation, it was not as efficient as ubiquitin fusion.…”

“…Cell Culture, Transfection, and siRNA depletion L/K b , EL4, EL4/NC NEDD8-ova, EL4/ova and EL4/SCRAP-GFP cell lines have been previously described (5,25,27). All EL4-derived cells were grown in RPMI 1640 supplemented with 10 mM HEPES, Glutamax, and 7.5% fetal calf serum (all from Thermo Fisher) and were incubated at 37°C and 6% CO2.…”

“…In order to study the effect of non-canonical NEDD8 binding on antigen presentation we created three OVA fusion proteins (Figure 1A), similar to previously described GFP constructs (25).…”

“…Our previous work with NEDD8 revealed that the fusion of NEDD8 to GFP caused the rapid degradation of GFP (25). This phenomenon was dependent on the proteasomal shuttling factor NEDD8 ultimate buster 1 (NUB1).…”

The Many Potential Fates of Non-Canonical Protein Substrates Subject to NEDDylation

“…N-terminal fusion proteins were created where the C-terminal glycine residues of NEDD8 were mutated to alanine which prevented removal of NEDD8 from the target protein, such as GFP or chicken ovalbumin. This modification resulted in an increased rate of degradation of the fusion protein demonstrating that N-terminal modification by NEDD8 can lead to a proteins degradation similar to N-terminal fusion of ubiquitin [78]. Interestingly, degradation of these substrates was blocked by both proteasome inhibition and by inhibition of autophagy, suggesting that there are multiple ways for cells to degrade NEDDylated proteins.…”