S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.     

A single MIU motif of MINDY‐1 recognizes K48‐linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs.     

Ubiquitin Ser65 phosphorylation affects ubiquitin structure,chain assembly and hydrolysis

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.     

Dimeric Ube2g2 simultaneously engages donor and acceptor ubiquitins to form Lys48‐linked ubiquitin chains

Cellular adaptation to proteotoxic stress at the endoplasmic reticulum (ER) depends on Lys48‐linked polyubiquitination by ER‐associated ubiquitin ligases (E3s) and subsequent elimination of ubiquitinated retrotranslocation products by the proteasome. The ER‐associated E3 gp78 ubiquitinates misfolded proteins by transferring preformed Lys48‐linked ubiquitin chains from the cognate E2 Ube2g2 to substrates. Here we demonstrate that Ube2g2 synthesizes linkage specific ubiquitin chains by forming an unprecedented homodimer: The dimerization of Ube2g2, mediated primarily by electrostatic interactions between two Ube2g2s, is also facilitated by the charged ubiquitin molecules. Mutagenesis studies show that Ube2g2 dimerization is required for ER‐associated degradation (ERAD). In addition to E2 dimerization, we show that a highly conserved arginine residue in the donor Ube2g2 senses the presence of an aspartate in the acceptor ubiquitin to position only Lys48 of ubiquitin in proximity to the donor E2 active site. These results reveal an unanticipated mode of E2 self‐association that allows the E2 to effectively engage two ubiquitins to specifically synthesize Lys48‐linked ubiquitin chains.     

Lysine 63‐linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48‐linked polyubiquitin chain. In contrast, modifications with the Lys63‐linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome‐independent cellular processes. Nevertheless, the ubiquitin chain‐type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin‐ligase in budding yeast, catalyzes the formation of Lys63‐linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63‐linked ubiquitinated substrate in vitro. To examine whether Lys63‐linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2‐p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63‐linkages, and the Lys63‐linked chains were sufficient for the proteasome‐binding and subsequent p120‐processing. In addition, Lys63‐linked chains as well as Lys48‐linked chains were detected in the 26S proteasome‐bound polyubiquitinated proteins. These results raise the possibility that Lys63‐linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.     

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys(48)), 38% (Lys(63)), 8% (Lys(29)), 2% (Lys(11)), and 0.5% or less for linear, Lys(6), Lys(27), and Lys(33) linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys(6), Lys(11), Lys(27), Lys(29), and Lys(48) mediate proteasomal degradation, whereas Lys(63) chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys(48), may also contribute to lysosomal targeting, as both Lys(63) and Lys(48) linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys(11), Lys(48), and Lys(63) but not Lys(29) linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.     

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63‐branched ubiquitin chains

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin‐conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin‐binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63‐linked polyubiquitin and facilitates the selective assembly of K48/K63‐branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.     

E2 conjugating enzyme selectivity and requirements for function of the E3 ubiquitin ligase CHIP

The transfer of ubiquitin (Ub) to a substrate protein requires a cascade of E1 activating, E2 conjugating, and E3 ligating enzymes. E3 Ub ligases containing U-box and RING domains bind both E2～Ub conjugates and substrates to facilitate transfer of the Ub molecule. Although the overall mode of action of E3 ligases is well established, many of the mechanistic details that determine the outcome of ubiquitination are poorly understood. CHIP (carboxyl terminus of Hsc70-interacting protein) is a U-box E3 ligase that serves as a co-chaperone to heat shock proteins and is critical for the regulation of unfolded proteins in the cytosol. We have performed a systematic analysis of the interactions of CHIP with E2 conjugating enzymes and found that only a subset bind and function. Moreover, some E2 enzymes function in pairs to create products that neither create individually. Characterization of the products of these reactions showed that different E2 enzymes produce different ubiquitination products, i.e. that E2 determines the outcome of Ub transfer. Site-directed mutagenesis on the E2 enzymes Ube2D1 and Ube2L3 (UbcH5a and UbcH7) established that an SPA motif in loop 7 of E2 is required for binding to CHIP but is not sufficient for activation of the E2～Ub conjugate and consequent ubiquitination activity. These data support the proposal that the E2 SPA motif provides specificity for binding to CHIP, whereas activation of the E2～Ub conjugate is derived from other molecular determinants.     

