cells Article Direct Conjugation of NEDD8 to the N-Terminus of a Model Protein Can Induce Degradation Kartikeya Vijayasimha, Marilyn Vo Tran, Amy L. Leestemaker-Palmer and Brian P. Dolan * Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA; [email protected] (K.V.); [email protected] (M.V.T.); [email protected] (A.L.L.-P.) * Correspondence: [email protected]; Tel.: +1-(541)-737-0828 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 Citation: Vijayasimha, K.; Tran, M.V.; protein substrate may result in the target proteins degradation. Leestemaker-Palmer, A.L.; Dolan, B.P. Direct Conjugation of NEDD8 to the Keywords: ubiquitination; NEDD8; protein degradation; proteasome N-Terminus of a Model Protein Can Induce Degradation. Cells 2021, 10, 854. https://doi.org/10.3390/ cells10040854 1. Introduction Intracellular protein turnover is an important physiological process. By degrading a Academic Editor: Kunihiro Sakuma subset of proteins and synthesizing different ones, a cell can alter its function in response to environmental changes [1] and remove old or damaged proteins, which prevents their Received: 9 March 2021 accumulation within cells and can lead to toxicity [2–5]. Protein degradation is also Accepted: 8 April 2021 Published: 9 April 2021 necessary for the recycling of amino acids [6–8] and for alerting the adaptive immune system to the presence of intracellular infections via the direct major histocompatibility Publisher’s Note: MDPI stays neutral (MHC) class I antigen presentation pathway [9–11]. A prominent pathway of protein with regard to jurisdictional claims in degradation involves the proteasome, a multi-subunit protein complex containing trypsin- published maps and institutional affil- like, chymotrypsin-like, and caspase-like protease activity [12]. To prevent non-specific iations. protease activity, proteins intended for proteasome-mediated degradation are modified by the addition of a small protein termed ubiquitin [13–15]. Once ubiquitinated, a targeted protein can be degraded via the proteasome or additional ubiquitin proteins can be added to the target protein leading to its subsequent destruction by the proteasome [16–18]. There are several other proteins within eukaryotic cells that are similar to ubiquitin Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and are termed ubiquitin-like proteins (UBLs) [19,20] and, like-ubiquitin, can be post- This article is an open access article translationally added to a variety of targets proteins resulting in changes to the proteins’ distributed under the terms and functions [21,22]. Unlike ubiquitin, most UBL-protein modifications do not result in a conditions of the Creative Commons protein’s destruction but rather alter protein function or localization [23]. There are a few Attribution (CC BY) license (https:// exceptions to this “rule” though, which suggest that in certain circumstances, UBL modifi- creativecommons.org/licenses/by/ cation may target a protein for destruction. FAT10 is a UBL with two ubiquitin domains 4.0/). and fusion of FAT10 to otherwise stable proteins resulted in their rapid degradation in Cells 2021, 10, 854. https://doi.org/10.3390/cells10040854 https://www.mdpi.com/journal/cells Cells 2021, 10, 854 2 of 23 the proteasome [24]. In addition, FAT10 can bind to regulatory units on the proteasome allowing for FAT10 conjugates to be degraded [25]. The UBL with the greatest similarity to ubiquitin is neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8). Similar to ubiquitin, the covalent linkage of NEDD8 to a target substrate requires an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. NAE1 is the E1 activating enzyme for NEDD8 [26,27], while both UBE2M and UBE2F are NEDD8 E2-conjugating enzymes [28–30]. Multiple E3 ligases have been identified, which can facilitate the transfer of NEDD8 to its substrate (reviewed in [31,32]). NEDDylation can be reversed through the action of the COP9 signalosome [33], which can remove NEDD8 from substrates. The canonical function of NEDD8 is to modify the scaffolding cullin family of