NEDD8: a new ataxin-3 interactor

Machado-Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.     

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.     

A dominant-negative UBC12 mutant sequesters NEDD8 and inhibits NEDD8 conjugation in vivo

NEDD8, a novel ubiquitin-like protein, has been shown to conjugate to proteins in a manner analogous to ubiquitination and sentrinization. Recently, human UBC12 was identified as a putative NEDD8 conjugation enzyme (E2). While investigating the in vivo function of UBC12, we found that the point mutant, UBC12(C111S), showed a dominant-negative effect on NEDD8 conjugation. This mutant, with a single Cys-to-Ser substitution at the conserved Cys residue in the E2 family, could specifically inhibit NEDD8 conjugation. We observed the dominant-negative effect on NEDD8 conjugation to substrates, including the C-terminal fragment of cullin-2 (Cul-2-DeltaN), full-length cullin-1, and also other uncharacterized target proteins. Interestingly, UBC12(C111S) formed a heterodimeric conjugate with NEDD8. This conjugate was stable under stringent conditions, including 6 m guanidine HCl, 8 m urea, 2% SDS, or 5% beta-mercaptoethanol. Our results are consistent with the hypothesis that UBC12(C111S) sequesters the NEDD8 monomer by forming a UBC12(C111S)-NEDD8 conjugate and, in turn, inhibits the subsequent transfer of NEDD8 to its targets. To examine the biological role of NEDD8 conjugation, this dominant-negative form of UBC12 was applied to a cell growth assay. Overexpression of UBC12(C111S) led to inhibition of growth in U2OS and HEK293 cells. Thus, this dominant-negative form of UBC12 could be useful in defining the role of NEDD8 modification in other biological systems.     

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.     

Interferon-induced Transmembrane Protein 3 Is a Type II Transmembrane Protein

The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.     

NEDD8 recruits E2-ubiquitin to SCF E3 ligase

NEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modifier that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modification of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our findings indicate that the NEDD8-modifying system accelerates the formation of the E2-E3 complex, which stimulates protein polyubiquitylation.     

The Saccharomyces Cerevisiae Spt8 Gene Encodes a Very Acidic Protein That Is Functionally Related to Spt3 and Tata-Binding Protein

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the β subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.     

Covalent NEDD8 Conjugation Increases RCAN1 Protein Stability and Potentiates Its Inhibitory Action on Calcineurin

Similar to ubiquitin, regulatory roles for NEDD8 (neural precursor cell-expressed developmentally down-regulated 8) are being clarified during cell growth, signal transduction, immune response, and development. However, NEDD8 targets and their functional alterations are not well known. Regulator of calcineurin 1 (RCAN1/DSCR1P1) is located near the Down syndrome critical region on the distal part of chromosome 21, and its gene product is an endogenous inhibitor of calcineurin signaling. RCAN1 is modified by ubiquitin and consequently undergoes proteasomal degradation. Here we report that NEDD8 is conjugated to RCAN1 (RCAN1-1S) via three lysine residues, K96, K104, and K107. Neddylation enhances RCAN1 protein stability without affecting its cellular location. In addition, we found that neddylation significantly inhibits proteasomal degradation of RCAN1, which may underlie the ability of NEDD8 to enhance RCAN1 stability. Furthermore, neddylation increases RCAN1 binding to calcineurin, which potentiates its inhibitory activity toward downstream NFAT signaling. The present study provides a new regulatory mechanism of RCAN1 function and highlights an important role for diverse RCAN1-involved cellular physiology.     

