nature structural & molecular biology volume 21 number 4 APrIl 2014 297

f o c u s o n u b i q u i t i n

Jacob E. Corn and Domagoj Vucic are at the Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA. e-mail: cornj@gene.com or domagoj@gene.com

assembly of the distal signaling components: the inhibitor of κB kinase (IKK) complex; the TGF-β– associated kinase TAK1 in complex with TAK1-binding protein 2 and 3 (TAB2 and TAB3); and the linear ubiquitin chain– assembly complex (LUBAC)9–12. LUBAC—which comprises SHANK-associated RH-domain interactor (SHARPIN), heme-oxidized IRP2 ubiquitin ligase 1 homolog (HOIL-1L) and the E3 HOIL-1–interacting protein (HOIP)—is recruited to the TNFR1 complex via binding to the autoubiquitinated c-IAP1 and c-IAP2 proteins to mediate the assembly of linear polyubiquitin chains on NF-κB essential modifier (NEMO) and RIP1 (refs. 13–15). TAB2 or TAB3 and IKK-γ or NEMO from the IKK complex bind poly-ubiquitin chains on RIP1 to enable IKK-β activation and subsequent phosphorylation of inhibitor of κB (IκB) (Fig. 1). Phosphorylated IκB is ubiquitinated with K48 linkages by the E3 ligase complex SCF–β-TrCP; this ubi-quitination promotes IκB degradation and allows translocation of NF-κB proteins to the nucleus. Similarly to TNF-induced signaling, ubiquitin-ligase activities of c-IAP1, c-IAP2 and LUBAC are critical for the signaling stimulated by a number of TNF family ligands including TWEAK, TL1A, CD40L and LT-β11.

TNF-stimulated signaling is negatively regulated by two DUBs with different linkage specificities: the DUB A20 removes K63-linked polyubiquitin chains from RIP1, whereas the ubiquitin thioesterase otulin disassembles lin-ear chains from RIP1 and NEMO16,17. The DUB activity of A20 and otulin restricts TNF-induced NF-κB and MAPK signaling and resulting gene

domains (which mostly dictate the selectiv-ity of ubiquitin-chain recognition, assembly and disassembly) and hundreds of E3 ligases (which provide substrate specificity) permit the vast combinatorial complexity of ubiquitin-ation processes7.

Ubiquitination in inflammatory pathwaysThe first line of defense against invading patho-gens is mediated by pathogen- recognition receptors8. This receptor family includes NOD-like receptors, Toll-like receptors (TLRs) and RIG-I–like receptors (RLRs), all of which can initiate NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways triggering the expression of proinflamma-tory cytokines and chemokines (Fig. 1). One of the major effectors of the immune response, tumor necrosis factor (TNF)-α induces strong inflammatory responses and promotes the recruitment of immune cells to sites of tissue damage or infection. TNF-α binds to the tumor necrosis factor receptor (TNFR) 1, a principal member of the TNFR superfamily, to trigger the assembly of the receptor-associated signaling complexes that activate NF-κB and MAPK signaling in a ubiquitin-dependent manner8. Initial engage-ment of the adaptor proteins TRADD and TRAF2 allows the recruitment of the ubiquitin ligases cellular inhibitors of apoptosis (c-IAPs) and their substrate receptor-interacting pro-tein 1 (RIP1) (Fig. 1). Within the TNFR1 complex, c-IAP proteins promote K11- and K63-linked polyubiquitination, mainly on RIP1 but also on TRAF2 and on them-selves, to generate a binding platform for the

Ubiquitination uses the activity of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin ligase, to attach either a single ubiquitin molecule (in monoubiquitination) or multiple ubiquitin adducts (in polyubiquitination) to a lysine of the substrate protein1. Each ubiquitin mol-ecule contains seven lysine residues and a free N terminus, thus allowing the formation of a variety of ubiquitin linkages to yield a struc-turally diverse array of polyubiquitin signals2. This diverse topology enables ubiquitination to transmit complex biological signals required for temporally and spatially controlled cellu-lar functions3. Lys63 (K63)-linked chains, N-terminally linked linear chains and, in some cellular pathways, Lys11 (K11)-linked chains provide scaffolding for the recruitment and assembly of signaling complexes4. In contrast, Lys48 (K48)-linked chains predominantly target substrates for proteasomal degrada-tion1. The assembly of ubiquitin chains can be reversed by deubiquitinating enzymes (DUBs), and several DUBs implicated in inflammatory signaling recognize specific ubiquitin-linkage chains, thus further fine-tuning the ubiqui-tination signal5. Finally, different ubiquitin moieties are recognized by a diverse group of ubiquitin-binding domains that interpret this information, thus resulting in varied biological outcomes6. Tens of different E2 and DUB enzymes and ubiquitin-binding

