Backbone and side chain 1H, 13C, and 15N assignments of the ubiquitin-like domain of human HOIL-1L, an essential component of linear ubiquitin chain assembly complex

HOIL-1L and its binding partner, HOIL-1L interacting protein (HOIP), are essential components of linear ubiquitin (Ub) chain assembly complex (LUBAC), a 600-kDa enzyme complex catalyzing elongation of a tandemly connected Ub chain, which serve as a regulator of NF-κB activation. Specific interaction between the N-terminal Ub-like domain (UBL) of HOIL-1L and the Ub-associated domain (UBA) located at the central region of HOIP is shown to be involved in the formation of LUBAC. For better understanding of the mechanisms underlying the generation of the linear Ub chains by LUBAC, it is necessary to characterize the UBL-UBA interaction on the basis of structural data, which, however, is not available to date. Here we report backbone and side chain NMR assignments of the UBL of human HOIL-1L. By inspection of chemical shift index, it was predicted that HOIL-1L-UBL assumes a Ub fold followed by an α-helical segment, offering the basis for determination its 3D structure and interaction with HOIP-UBA in solution.     

Biophysical and biological evaluation of optimized stapled peptide inhibitors of the linear ubiquitin chain assembly complex (LUBAC)

Linear ubiquitylation, in which ubiquitin units are covalently linked through N- and C-terminal amino acids, is a unique cellular signaling mechanism. This process is controlled by a single E3 ubiquitin ligase, the linear ubiquitin chain assembly complex (LUBAC), which is composed of three proteins – HOIL-1L, HOIP and SHARPIN. LUBAC is involved in the activation of the canonical NF-κB pathway and has been linked to NF-κB dependent malignancies. In this work, we present HOIP-based stapled alpha-helical peptides designed to inhibit LUBAC through the disruption of the HOIL-1L-HOIP interaction and loss of the functional complex. We find our HOIP peptides to be active LUBAC ubiquitylation inhibitors in vitro, though through interaction with HOIP rather than HOIL. Active peptides were shown to have inhibitory effects on cell viability, reduced NF-κB activity and decreased production of NF-κB related gene products. This work further demonstrates the potential of LUBAC as a therapeutic target and of the use of stapled peptides as inhibitors of protein-protein interactions.     

Linear Ubiquitination of NEMO Negatively Regulates the Interferon Antiviral Response through Disruption of the MAVS-TRAF3 Complex

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.     

Linear ubiquitin assembly complex negatively regulates RIG-I- and TRIM25-mediated type I interferon induction

Upon detection of viral RNA, retinoic acid-inducible gene I (RIG-I) undergoes TRIM25-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that the linear ubiquitin assembly complex (LUBAC), comprised of two RING-IBR-RING (RBR)-containing E3 ligases, HOIL-1L and HOIP, independently targets TRIM25 and RIG-I to effectively suppress virus-induced IFN production. RBR E3 ligase domains of HOIL-1L and HOIP bind and induce proteasomal degradation of TRIM25, whereas the NZF domain of HOIL-1L competes with TRIM25 for RIG-I binding. Consequently, both actions by the HOIL-1L/HOIP LUBAC potently inhibit RIG-I ubiquitination and antiviral activity, but in a mechanistically separate manner. Conversely, the genetic deletion or depletion of HOIL-1L and HOIP robustly enhances virus-induced type I IFN production. Taken together, the HOIL-1L/HOIP LUBAC specifically suppresses RIG-I ubiquitination and activation by inducing TRIM25 degradation and inhibiting TRIM25 interaction with RIG-I, resulting in the comprehensive suppression of the IFN-mediated antiviral signaling pathway.     

Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

The linear ubiquitin chain assembly complex (LUBAC) ligase, consisting of HOIL-1L, HOIP, and SHARPIN, specifically generates linear polyubiquitin chains. LUBAC-mediated linear polyubiquitination has been implicated in NF-κB activation. NEMO, a component of the IκB kinase (IKK) complex, is a substrate of LUBAC, but the precise molecular mechanism underlying linear chain-mediated NF-κB activation has not been fully elucidated. Here, we demonstrate that linearly polyubiquitinated NEMO activates IKK more potently than unanchored linear chains. In mutational analyses based on the crystal structure of the complex between the HOIP NZF1 and NEMO CC2-LZ domains, which are involved in the HOIP-NEMO interaction, NEMO mutations that impaired linear ubiquitin recognition activity and prevented recognition by LUBAC synergistically suppressed signal-induced NF-κB activation. HOIP NZF1 bound to NEMO and ubiquitin simultaneously, and HOIP NZF1 mutants defective in interaction with either NEMO or ubiquitin could not restore signal-induced NF-κB activation. Furthermore, linear chain-mediated activation of IKK2 involved homotypic interaction of the IKK2 kinase domain. Collectively, these results demonstrate that linear polyubiquitination of NEMO plays crucial roles in IKK activation and that this modification involves the HOIP NZF1 domain and recognition of NEMO-conjugated linear ubiquitin chains by NEMO on another IKK complex.     