Stress,specificity and the NEDD8 proteome

Comment on: Leidecker O, et al. Cell Cycle 2012; 1142–50In an exciting and surprising paper in a recent issue of Cell Cycle, Leidecker et al. show that the balance between protein modification by ubiquitin or the ubiquitin like protein NEDD8 is dramatically altered by cellular stress. In a variety of conditions that reduce the concentration of free ubiquitin, a very dramatic increase in protein modification by neddylation is revealed. Importantly, this process is shown to arise as NEDD8 is activated under these conditions by the ubiquitin-activating enzyme Ube1 and not by the typical NEDD8 specific EI enzyme, NAE. This results in many proteins in stressed cells being modified by mixed ubiquitin NEDD8 chains, which is highly relevant in the development of novel cancer therapeutics, as the NAE specific inhibitor MLN4924²does not block this new pathway despite its promising anticancer activity.Initial comparative studies on the ubiquitin and ubiquitin-like (Ubl) protein pathways have established that each pathway has separate and specific enzymes both for activating the Ubl and for removing it.³ In the case of NEDD8, the E1 is NAE; the E2s are Ubc12 and Ube2F, and the E3s include the Rbx1 and Rbx2 RING finger proteins as well as members of the DCN family of proteins. The first studies of the NEDD8 system suggested that there were very few substrates for this modification, with most emphasis placed on the cullin proteins. The cullins are components of the cullin-RING ligases (CRLs) that are responsible for the ubiquitylation of many critical substrates, for example, oncoproteins such as cyclin E and c-myc. The cullins are modified by neddylation, which increases the E3 activity of the CRLs, probably through structural alterations that free the Ring domain of the E3 and/or by blocking the binding of inhibitory proteins such as CAND 1.^4,5 Recently, many new substrates and E3 ligases for NEDD8 have been uncovered, with initial studies identifying p53 and Mdm2 as substrates for neddylation, and Mdm2 as a E3 ligase for both NEDD8 and ubiquitin.⁶ Proteomic approaches have now identified many more substrates, notable among them being the ribosomal proteins involved in signaling to p53.^7,8 In the current study, the authors found that a high level of NEDD8-conjugated proteins were rapidly induced by proteasome inhibition with MG132, but that this reaction was not inhibited by MLN4924, even while the same compound was blocking cullin neddylation. This meant that another E1 had to be in play for the neddylation of these new substrates, and knockdown of Ube1 (which was known to be able to activate NEDD8 in vitro)⁹ showed that it was, indeed, responsible. Exploring further stress signals showed that this increased neddylation response was induced by heat shock and by elevated levels of reactive oxygen species (ROS). Since all of these stress pathways reduce free ubiquitin levels, the authors asked if NAE-independent neddylation could be triggered simply by reducing free ubiquitin levels. The clearly positive results of this study suggested that competition with ubiquitin for Ube1 may normally limit Ube1 activation of NEDD8 and the neddylation of non-cullin substrates (Fig. 1). Open in a separate windowFigure 1. Nedd8 pathway and stress. (A) In unstressed cells, two parallel and non-overlapping pathways are in play. Nedd8 activation is through the action of NAE, while ubiquitin is activated by Ube1. Substrate selectivity of the E2 and E3 results in many proteins being ubiquitinated, but few are Nedd8-modified, notably, the cullins. (B) Low free ubiquitin levels in stress conditions results in Nedd8 being activated by the ubiquitin Ube1 as well as NAE1. This, in turn, results in a large increase in the variety of protein substrates that are NEDD8-modified, in addition to the cullins.In stress conditions then, when free ubiquitin levels fall, Ube1 acts as a sensor of this state and neddylation increases. Why would this be useful? The speculation is that the modification of substrate proteins by NEDD8 may help the cell to cope with stress signals, for example, by promoting cell survival through inhibition of the degradation of very labile pro-survival proteins, such as Mcl-1. After the stress signal abates, the many effective de-ubiquitinating and de-neddylating enzymes can come into play to restore homeostasis. Improved mass spectrometry methods developed in this paper using Lys-C to digest neddylated proteins allow one to distinguish NEDD8 modification from ubiquitination. This helps to further refine our knowledge of this fascinating system, but, meanwhile, protein neddylation may provide a new biomarker for cellular stress. Many critical issues remain to be resolved: are there proteins with ubiquitin/NEDD8 binding domains that specifically recognize the ubiquitin NEDD8 hybrid chains that result from these stress signals? Which E2s and E3s are responsible for stress-induced neddylation? Should Ube1 inhibitors be developed to complement the NAE inhibitor in cancer treatments, or would they prove too toxic? The next few years promise to reveal critical insights into the crosstalk between the different Ubl pathways.     

