The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8

Abstract

Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites.

NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Promotes Transfer of NEDD8 to Proteasome for Degradation through the P97UFD1/NPL4 Complex

“…Analogously, the Asn-51 residue of NEDD8 also plays an important role in recognition by the NEDD8-specific proteases, such as DEN1 (66) and SENP8 (67). It was also reported that the N-terminal UBQ1 domain of FAT10, but not the C-terminal UBQ2 domain, interacts with NUB1L (24).…”

DENEDDYLASE1 Deconjugates NEDD8 from Non-Cullin Protein Substrates in Arabidopsis thaliana

“…Mature NEDD8 is derived from these precursors through the N-and C-terminal processing of these extensions. DEN1/NEDP1/SENP8 (DENEDDYLASE1/NEDD8-SPECIFIC PROTEASE1/ SENTRIN-SPECIFIC PROTEASE8; hitherto DEN1) was originally described from Drosophila melanogaster and mammals as a NEDD8-specific processing enzyme Mendoza et al, 2003;Wu et al, 2003;Shen et al, 2005;Chan et al, 2008;Shin et al, 2011). In addition, several ubiquitin C-terminal hydrolases from animals and yeasts were shown to possess a dual specificity for ubiquitin and NEDD8 processing (Wada et al, 1998;Johnston et al, 1999;Linghu et al, 2002;Hemelaar et al, 2004;Frickel et al, 2007;Yu et al, 2007).…”

“…DEN1 was originally identified as an enzyme required for NEDD8 precursor processing Mendoza et al, 2003;Wuetal, 2003;Shen et al, 2005;Chan et al, 2008;Shin et al, 2011). Since the processing of the Arabidopsis NEDD8 RUB precursors was not detectably affected in the den1 mutant, we tested the enzymatic activity of DEN1 toward an artificial recombinant substrate, UB:NEDD8:His.…”

Identifying de‐NEDDylation inhibitors: Virtual high‐throughput screens targeting SENP8

“…SENP8 also removes excess NEDD8 from hyper‐NEDDylated proteins (Gan‐Erdene et al., ; Mendoza et al., ; Wu et al., ). The NEDDylation process stabilizes proteins or modifies protein interaction (Broemer et al., ; Shin, Tang, Chen, Liao, & Chang, ).…”

“…With control over systems involved in various diseases, NEDD8 emerged as a promising therapeutic target, especially for cancer (Soucy et al, 2009). Successful development of the FDA approved drug bortezomib (Velcade™) (Field-Smith, Morgan, & Davies, 2006;Kane, Bross, Farrell, & Pazdur, 2003;Kane et al, 2007) targeting ubiquitin, a similarly structured protein also involved in protein modification (Hochstrasser, 2000;Shin et al, 2011;Wu et al, 2003), inspired others to search for a similar avenue of treatment with NEDD8. The search resulted in MLN4924 (Pevonedistat), a selective small-molecule inhibitor of NAE, the activating enzyme of NEDD8, and the first step in the NEDDylation process (Soucy et al, 2009).…”