RING‐between‐RINGs—keeping the safety on loaded guns

EMBO J (2012) 31 19, 3833–3844 doi:10.1038/emboj.2012.217; published online September072012EMBO Rep (2012) 13 9, 840–846 doi:10.1038/embor.2012.105; published online September072012The ‘RING-between-RING''-type E3 ubiquitin ligase HOIP acts via a novel RING/HECT-hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non-covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto-inhibited. This explains why assembly of either HOIP/HOIL-1L or HOIP/SHARPIN is required to catalyse linear chain formation.Post-translational modification of a protein with Ubiquitin (Ub) requires the activity of three enzymes: a Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub ligase (E3). Final Ub transfer is performed by an E3 enzyme, which mediates the ligation of Ub from an E2∼Ub conjugate (‘∼'' denotes a thioester) onto a substrate. E3s are commonly divided into two mechanistic classes: RING/U-box E3s and HECT E3s. RING/U-box E3s facilitate the transfer of Ub from the E2∼Ub directly onto a substrate amino group. In contrast, HECTs transfer Ub from the E2∼Ub to the substrate via a HECT∼Ub intermediate. This mechanistic difference leads to an important distinction regarding what determines the type of Ub product (i.e., the specific Ub-chain linkage) formed: in ubiquitination pathways involving RING-type E3 ligases, the E2 determines the product formed, whereas for HECT-catalysed pathways, the E3 governs product formation (Christensen et al, 2007; Kim and Huibregtse, 2009).RING-between-RING (RBR) E3s comprise a class of E3s that appear to have special properties. Although RBR E3s have been considered as a subfamily of RING E3s, the RBR E3 HHARI (Human Homologue of ARIadne) was recently shown to form a HECT-like E3∼Ub intermediate (Wenzel et al, 2011). Two other members of the RBR family, HOIL-1 and HOIP, form the Linear Ub Chain Assembly Complex (LUBAC), the only E3 ligase known to catalyse the synthesis of linear Ub chains (Kirisako et al, 2006). Linear Ub chains are produced by head-to-tail conjugation of Ub molecules through their N- and C-termini and have been shown to activate the canonical NF-κB pathway (Tokunaga et al, 2009).Two studies by the Rittinger and Sixma groups now reveal important insights regarding the formation of linear Ub chains by the dimeric RBR E3 complex HOIP/HOIL-1L (Smit et al, 2012; Stieglitz et al, 2012). Results from these studies highlight three emerging themes among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions.The RBR E3 ligase domain consists of two distinct RING domains, called RING1 and RING2, connected by an IBR (In-Between-Ring) domain. Despite its name, RING2 is not a canonical RING domain as it contains an active site Cysteine (Cys), which has recently been shown to form a thioester E3∼Ub intermediate, as directly detected for the RBR E3 HHARI. Although the Ub-loaded species could not be detected for the RBR E3 parkin, mutation of the analogous cysteine residue abrogated parkin''s ligase activity implying that it works via the same mechanism. On the basis of these observations, Wenzel et al (2011) proposed that the RBR E3s are a family of RING/HECT hybrids that use RING1 to bind an E2 (RING-like) and RING2 to present the active site Cys (HECT-like) as shown schematically in Figure 1. Both Smit et al (2012) and Stieglitz et al (2012) observed a HOIP∼Ub thioester, confirming that HOIP also acts via a RING/HECT-hybrid mechanism. Furthermore, Smit et al (2012) used a clever strategy to uncouple the first transfer event (E2∼Ub to E3) from the final transfer event (E3∼Ub to substrate Ub) to verify that the E3∼Ub intermediate is a prerequisite for Ub transfer onto a substrate and not just a serendipitous side product. The results extend the number of RBR E3s for which a thioester intermediate has been observed and support the notion that RBR E3s are indeed RING/HECT hybrids.Open in a separate windowFigure 1Three common themes are emerging among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions. RBR E3s are characterized by their RBR domain that consists of two distinct RING domains, RING1 that binds the E2, and RING2 that harbours the active site Cys. Two new studies on the RBR E3 HOIP show that (a) domain(s) in HOIP''s N-terminal region inhibits its ligase activity and (b) a domain C-terminal to HOIP''s RBR binds and orients an acceptor Ub to direct linear Ub-chain formation (‘Linear Ub chain Determining Domain'' or LDD). (A)Three ways in which auto-inhibition might occur are illustrated: (1) inhibition of E2∼Ub binding by RING1, (2) obstruction of the active site cysteine on RING2, and/or (3) occlusion of acceptor Ub binding on the LDD. (B) A possible flow of events that occur once auto-inhibition released is shown. Details of each step and how specifically auto-inhibition is released are still unknown.Previous studies have established that HOIP Ub ligase activity and subsequent activation of NF-κB require either the RBR-containing protein, HOIL-1L, or SHARPIN, an adaptor protein associated with LUBAC (Ikeda et al, 2011; Tokunaga et al, 2011). The two current studies now show that although full-length HOIP exhibits very low activity on its own, removal of the N-terminal ∼700 residues results in robust ligase activity. Thus, HOIP appears to be auto-inhibited in the absence of a binding partner. Further analysis revealed that HOIP''s UBA (Ub-Associated) domain is partly responsible for auto-inhibition, although additional N-terminal domains appear to have auto-inhibitory effects as well. SHARPIN, which contains a UBL (Ub-Like) domain, can relieve auto-inhibition of HOIP. Similarly, the addition of the HOIL-1L UBL domain, previously shown to interact with the HOIP UBA domain (Yagi et al, 2012), relieves inhibition. Interestingly, the addition of full-length HOIL-1L results in even greater ubiquitination activity.Stieglitz et al (2012) show that the RBR E3 HOIL-1L has very low E3 activity on its own. Intriguingly, they