Evolutionally conserved intermediates between ubiquitin and NEDD8

The investigation of common structural motifs provides additional information on why proteins conserve similar topologies yet may have non-conserved amino acid sequences. Proteins containing the ubiquitin superfold have similar topologies, although the sequence conservation is rather poor. Here, we present novel similarities and differences between the proteins ubiquitin and NEDD8. They have 57% identical sequence, almost identical backbone topology and similar functional strategy, although their physiological functions are mutually different. Using variable pressure NMR spectroscopy, we found that the two proteins have similar conformational fluctuation in the evolutionary conserved enzyme-binding region and contain a structurally similar locally disordered conformer (I) in equilibrium with the basic folded conformer (N). A notable difference between the two proteins is that the equilibrium population of I is far greater for NEDD8 (DeltaG(0)(NI)<5 kJ/mol) than for ubiquitin (DeltaG(0)(NI)=15.2(+/-1.0) kJ/mol), and that the tendency for overall unfolding (U) is also far higher for NEDD8 (DeltaG(0)(NU)=11.0(+/-1.5) kJ/mol) than for ubiquitin (DeltaG(0)(NU)=31.3(+/-4.7) kJ/mol). These results suggest that the marked differences in thermodynamic stabilities of the locally disordered conformer (I) and the overall unfolding species (U) are a key to determine the functional differences of the two structurally similar proteins in physiology.     

Stress,specificity and the NEDD8 proteome

Comment on: Leidecker O, et al. Cell Cycle 2012; 1142–50     

Role of NEDD8 in HIV-associated lipodystrophy

The pathogenetic bases of HAART-associated lipodystrophy are still poorly known, even if it is clear that adipose tissue and its metabolism are sensitive to antiretroviral therapy alone and/or in combination with HIV infection. The NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression, morphogenesis and tumorigenesis. We investigated the possible involvement of NEED8 in adipogenesis and, consequently, in HIV-related lipodystrophy.     

The Caspase-8 Dimerization/Dissociation Balance Is a Highly Potent Regulator of Caspase-8, -3, -6 Signaling

Apoptosis is driven by positive feedback activation between aspartate-specific cysteinyl proteases (caspases). These feedback loops ensure the swift and efficient elimination of cells upon initiation of apoptosis execution. At the same time, the signaling network must be insensitive to erroneous, mild caspase activation to avoid unwanted, excessive cell death. Sublethal caspase activation in fact was shown to be a requirement for the differentiation of multiple cell types but might also occur accidentally during short, transient cellular stress conditions. Here we carried out an in silico comparison of the molecular mechanisms that so far have been identified to impair the amplification of caspase activities via the caspase-8, -3, -6 loop. In a systems model resembling HeLa cervical cancer cells, the dimerization/dissociation balance of caspase-8 potently suppressed the amplification of caspase responses, surprisingly outperforming or matching known caspase-8 and -3 inhibitors such as bifunctional apoptosis repressor or x-linked inhibitor of apoptosis protein. These findings were further substantiated in global sensitivity analyses based on combinations of protein concentrations from the sub- to superphysiological range to screen the full spectrum of biological variability that can be expected within cell populations and between distinct cell types. Additional modeling showed that the combined effects of x-linked inhibitor of apoptosis protein and caspase-8 dimerization/dissociation processes can also provide resistance to larger inputs of active caspases. Our study therefore highlights a central and so far underappreciated role of caspase-8 dimerization/dissociation in avoiding unwanted cell death by lethal amplification of caspase responses via the caspase-8, -3, -6 loop.     

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.     

Kinesin-8 Is a Low-Force Motor Protein with a Weakly Bound Slip State

During the cell cycle, kinesin-8s control the length of microtubules by interacting with their plus ends. To reach these ends, the motors have to be able to take many steps without dissociating. However, the underlying mechanism for this high processivity and how stepping is affected by force are unclear. Here, we tracked the motion of yeast (Kip3) and human (Kif18A) kinesin-8s with high precision under varying loads using optical tweezers. Surprisingly, both kinesin-8 motors were much weaker compared with other kinesins. Furthermore, we discovered a force-induced stick-slip motion: the motor frequently slipped, recovered from this state, and then resumed normal stepping motility without detaching from the microtubule. The low forces are consistent with kinesin-8s being regulators of microtubule dynamics rather than cargo transporters. The weakly bound slip state, reminiscent of a molecular safety leash, may be an adaptation for high processivity.