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.     

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.     

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.     

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.     

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.     

Structural and functional analysis of ataxin-2 and ataxin-3.

Spinocerebellar ataxia types 2 (SCA2) and 3 (SCA3) are autosomal-dominantly inherited, neurodegenerative diseases caused by CAG repeat expansions in the coding regions of the genes encoding ataxin-2 and ataxin-3, respectively. To provide a rationale for further functional experiments, we explored the protein architectures of ataxin-2 and ataxin-3. Using structure-based multiple sequence alignments of homologous proteins, we investigated domains, sequence motifs, and interaction partners. Our analyses focused on presumably functional amino acids and the construction of tertiary structure models of the RNA-binding Lsm domain of ataxin-2 and the deubiquitinating Josephin domain of ataxin-3. We also speculate about distant evolutionary relationships of ubiquitin-binding UIM, GAT, UBA and CUE domains and helical ANTH and UBX domain extensions.     

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.     

A Metal-Based Inhibitor of NEDD8-Activating Enzyme

A cyclometallated rhodium(III) complex [Rh(ppy)₂(dppz)]⁺ (1) (where ppy = 2-phenylpyridine and dppz = dipyrido[3,2-a:2′,3′-c]phenazine dipyridophenazine) has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE). The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.     

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     

Site-specific mutagenesis in a homogeneous polyglutamine tract: application to spinocerebellar ataxin-3

In recent years, nine neurodegenerative diseases have been found to be caused by the expansion of a CAG-triplet repeat in the coding region of the respective genes, resulting in lengthening of an otherwise harmless polyglutamine tract in the gene products. To facilitate structural studies of these disease mechanisms, a general protocol is described that allows site-specific mutations to be introduced into the polyglutamine tract. Based on 'cassette mutagenesis', this protocol involves engineering unique restriction sites into the flanking regions of the CAG repeat and subsequently replacing the wild-type CAG repeat with a double-stranded synthetic DNA fragment containing the desired mutations. This method was applied to the spinocerebellar ataxin-3 protein, such that the wild-type amino acid sequence -Q(3)KQ(22)- was replaced by a -Q(9)CQ(9)- sequence. In this case, the incorporated cysteine residue can be exploited for various chemical modifications, lending the host glutamine repeat to many structural and biophysical techniques for the resolution of a specific residue. The method reported here bypasses many problems that can arise from PCR-based mutagenesis methods.     

Temperature profoundly affects ataxin-3 fibrillogenesis

Ataxin-3 (AT3) triggers spinocerebellar ataxia type 3 when it carries a polyglutamine stretch expanded beyond a critical threshold. By Fourier transform infrared spectroscopy and atomic force microscopy we previously showed that a normal (AT3Q24) and an expanded (AT3Q55) variant were capable of evolving into oligomers and protofibrils at 37 °C, whereas only the expanded form generated irreversibly aggregated fibrils that also were associated with a network of side-chain glutamine hydrogen bonding [Natalello et al. (2011) PLoS One. 6:e18789]. We report here that AT3Q24, when gradually heated up to 85 °C, undergoes aggregation similar to that observed at 37 °C; in contrast, AT3Q55 only generates large, amorphous aggregates. We propose a possible interpretation of the mechanism by which temperature affects the outcome of fibrillogenesis.     

Autophagy mediates SUMO-induced degradation of a polyglutamine protein ataxin-3

Previously, we reported that small ubiquitin-like modifier-1 (SUMO-1) promotes the degradation of a polyglutamine (polyQ) protein ataxin-3 and proposed that proteasomes mediate the proteolysis. Here, we present evidence that autophagy is also responsible for SUMO-induced degradation of this polyQ protein. The autophagy inhibitor 3-MA increased the steady-state level of ataxin-3 and stabilized SUMO-modified ataxin-3 more prominently than the proteasome inhibitor MG132. Interestingly, SUMO-1 overexpression enhanced the co-localization of ataxin-3 and autophagy marker LC3 without increasing LC3 puncta formation suggesting that SUMO-1 is involved in the substrate recruitment rather than the induction of autophagy. To assess the importance of a putative SUMO-interacting motif (SIM) in ataxin-3 for SUMO-induced degradation, we constructed a SIM mutant of ataxin-3. Substitution of putative SIM (V165G) facilitated the degradation of polyQ-expanded ataxin-3, which is more resistant to SUMO-induced degradation than the normal ataxin-3. These results together indicate that SUMO-1 promotes the degradation of ataxin-3 via autophagy and the putative SIM of ataxin-3 plays a role in this process.