found that mutation of the HOIL-1L RING2 active site Cys (C460A) reduced activity of the HOIP/HOIL-1L complex back to levels comparable to HOIP activity in presence of HOIL UBL alone. This suggests a more active, catalytic role for HOIL-1L in linear Ub-chain formation than previously appreciated. The details regarding this role must await further studies, but involvement of an active site Cys residue on a second RING2 domain suggests a possible reciprocal transfer mechanism. Perhaps linear chains can be pre-built via such a mechanism and passed en bloc to substrate, similarly to mechanisms used by some HECT-type bacterial E3 ligases (Levin et al, 2010).Parkin, another RBR E3, also exhibits auto-inhibition (Chaugule et al, 2011), but the auto-inhibitory mechanism and the release thereof differ from HOIP. Unlike parkin''s N-terminal UBL, which is thought to interact within the RBR domain at RING2, HOIP''s UBA does not bind detectably in trans to any region in the RBR domain (Stieglitz et al, 2012). Furthermore, addition of its UBA in trans does not inhibit the activity of HOIP RBR E3 as was seen with parkin and its UBL domain. The auto-inhibition of parkin is likely released by substrate binding, because addition of either the UIM of Eps15 or the SH3 domain of endophilin-A, both known to bind the parkin UBL, can restore the activity of parkin (Chaugule et al, 2011). In addition, phosphorylation of Ser65 within the UBL of parkin by PINK-1 activates parkin, presumably by releasing the UBL from RING2 (Kondapalli et al, 2012). In contrast, HOIP overcomes its auto-inhibition through binding either HOIL-1L or SHARPIN. There is no additive effect when both binding partners are present, consistent with the notion that both proteins act via their UBL domains, although this remains to be demonstrated for SHARPIN. The activity of either SHARPIN/HOIP or HOIL-1L/HOIP can activate NF-κB (Ikeda et al, 2011; Tokunaga et al, 2011), but how the protein complexes differ in their cellular roles remains to be further analysed.The finding that HOIP and parkin exhibit auto-inhibition raises the question whether there is something special about the RBR E3s that require auto-inhibition. In this regard, we note that RBR E3s bind the E2 UbcH7 with significantly tighter affinity than canonical RING E3s bind their E2s (Dove and Klevit, unpublished). In the absence of a substrate, RING1 loaded with UbcH7∼Ub would lead to non-productive transfer of Ub from UbcH7∼Ub to the active site of RING2. Occlusion of the active site by auto-inhibition may therefore act as a safety check until its activity is required for transfer of Ub to a substrate. As yet, there is no evidence to indicate whether substrate binding will release HOIP auto-inhibition, as it does for parkin, but this remains a possibility.The revelation that removal of all domains N-terminal to the HOIP RING1 domain yields a highly active ligase allowed both groups to explore questions pertaining to how linear chains are built. Remarkably, constructs comprised of only the RBR domain through the C-terminus of HOIP are sufficient to specify linear Ub chains. (The two groups use HOIP constructs that differ by only two N-terminal residues (697/699–1072) but Stieglitz et al call their construct RBR whereas Smit et al call it RBR-LDD.) (Smit et al, 2012; Stieglitz et al, 2012). Smit et al (2012) demonstrate that the region immediately C-terminal to RING2 is required for linear chain building activity and name the region the ‘LDD'' (Linear Ub chain Determining Domain). Their results indicate that the LDD binds and orients the acceptor Ub to promote transfer of the donor Ub from the RING2 active site to the N-terminus of the acceptor Ub (Figure 1). Parkin has also been suggested to bind free Ub. Details about whether parkin binds acceptor or donor Ub and whether Ub binding determines Ub-chain specificity are still unknown.There is precedence for acceptor Ub binding by HECT E3s and this interaction is essential for chain formation by NEDD4 and its yeast orthologue Rsp5 (Kim et al, 2011; Maspero et al, 2011). In another example, the inactive E2 variant MMS2 binds an acceptor Ub and orients the Ub-Lys63 into the active site of Ubc13 thereby guaranteeing K63-linked chain formation by the E2 (Eddins et al, 2006). Besides proper orientation of the acceptor Ub, chemical differences between α- and ɛ-amino groups likely contribute to linear Ub-chain specificity. For example, E2s known to be active with RING-type E3s can transfer Ub onto the amino acid lysine, but not the other amino acids containing α-amino groups indicating specificity towards the ɛ-amino of lysine (Wenzel et al, 2011).Catalysed by the unexpected discovery that HHARI is a HECT/RING hybrid E3, details about how the RBR class of E3s function are beginning to emerge. We now know, either directly or indirectly, that at least 4 RBR E3s of the 13 identified in humans (HHARI, HOIL, HOIP, and parkin) require a trans-thiolation event using an active site cysteine within RING2. Conservation of this cysteine among all RBR E3s strongly suggests that the RING/HECT-hybrid mechanism is conserved and therefore defines the class. The hybrid mechanism also offers an explanation for the heretofore puzzling observation that, despite being categorized as a RING E3, HOIP determines the type of Ub chain formed. The ability to bind an acceptor Ub close to the RING2 active site likely contributes to how the RBR E3s dictate the type of product they produce. Finally, both HOIP and parkin are auto-inhibited. It remains to be seen whether HOIP''s auto-inhibitory domains work via inhibition of E2∼Ub binding by RING1, obstruction of the active site cysteine on RING2, and/or occlusion of acceptor Ub binding on the LDD (Figure 1). Regardless of the mechanistic details, the ability to modulate their activity may be a common trait of the RBR E3s. Given recent rapid progress, our understanding of this special class of E3s will continue to grow apace.     