Ubiquitin in inflammation: the right linkage makes all the differenceJacob E Corn & Domagoj Vucic

The immune system must operate in an effective, precise and safe manner to defend against diverse pathogens while avoiding attacking the body itself and commensal bacteria. Inflammatory pathways mediated by NOD-like, Toll-like, RIG-I–like and tumor-necrosis-factor receptor families are tightly regulated by ubiquitination, especially by Lys63-linked and linear polyubiquitin chains. Here we discuss the human ubiquitin-mediated inflammatory signaling system, emphasizing the interactions and activities whose coordination ensures timely, accurate regulation of inflammatory responses.

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ways (Fig. 1). Activation of TLR4 by lipopoly-saccharide (LPS) leads to initial recruitment of the adaptor protein MyD88 and, through MyD88, the interleukin-1 receptor–associated kinases IRAK1 and IRAK4. Phosphorylation of IRAK1 by IRAK4 enables recruitment and subsequent dimerization of TRAF6, thus resulting in the induction of TRAF6 E3 activity. In collaboration with the E2 complex Ubc13–Uev1, TRAF6 promotes K63-linked polyubiquitination leading to the activation of NF-κB and MAPK signaling21. A20 is involved in TLR signaling, and its DUB activity is required for the regulation of TLR responses22. The importance of other ubiquitin-modifying enzymes is not clear at the moment, although LUBAC and linear ubiquitination have been

promotes K63-linked polyubiquitination of RIP2 (ref. 18). This post-translational modi-fication then brings LUBAC to RIP2, thus resulting in linear polyubiquitination of RIP2 and subsequent activation of the IKK com-plex and induction of proinflammatory cyto-kine and chemokine expression (Fig. 1)19. In analogous fashion to TNF signaling, A20 and otulin negatively regulate NOD2 signaling by removing K63-linked and linear chains from RIP2 (refs. 19,20). Therefore, the assembly and disassembly of K63-linked and linear polyubi-quitin chains is instrumental for the execution and regulation of TNF and NOD signaling.

Other proinflammatory stimuli rely pre-dominantly on K63-linked polyubiquitin chains for the activation of signaling path-

expression, thus demonstrating the importance of K63-linked and linear ubiquitin chains for TNF-stimulated proinflammatory signaling.

NOD-like-receptor signaling relies on K63-linked and linear polyubiquitination in a comparable manner to TNFR1 signaling8. Activation of nucleotide-binding oligomer-ization domain containing 2 (NOD2) by the bacterial cell-wall product muramyl dipep-tide recruits RIP2 via homotypic interaction between caspase activation and recruitment domains (CARDs) to stimulate NF-κB and MAPK signaling. Again, the activation of sig-naling requires ubiquitin ligases. However, unlike the TNFR1 complex that relies on c-IAPs, the NOD2–RIP2 complex recruits X chromosome–linked IAP, which in turn

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Figure 1 Ubiquitination is an integral part of inflammatory signaling. In TNFR-mediated signaling, c-IAP proteins ubiquitinate RIP1 and themselves (K63- and K11-linked polyubiquitination), and LUBAC directs NEMO ubiquitination (linear polyubiquitination). In NOD2-mediated signaling, X chromosome–linked IAP (XIAP) and LUBAC promote K63-linked and linear polyubiquitination of RIP2, thus allowing the activation of the IKK complex and proteasomal degradation of the NF-κB–inhibitory subunit IκB to enable translocation of the NF-κB proteins p50 and RelA to the nucleus. TLR4 and RIG-I signaling pathways are regulated by TRAF6 and TRIM25, which promote K63-linked polyubiquitination on TRAF6 and RIG-I to stimulate NEMO recruitment and IKK activation. NEMO is a substrate for linear ubiquitination, but it also binds polyubiquitin chains composed of various linkages, thereby providing a physical link between upstream signaling complexes (often receptor associated) and downstream complexes whose kinase activation leads to stimulation of NF-κB–mediated expression of inflammatory cytokines. MDP, muramyl dipeptide; P, phosphate; Ub, ubiquitin.