proteins. Cullins associate with RING-box proteins to form Cullin-Ring-Ligases (CRLs), the largest family of E3 ubiquitin ligases. The modification of cullins by a single NEDD8 domain brings about a conformational change that facilitates the transfer of ubiquitin from a recruited E2-ubiquitin conjugating enzyme to the substrate of the CRL [34–36]. However, NEDD8 is known to covalently link to other proteins, such as TRAF6, VHL, p53, BCA3, and ribosomal protein L11 to name a few [37–39], and the consequences of protein NEDDylation are often unknown [31]. Several recent reports have highlighted the potential for non-canonical NEDDylation to result in a target protein’s destabilization and destruction. The first substrate identified that could be destabilized and destroyed following NEDD8 conjugation was the epithelial growth factor receptor (EGFR) [40,41]. Other proteins soon joined the list of substrates that could be destabilized and degraded following NEDDylation including HDAC, JunB, SMF2, c-Src, SRSF3, Ajuba, and E2F-1 [42–49]. In some instances, degradation was mediated by the ubiquitin-proteasome system [42–44,47–49]. Additionally, the proteasome-associated protein, NEDD8 ultimate buster 1 (NUB1), has been shown to interact with NEDD8-modified proteins, facilitating their degradation [50]. Considering its similarity to ubiquitin, it is possible that non-canonical NEDD8-modification can serve as a signal for protein degradation. Here we test this hypothesis by creating a non-cleavable form of NEDD8 appended to the N-terminus of GFP. Fusion of ubiquitin and UBLs to the N-terminus of a protein of interest is a powerful tool for identifying UBLs, such as FAT10, that can target a protein for degradation [24,51]. Ubiquitin-fusion has also been extensively used for the study of direct antigen presentation of peptides via the MHC class I antigen presentation pathway [52–55]. We find the fusion of NEDD8 resulted in the rapid degradation of GFP by both autophagy and the proteasome. Degradation required ubiquitination and was partially dependent on the action of NUB1. Interestingly, the N-terminal addition of NEDD8 to GFP prevented fluorescence in eukaryotic cells, though restoring protein folding and fluorescence was not sufficient to prevent rapid protein degradation. While the addition of NEDD8 to a protein could serve as a signal for protein degradation, eliminating the NEDD8 activity in the cell by treatment with MLN4924 did not alter the overall degradation of another rapidly degraded protein. These data indicate that under certain circumstances, the non-canonical addition of NEDD8 to a target protein may result in the proteins’ destruction. 2. Materials and Methods 2.1. Plasmids, Antibodies, and Reagents All primers were from Integrated DNA Technologies (IDT, Coralville, IA, USA) and PCR reactions amplified using a Veriti thermocycler (Applied Biosystems, Foster City, CA, USA). To generate pCAGGS-IRES-Thy1.1, we PCR amplified the IRES-Thy1.1 cassette from pMSCV Thy1.1 using the forward primer 50-ATATATGCTAGCTGATCAGCGGCCGCACCG GTTTCGAACCCCCCCCCCTAACGTTACTGGCCGAA-30 and reverse primer 50-ATAAGA TCTTCACAGAGAAATGAAGTCCAGGGCTTGGAGGAG-30. The resulting PCR product was subcloned into pCAGGS using the NheI and BglII restriction sites and confirmed by Sanger DNA sequencing at the Oregon State University Center for Genome Research and Biocomputing. DNA sequences encoding fusion proteins (wildtype human NEDD8 Cells 2021, 10, 854 3 of 23 or non-cleavable (NC) NEDD8) fused in frame with either enhanced GFP (hereafter GFP) or a cytosolic form of ovalbumin (hereafter OVA) were generated as Gene Blocks by IDT and contained an N-terminal EcoRI site and a C-terminal NheI site for subse- quent cloning into pCAGGS-IRES-Thy1.1. For NC NEDD8-constructs, the three terminal glycines of NEDD8 were changed to alanine and fused in frame with the protein of inter- est. To generate the NC NEDD8-SL8-GFP construct, the amino acid sequence SIINFEKL was introduced between the terminal amino acid for NC NEDD8 and the first amino acid of GFP. Sanger sequencing was