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.     

Solution structure and dynamics of human ubiquitin conjugating enzyme Ube2g2

Ube2g2 is an E2 enzyme which functions as part of the endoplasmic reticulum‐associated degradation (ERAD) pathway responsible for identification and degradation of misfolded proteins in the endoplasmic reticulum. In tandem with a cognate E3 ligase, Ube2g2 assembles K48‐linked polyubiquitin chains and then transfers them to substrate, leading ultimately to proteasomal degradation of the polyubiquitin‐tagged substrate. We report here the solution structure and backbone dynamics of Ube2g2 solved by nuclear magnetic resonance spectroscopy. Although the solution structure agrees well with crystallographic structures for the E2 core, catalytically important loops (encompassing residues 95–107 and 130–135) flanking the active site cysteine are poorly defined. ¹⁵N spin relaxation and residual dipolar coupling analysis directly demonstrates that these two loops are highly dynamic in solution. These results suggest that Ube2g2 requires one or more of its protein partners, such as cognate E3, acceptor ubiquitin substrate or thiolester‐linked donor ubiquitin, to assume its catalytically relevant conformation. Within the NMR structural ensemble, interactions were observed between His94 and the highly mobile loop residues Asp98 and Asp99, supporting a possible role for His94 as a general base activated by the carboxylate side‐chains of Asp98 or Asp99. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Allosteric regulation of E2:E3 interactions promote a processive ubiquitination machine

RING finger proteins constitute the large majority of ubiquitin ligases (E3s) and function by interacting with ubiquitin‐conjugating enzymes (E2s) charged with ubiquitin. How low‐affinity RING–E2 interactions result in highly processive substrate ubiquitination is largely unknown. The RING E3, gp78, represents an excellent model to study this process. gp78 includes a high‐affinity secondary binding region for its cognate E2, Ube2g2, the G2BR. The G2BR allosterically enhances RING:Ube2g2 binding and ubiquitination. Structural analysis of the RING:Ube2g2:G2BR complex reveals that a G2BR‐induced conformational effect at the RING:Ube2g2 interface is necessary for enhanced binding of RING to Ube2g2 or Ube2g2 conjugated to Ub. This conformational effect and a key ternary interaction with conjugated ubiquitin are required for ubiquitin transfer. Moreover, RING:Ube2g2 binding induces a second allosteric effect, disrupting Ube2g2:G2BR contacts, decreasing affinity and facilitating E2 exchange. Thus, gp78 is a ubiquitination machine where multiple E2‐binding sites coordinately facilitate processive ubiquitination.     

Induced TRIM21 ISGylation by IFN-β enhances p62 ubiquitination to prevent its autophagosome targeting

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-β also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-β.Subject terms: Proteins, Autophagy, Innate immunity, Post-translational modifications     

Ubiquitin conjugation to cytochromes c. Structure of the yeast iso-1 conjugate and possible recognition determinants.

Saccharomyces cerevisiae iso-1-cytochrome c was conjugated with ubiquitin (Ub) in vitro in a rabbit reticulocyte extract (Fraction II). By N-terminal protein sequencing, it was found for both the mono- and diubiquitinated products that the major Ub attachment site is on Lys4 (residue 9) of the cytochrome c. Thus, the residue ubiquitinated in iso-1-cytochrome c is identical with that previously determined for the yeast iso-2 form (Sokolik, C. W., and Cohen, R. E. (1991) J. Biol. Chem. 266, 9100-9107). For both cytochromes c, the proportions of diubiquitinated and higher order conjugates are drastically reduced when Ub is replaced with a Lys48----Arg variant, suggesting that the Ub-Ub moieties are linked predominantly through Lys48. Despite close similarities in structure and ubiquitination sites, conjugation to iso-2-cytochrome c is approximately 5-fold faster than for the iso-1 form; vertebrate cytochromes c are even poorer substrates, being ubiquitinated at only approximately 5% of the rate of the iso-2 protein. Comparison of several cytochrome c variants excludes alpha-N-acetylation or the identity of the N-terminal amino acid as the important recognition determinants in these reactions. The results, which include the finding that ferro and ferri-iso-2-cytochromes c are ubiquitinated equally, also are evidence against a simple correlation between ubiquitination efficiency and thermodynamic stability. Rather, the presence of a pair of lysines (Lys4-Lys5) within the relatively unstructured N-terminal extension of the yeast cytochromes c may be responsible for their preferential ubiquitination.     