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disassembles K48-linked chains, whereas the full-length protein may remove K63-linked chains from TRAF6 in a single step by cutting the linkage between the proximal ubiquitin and the substrate lysine32. Structural and mecha-nistic insights into this activity have so far been elusive. CYLD is an USP family DUB that is unusual among other DUBs of this type in its specificity for K63-linked chains33. Unlike that of A20, CYLD activity is directed toward endo-linkages in ubiquitin chains, thus indicating that

role in the formation of K63-linked chains, because the Glu64 of ubiquitin is adjacent to the attacking K63 in the structure of Mms2–Ubc13, and mutation of this residue negatively affects the formation of K63 linkages29.

Removal of K63-linked chains from immune signaling complexes is accomplished by A20 and CYLD DUBs. A20 is a member of the OTU fam-ily of DUBs, and it contains several regulatory domains that modulate its activity. For exam-ple, the catalytic domain of A20 in isolation

reported to modulate IKK phosphorylation in LPS-stimulated TLR4 signaling23.

K63-linked polyubiquitination is also crucial for RLR signaling. Upon viral infection, the E3 TRIM25 assembles K63-linked polyubiquitin chains on the RNA helicase RIG-I, thus leading to RIG-I oligomerization and association with the mitochondrial protein MAVS (also known as VISA or IPS-1)24. This complex recruits TRAF3 and TBK1, to stimulate interferon production, as well as TRAF6, to induce NF-κB activation. K63-linked polyubiquitination in RLR signal-ing is counteracted by the DUB cylindroma-tosis (CYLD), which can remove K63-linked chains from RIG-I and thereby prevent RIG-I binding to MAVS25. Somewhat unexpectedly, linear ubiquitination appears to negatively reg-ulate RIG-I signaling by obstructing TRIM25’s interaction with, and ubiquitination of, RIG-I26. Thus, RLR proinflammatory signaling appears to rely exclusively on K63-linked polyubiqui-tination for mounting antiviral responses and producing inflammatory cytokines.

Molecular mechanisms for modulating inflammatory ubiquitin signalingUnderstanding of the mechanism by which E1-E2-E3 cascades generate the ubiquitin chains recognized during inflammatory signal-ing, as well as the means by which DUBs cleave these chains, has greatly advanced in the past several years. The discovery that K63-linked and linear ubiquitin chains can adopt distinct extended conformations27 has highlighted these chains’ structural differences from com-pact K48-linked chains28 and has implied that they may be synthesized, recognized and degraded in unique fashions.

A nearly complete picture of the formation of K63-linked chains is available from a com-bination of the structures of Mms2 (a K63-specific heterodimeric E2 conjugating enzyme, and the yeast homolog of UEV1A) and Ubc13 bound to a crystallographic ubiquitin dimer29 and the TRAF6 E3 ligase30. The ‘donor’ ubi-quitin contacts solely Ubc13, and the donor’s C-terminal glycine is covalently attached to the catalytic cysteine of Ubc13 (a serine in the structure) (Fig. 2). The ‘acceptor’ ubi-quitin is held by both Ubc13 and Mms2, such that K63 is positioned to attack the thioester bond. Intriguingly, early structural analysis of E2 conjugating enzymes failed to identify an obvious residue involved in deprotonating the ubiquitin lysine involved in attack on the thioester bond. Experimentally driven model-ing of a monomeric K11-specific E2 indicated that this role is fulfilled by an acidic residue on ubiquitin itself31, in a mechanism known as substrate-assisted catalysis. In retrospect, substrate-assisted catalysis may also have a

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Figure 2 Structures of molecules that make and break ubiquitin chains involved in inflammatory signaling. (a) Ubc13 and Mms2 (UEV1A in humans) form a heterodimeric E2 for creating K63-linked chains with TRAF6. Coordination of the acceptor ubiquitin uniquely positions K63 for attack on the thioester formed between Ubc13 and the donor ubiquitin (PDB 2GMI)29. WT, wild type. (b) HOIP is an RBR-type E3 ligase that generates linear ubiquitin chains. Binding platforms for both the body of the acceptor ubiquitin and the tail of the donor ubiquitin orient these moieties to ensure specificity (PDB 4LJO)36. (c) Otulin is a DUB that cuts only linear chains. Unique binding of the acceptor and donor ubiquitins, as well as substrate-assisted catalysis mediated by ubiquitin’s Glu16, ensure exquisite specificity for linear chains (PDB 3ZNZ)17.