Mutual regulation of conventional protein kinase C and a ubiquitin ligase complex

Several isoforms of protein kinase C (PKC) are degraded by the ubiquitin-proteasome pathway after phorbol ester-mediated activation. However, little is known about the ubiquitin ligase (E3) that targets activated PKCs. We recently showed that an E3 complex composed of HOIL-1L and HOIP (LUBAC) generates linear polyubiquitin chains and induces the proteasomal degradation of a model substrate. HOIL-1L has also been characterized as a PKC-binding protein. Here we show that LUBAC preferentially binds activated conventional PKCs and their constitutively active mutants. LUBAC efficiently ubiquitinated activated PKC in vitro, and degradation of activated PKCalpha was delayed in HOIL-1L-deficient cells. Conversely, PKC activation induced cleavage of HOIL-1L and led to downregulation of the ligase activity of LUBAC. These results indicate that LUBAC is an E3 for activated conventional PKC, and that PKC and LUBAC regulate each other for proper PKC signaling.     

Structural insights into specificity and diversity in mechanisms of ubiquitin recognition by ubiquitin-binding domains

UBDs [Ub (ubiquitin)-binding domains], which are typically small protein motifs of <50 residues, are used by receptor proteins to transduce post-translational Ub modifications in a wide range of biological processes, including NF-κB (nuclear factor κB) signalling and proteasomal degradation pathways. More than 20 families of UBDs have now been characterized in structural detail and, although many recognize the canonical Ile44/Val70-binding patch on Ub, a smaller number have alternative Ub-recognition sites. The A20 Znf (A20-like zinc finger) of the ZNF216 protein is one of the latter and binds with high affinity to a polar site on Ub centred around Asp58/Gln62. ZNF216 shares some biological function with p62, with both linked to NF-κB signal activation and as shuttle proteins in proteasomal degradation pathways. The UBA domain (Ub-associated domain) of p62, although binding to Ub through the Ile44/Val70 patch, is unique in forming a stable dimer that negatively regulates Ub recognition. We show that the A20 Znf and UBA domain are able to form a ternary complex through independent interactions with a single Ub molecule, supporting functional models for Ub as a 'hub' for mediating multi-protein complex assembly and for enhancing signalling specificity.     

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.     

Target Specificity of the E3 Ligase LUBAC for Ubiquitin and NEMO Relies on Different Minimal Requirements

The ubiquitination of NEMO with linear ubiquitin chains by the E3-ligase LUBAC is important for the activation of the canonical NF-κB pathway. NEMO ubiquitination requires a dual target specificity of LUBAC, priming on a lysine on NEMO and chain elongation on the N terminus of the priming ubiquitin. Here we explore the minimal requirements for these specificities. Effective linear chain formation requires a precise positioning of the ubiquitin N-terminal amine in a negatively charged environment on the top of ubiquitin. Whereas the RBR-LDD region on HOIP is sufficient for targeting the ubiquitin N terminus, the priming lysine modification on NEMO requires catalysis by the RBR domain of HOIL-1L as well as the catalytic machinery of the RBR-LDD domains of HOIP. Consequently, target specificity toward NEMO is determined by multiple LUBAC components, whereas linear ubiquitin chain elongation is realized by a specific interplay between HOIP and ubiquitin.     