Two Mms2 residues cooperatively interact with ubiquitin and are critical for Lys63 polyubiquitination in vitro and in vivo

Recent structural analyses support a model whereby Mms2 interacts with and orientates Ub to promote Ubc13-mediated Lys63 chain formation. However, residues of the hMms2-Ub interface have not been addressed. We found two hMms2 residues to be critical for binding and polyUb conjugation. Surprisingly, while each single mutation reduces the binding affinity, the double mutation causes significant reduction of Ub binding and abolishes polyUb chain formation. Furthermore, the corresponding yeast mms2 double mutant exhibited an additive phenotype that caused a complete loss of MMS2 function. Taken together, this study identifies key residues of the Mms2-Ub interface and provides direct experimental evidence that Mms2 physical association with Ub is correlated with its ability to promote Lys63-linked Ub chain assembly.     

Activation of Duck RIG-I by TRIM25 Is Independent of Anchored Ubiquitin

Retinoic acid inducible gene I (RIG-I) is a viral RNA sensor crucial in defense against several viruses including measles, influenza A and hepatitis C. RIG-I activates type-I interferon signalling through the adaptor for mitochondrial antiviral signaling (MAVS). The E3 ubiquitin ligase, tripartite motif containing protein 25 (TRIM25), activates human RIG-I through generation of anchored K63-linked polyubiquitin chains attached to lysine 172, or alternatively, through the generation of unanchored K63-linked polyubiquitin chains that interact non-covalently with RIG-I CARD domains. Previously, we identified RIG-I of ducks, of interest because ducks are the host and natural reservoir of influenza viruses, and showed it initiates innate immune signaling leading to production of interferon-beta (IFN-β). We noted that K172 is not conserved in RIG-I of ducks and other avian species, or mouse. Because K172 is important for both mechanisms of activation of human RIG-I, we investigated whether duck RIG-I was activated by TRIM25, and if other residues were the sites for attachment of ubiquitin. Here we show duck RIG-I CARD domains are ubiquitinated for activation, and ubiquitination depends on interaction with TRIM25, as a splice variant that cannot interact with TRIM25 is not ubiquitinated, and cannot be activated. We expressed GST-fusion proteins of duck CARD domains and characterized TRIM25 modifications of CARD domains by mass spectrometry. We identified two sites that are ubiquitinated in duck CARD domains, K167 and K193, and detected K63 linked polyubiquitin chains. Site directed mutagenesis of each site alone, does not alter the ubiquitination profile of the duck CARD domains. However, mutation of both sites resulted in loss of all attached ubiquitin and polyubiquitin chains. Remarkably, the double mutant duck RIG-I CARD still interacts with TRIM25, and can still be activated. Our results demonstrate that anchored ubiquitin chains are not necessary for TRIM25 activation of duck RIG-I.     

Assembly of different length of polyubiquitins on the catalytic cysteine of E2 enzymes without E3 ligase; a novel application of non-reduced/reduced 2-dimensional electrophoresis

In this study using non-reduced/reduced 2-dimensional electrophoresis (NR/R-2DE), we clearly demonstrated that E3-independent ubiquitination by Ube2K produced not only unanchored but also Ube2K-linked polyubiquitins through thioester and isopeptide bonds. E3-independent assembly of polyubiquitins on the catalytic cysteine of Ube2K strongly supports the possibility of ‘en bloc transfer’ for polyubiquitination. From the same analyses of E3-independent ubiquitination products by other E2s, we also found that different lengths of polyubiquitins were linked to different E2s through thioester bond; longer chains by Cdc34 like Ube2K, short chains by Ube2g2, and mono-ubiquitin by UbcH10. Our results suggest that E2s possess the different intrinsic catalytic activities for polyubiquitination.     

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production.