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ACKNOWLEDGMENTSWe thank N. Kayagaki for helpful comments.

COMPETING FINANCIAL INTERESTSThe authors declare competing financial interests: details are available in the online version of the paper (doi:10.1038/nsmb.2808).

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other chain types27,38. By contrast, otulin (also known as gumby and Fam105b) is an OTU family DUB that degrades exclusively linear chains17,39. At least some of this specificity is probably imparted by otulin’s high affinity for linear chains (Kd of ~100 nM) versus other chains (for example, K63-linked diubiquitin, with Kd of ~10 μM). Structures of otulin com-plexed with linear diubiquitin showed that this tight binding is accomplished by contacts between otulin and both the distal and proxi-mal ubiquitins that presumably can accommo-date only a linear chain17,39.

In addition to this thermodynamic con-straint on specificity, otulin also kinetically imparts exclusivity for linear chains via the organization of its active site. The catalytic center of otulin resembles other OTUs, with a catalytic cysteine that is activated by a histidine, which in turn is polarized by an asparagine. In the otulin– diubiquitin complex, Glu16 from ubiquitin itself reaches into the active site and engages the active site asparagine, thereby fur-ther helping to align the activating histidine. These contacts are functionally critical, because mutation of ubiquitin’s Glu16 reduces the kcat by over 200-fold. Because this mutation has little effect on Km, otulin appears to use substrate-assisted catalysis for its activity, in a manner that is likely to be specific for linear chains17.

OutlookUbiquitination is a multifunctional post- translational modification that can convey a wealth of information via several unique link-age types. Inflammatory signaling makes use of several of these linkages, especially K63-linked and linear chains. Understanding of ubiqui-tin’s role in inflammation has greatly increased within the last few years, but several questions still remain. Future efforts directed at defining the exact composition of signaling polyubi-quitin chains (e.g., whether uniform, branched or mixed K63-linked and linear chains have a predominant role) and determination of the exact timing of their assembly and cleavage should enhance understanding and usher in new strategies for therapeutic intervention in pathophysiological inflammatory conditions regulated by ubiquitination.

CYLD trims back K63-linked chains instead of removing them wholesale.

The addition of linear ubiquitin chains (linked to one another via the α-amino group of Met1 and the carboxylate group of Gly76) onto substrate proteins by a specific ubiquitin ligase is a relatively new discovery34. Indeed, because ubiquitin is genomically encoded as a linear fusion of ubiquitin monomers, the existence of a linear ligase seemed redundant. However, LUBAC is now understood to have a role in immune signaling via the synthesis of linear chains34. The LUBAC subunit HOIP belongs to a subclass of RING E3s known as RBR ligases, which use a RING1 subdomain to recognize ubiquitin-loaded E2, thereby trans-ferring the incoming ubiquitin onto a RING2 subdomain that forms a covalent thioester between its catalytic cysteine (activated by a neighboring histidine) and the ubiquitin35. Ubiquitin is finally transferred onto a substrate via nucleophilic attack of the substrate lysine that displaces the ubiquitin-ligase thioester.

A recent structure of HOIP has both shed new light on this unusual class of ligases and revealed the mechanism by which LUBAC specifically synthesizes linear chains36. HOIP structurally resembles other crystallized RBRs, such as Parkin and HHARI37, but it also con-tains an unusual ubiquitin-binding module. This region coordinates the acceptor ubiquitin (which is already part of the chain) and orients ubiquitin’s N terminus so that it points toward the thio ester that would be formed between HOIP and the C terminus of the donor ubi-quitin (Fig. 2). An acidic residue of ubiquitin (Glu16) is again located in proximity to the attacking amino group (ubiquitin’s N terminus). However, additional direct contacts between Glu16 and HOIP make it unclear whether reductions in catalysis upon mutation of this residue stem from a substrate-mediated cata-lytic mechanism, as suggested for E2 transfer complexes. Future work will, we hope, yield new insight into the means by which RBR ligases such as HOIP are activated by E2 enzymes.

Removal and degradation of linear ubiquitin chains is accomplished by a new addition to the list of known DUBs. Most DUBs either do not cut linear chains or do so equivalently with

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