Direct interactions between NEDD8 and ubiquitin E2 conjugating enzymes upregulate cullin-based E3 ligase activity

Although cullin-1 neddylation is crucial for the activation of SCF ubiquitin E3 ligases, the underlying mechanisms for NEDD8-mediated activation of SCF remain unclear. Here we demonstrate by NMR and mutational studies that NEDD8 binds the ubiquitin E2 (UBC4), but not NEDD8 E2 (UBC12). Our data imply that NEDD8 forms an active platform on the SCF complex for selective recruitment of ubiquitin-charged E2s in collaboration with RBX1, and thereby upregulates the E3 activity.     

Identification of NEDD8-conjugation site in human cullin-2

NEDD8 is a novel ubiquitin-like protein that has been shown to conjugate to nuclear proteins in a manner analogous to ubiquitination and sentrinization. Recently, human cullin-4A was reported to be conjugated by a single molecule of NEDD8. Here, we show that human cullin-2 is also conjugated by a single molecule of the NEDD8. The C-terminal 171-amino-acid residues in human cullin-2 are sufficient for NEDD8-conjugation. In addition, the equivalent C-terminal fragments of other cullins have been shown to be conjugated by NEDD8. Mapping of the NEDD8-conjugation site revealed that Lys-689 in human cullin-2 is conjugated by NEDD8. Interestingly, the Lys residue at position 689 in cullin-2 is conserved in all cullin family members, including human cullin-1, -2, -3, -4A, -4B, and -5 and yeast cullin (Cdc53), suggesting the possibility that other cullin family members are conjugated by NEDD8/Rub1 at a Lys residue of equivalent position.     

Covalent modifier NEDD8 is essential for SCF ubiquitin-ligase in fission yeast

A ubiquitin-like modifier, NEDD8, is covalently attached to cullin-family proteins, but its physiological role is poorly understood. Here we report that the NEDD8-modifying pathway is essential for cell viability and function of Pcu1 (cullin-1 orthologue) in fission yeast. Pcu1 assembled on SCF ubiquitin-ligase was completely modified by NEDD8. Pcu1(K713R) defective for NEDD8 conjugation lost the ability to complement lethality due to pcu1 deletion. Forced expression of Pcu1(K713R) or depletion of NEDD8 in cells resulted in impaired cell proliferation and marked stabilization of the cyclin-dependent kinase inhibitor Rum1, which is a substrate of the SCF complex. Based on these findings, we propose that covalent modification of cullin-1 by the NEDD8 system plays an essential role in the function of SCF in fission yeast.     

The von Hippel-Lindau tumor suppressor gene product promotes, but is not essential for, NEDD8 conjugation to cullin-2

We have previously shown that human cullin-2 (Cul-2) is covalently modified at Lys-689 by NEDD8 (Wada, H., Yeh, E. T. H., and Kamitani, T. (1999) Biochem. Biophys. Res. Commun. 257, 100-105). Cul-2 has also been reported to form a multiprotein complex, Cul-2.VBC, with the von Hippel-Lindau tumor suppressor gene product (pVHL) and elongins B and C. In this study, using an in vivo coexpression system in COS cells, we show that NEDD8 conjugation to Cul-2 is promoted by coexpression with wild-type pVHL and elongins B and C. Interestingly, tumorigenic mutants and deletion mutants of pVHL, which are unable to form a Cul-2.VBC complex, do not have the activity to promote NEDD8 conjugation to Cul-2. These results suggest that the complex formation is required for NEDD8 conjugation to Cul-2. Furthermore, we used a pVHL-deficient cell line, 786-0, to show that Cul-2 is poorly but clearly conjugated by NEDD8, indicating that pVHL is not the only molecule that promotes NEDD8 conjugation to Cul-2. Taken together, the VBC complex appears to have ligase activity in the conjugation of NEDD8 to Cul-2.     

The NEDD8 pathway is essential for SCF(beta -TrCP)-mediated ubiquitination and processing of the NF-kappa B precursor p105

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.     

The molecular determinants of NEDD8 specific recognition by human SENP8

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.     

A unique E1-E2 interaction required for optimal conjugation of the ubiquitin-like protein NEDD8

Ubiquitin-like proteins (UBLs) such as NEDD8 are transferred to their targets by distinct, parallel, multienzyme cascades that involve the sequential action of E1, E2 and E3 enzymes. How do enzymes within a particular UBL conjugation cascade interact with each other? We report here that the unique N-terminal sequence of NEDD8's E2, Ubc12, selectively recruits NEDD8's E1 to promote thioester formation between Ubc12 and NEDD8. A peptide corresponding to Ubc12's N terminus (Ubc12N26) specifically binds and inhibits NEDD8's E1, the heterodimeric APPBP1-UBA3 complex. The structure of APPBP1-UBA3- Ubc12N26 reveals conserved Ubc12 residues docking in a groove generated by loops conserved in UBA3s but not other E1s. These data explain why the Ubc12-UBA3 interaction is unique to the NEDD8 pathway. These studies define a novel mechanism for E1-E2 interaction and show how enzymes within a particular UBL conjugation cascade can be tethered together by unique protein-protein interactions emanating from their common structural scaffolds.     

The ubiquitin E1 enzyme Ube1 mediates NEDD8 activation under diverse stress conditions

Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of “free” ubiquitin. A decrease in “free” ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under stress reveals the existence of mixed chains between NEDD8 and ubiquitin. We further show that NEDDylation of the p53 tumor suppressor upon stress is mediated mainly through Ube1. Our studies reveal an unprecedented interplay between NEDD8 and ubiquitin pathways operating in diverse cellular stress conditions.     

NEDD8 overexpression results in neddylation of ubiquitin substrates by the ubiquitin pathway

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway.     

Regulation of the NEDD8 conjugation system by a splicing variant,NUB1L

NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to target proteins. We previously identified a negative regulator of the NEDD8 conjugation system, NUB1, which works by recruiting NEDD8 and its conjugates to the proteasome for degradation. Recently, we found its splicing variant, NUB1L. It possesses an insertion of 14 amino acids that codes for a UBA domain. Structural study revealed that NUB1 has a NEDD8-binding site at the C terminus, whereas NUB1L has an additional site at the newly generated UBA domain. Interestingly, the sequence A(X4)L(X10)L(X3)L was conserved in these NEDD8-binding sites among human and other mammals. Mutational studies revealed that at least three Leu residues in the conserved sequence are required for binding with NEDD8. Functional study suggested that the NEDD8-binding ability at the C terminus of NUB1 and NUB1L is mainly involved in the down-regulation of NEDD8, but the NEDD8-binding ability at the UBA2 domain of NUB1L is minimally or not involved at all. The NEDD8-binding ability at the UBA2 domain might be required for an unknown function of NUB1L.     

Changes in the ratio of free NEDD8 to ubiquitin triggers NEDDylation by ubiquitin enzymes

Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.     

Identification of rabbit reticulocyte E217K as a UBC7 homologue and functional characterization of its core domain loop.

The structural basis by which ubiquitin (Ub)-conjugating enzymes (E2s) determine substrate specificity remains unclear. We cloned rabbit reticulocyte E217K because unlike the similarly sized class I E2s, E214K and UBC4, it is unable to support ubiquitin-protein ligase (E3)-dependent conjugation to endogenous proteins. RNA analysis revealed that this E2 was expressed in all tissues tested, with higher levels in the testis. Analysis of testis RNA from rats of different ages showed that E217K mRNA was induced from days 15 to 30. The predicted amino acid sequence indicates that E217K is a 19. 5-kDa class I E2 but differs from other class I enzymes in possessing an insertion of 13 amino acids distal to the active site cysteine. E217K shows 74% amino acid identity with Saccharomyces cerevisiae UBC7, and therefore, we rename it mammalian UBC7. Yeast UBC7 crystal structure indicates that this insertion forms a loop out of the otherwise conserved folding structure. Sequence analysis of E2s had previously suggested that this loop is a hypervariable region and may play a role in substrate specificity. We created mutant UBC7 lacking the loop (ubc7Deltaloop) and a mutant E214k with an inserted loop (E214k+loop) and characterized their biochemical functions. Ubc7Deltaloop had higher affinity for the E1-Ub thiol ester than native UBC7 and permitted conjugation of Ub to selected proteins in the testis but did not permit the broad spectrum E3-dependent conjugation to endogenous reticulocyte proteins. Surprisingly, E214k+loop was unable to accept Ub from ubiquitin-activating enzyme (E1) but was able to accept NEDD8 from E1. E214k+loop was able to support conjugation of NEDD8 to endogenous reticulocyte proteins but with much lower efficiency than E214k. Thus, the loop can influence interactions of the E2 with charged E1 as well as with E3s or substrates, but the exact nature of these interactions depends on divergent sequences in the remaining conserved core domain.     

Inhibition of a NEDD8 Cascade Restores Restriction of HIV by APOBEC3G

Cellular restriction factors help to defend humans against human immunodeficiency virus (HIV). HIV accessory proteins hijack at least three different Cullin-RING ubiquitin ligases, which must be activated by the small ubiquitin-like protein NEDD8, in order to counteract host cellular restriction factors. We found that conjugation of NEDD8 to Cullin-5 by the NEDD8-conjugating enzyme UBE2F is required for HIV Vif-mediated degradation of the host restriction factor APOBEC3G (A3G). Pharmacological inhibition of the NEDD8 E1 by MLN4924 or knockdown of either UBE2F or its RING-protein binding partner RBX2 bypasses the effect of Vif, restoring the restriction of HIV by A3G. NMR mapping and mutational analyses define specificity determinants of the UBE2F NEDD8 cascade. These studies demonstrate that disrupting host NEDD8 cascades presents a novel antiretroviral therapeutic approach enhancing the ability of the immune system to combat HIV.     

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

The evolutionarily conserved 8-kD protein NEDD8 (NEURAL PRECURSOR CELL EXPRESSED, DEVELOPMENTALLY DOWN-REGULATED8) belongs to the family of ubiquitin-like modifiers. Like ubiquitin, NEDD8 is conjugated to and deconjugated from target proteins. Many targets and functions of ubiquitylation have been described; by contrast, few targets of NEDD8 have been identified. In plants as well as in non-plant organisms, the cullin subunits of cullin-RING E3 ligases are NEDD8 conjugates with a demonstrated functional role for the NEDD8 modification. The existence of other non-cullin NEDD8 targets has generally been questioned. NEDD8 is translated as a precursor protein and proteolytic processing exposes a C-terminal glycine required for NEDD8 conjugation. In animals and yeast, DENEDDYLASE1 (DEN1) processes NEDD8. Here, we show that mutants of a DEN1 homolog from Arabidopsis thaliana have no detectable defects in NEDD8 processing but do accumulate a broad range of NEDD8 conjugates; this provides direct evidence for the existence of non-cullin NEDD8 conjugates. We further identify AUXIN RESISTANT1 (AXR1), a subunit of the heterodimeric NEDD8 E1 activating enzyme, as a NEDD8-modified protein in den1 mutants and wild type and provide evidence that AXR1 function may be compromised in the absence of DEN1 activity. Thus, in plants, neddylation may serve as a regulatory mechanism for cullin and non-cullin proteins.     

Another tier for caspase regulation: IAPs as NEDD8 E3 ligases

Many inhibitor of apoptosis proteins (IAPs) function as E3 ligases to ubiquitinate important cell death proteins, including caspases. Broemer et?al. (2010) report recently in Molecular Cell that IAPs can also inhibit caspases by promoting conjugation of the UBL NEDD8.     

Systematic in vivo RNAi analysis identifies IAPs as NEDD8-E3 ligases

The intimate relationship between mediators of the ubiquitin (Ub)-signaling system and human diseases has sparked profound interest in how Ub influences cell death and survival. While the consequence of Ub attachment is intensely studied, little is known with regards to the effects of other Ub-like proteins (UBLs), and deconjugating enzymes that remove the Ub or UBL adduct. Systematic in vivo RNAi analysis identified three NEDD8-specific isopeptidases that, when knocked down, suppress apoptosis. Consistent with the notion that attachment of NEDD8 prevents cell death, genetic ablation of deneddylase 1 (DEN1) suppresses apoptosis. Unexpectedly, we find that Drosophila and human inhibitor of apoptosis (IAP) proteins can function as E3 ligases of the NEDD8 conjugation pathway, targeting effector caspases for neddylation and inactivation. Finally, we demonstrate that DEN1 reverses this effect by removing the NEDD8 modification. Altogether, our findings indicate that IAPs not only modulate cellular processes via ubiquitylation but also through attachment of NEDD8, thereby extending the complexity of IAP-mediated signaling.