The E3 ligase HOIP specifies linear ubiquitin chain assembly through its RING-IBR-RING domain and the unique LDD extension

Activation of the NF-κB pathway requires the formation of Met1-linked ‘linear'' ubiquitin chains on NEMO, which is catalysed by the Linear Ubiquitin Chain Assembly Complex (LUBAC) E3 consisting of HOIP, HOIL-1L and Sharpin. Here, we show that both LUBAC catalytic activity and LUBAC specificity for linear ubiquitin chain formation are embedded within the RING-IBR-RING (RBR) ubiquitin ligase subunit HOIP. Linear ubiquitin chain formation by HOIP proceeds via a two-step mechanism involving both RING and HECT E3-type activities. RING1-IBR catalyses the transfer of ubiquitin from the E2 onto RING2, to transiently form a HECT-like covalent thioester intermediate. Next, the ubiquitin is transferred from HOIP onto the N-terminus of a target ubiquitin. This transfer is facilitated by a unique region in the C-terminus of HOIP that we termed ‘Linear ubiquitin chain Determining Domain'' (LDD), which may coordinate the acceptor ubiquitin. Consistent with this mechanism, the RING2-LDD region was found to be important for NF-κB activation in cellular assays. These data show how HOIP combines a general RBR ubiquitin ligase mechanism with unique, LDD-dependent specificity for producing linear ubiquitin chains.     

Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger?4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.     

ARD1 binding to RIP1 mediates doxorubicin-induced NF-κB activation

NF-κB is activated by several cellular stresses. Of these, the TNFα-induced activation pathway has been examined in detail. It was recently reported that receptor-interacting protein 1 (RIP1) is involved in DNA damage-induced NF-κB activation by forming a complex with the p53 interacting death domain protein (PIDD) and NF-κB essential modulator (NEMO) in the nucleus, although the underlying mechanism of this interaction has yet to be clarified. This study shows that siRNA knock-down of arrest-defective 1 protein (ARD1) abrogated doxorubicin- but not TNFα-induced activation. Conversely, the over-expression of ARD1 greatly enhanced NF-κB activation induced by doxorubicin. Immunoprecipitation experiments revealed that ARD1 interacted with RIP1 via the acetyltransferase domain. Furthermore, the over-expression of several domain-deleted ARD1 constructs demonstrated that the N-terminal and acetyltransferase domains of ARD1 were required for doxorubicin-induced NF-κB activation. Treatment of deacetylase inhibitor, trichostatin A, significantly increased doxorubicin-induced NF-κB activation in the presence of ARD1 but not acetyltransferase-defective ARD1 mutant. Moreover, N-terminal domain-deleted ARD1 could not be localized in the nucleus in response to doxorubicin treatment. These data indicate that the interaction between ARD1 and RIP1 plays an important role in the DNA damage-induced NF-κB activation, and that the acetyltransferase activity of ARD1 and its localization in to the nucleus are involved in such stress response.     

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.     

NF-κB activation and polyubiquitin conjugation are required for pulmonary inflammation-induced diaphragm atrophy

Loss of diaphragm muscle strength in inflammatory lung disease contributes to mortality and is associated with diaphragm fiber atrophy. Ubiquitin (Ub) 26S-proteasome system (UPS)-dependent protein breakdown, which mediates muscle atrophy in a number of physiological and pathological conditions, is elevated in diaphragm muscle of patients with chronic obstructive pulmonary disease. Nuclear factor kappa B (NF-κB), an essential regulator of many inflammatory processes, has been implicated in the regulation of poly-Ub conjugation of muscle proteins targeted for proteolysis by the UPS. Here, we test if NF-κB activation in diaphragm muscle and subsequent protein degradation by the UPS are required for pulmonary inflammation-induced diaphragm atrophy. Acute pulmonary inflammation was induced in mice by intratracheal lipopolysaccharide instillation. Fiber cross-sectional area, ex vivo tyrosine release, protein poly-Ub conjugation, and inflammatory signaling were determined in diaphragm muscle. The contribution of NF-κB or the UPS to diaphragm atrophy was assessed in mice with intact or genetically repressed NF-κB signaling or attenuated poly-Ub conjugation, respectively. Acute pulmonary inflammation resulted in diaphragm atrophy measured by reduced muscle fiber cross-sectional area. This was accompanied by diaphragm NF-κB activation, and proteolysis, measured by tyrosine release from the diaphragm. Poly-Ub conjugation was increased in diaphragm, as was the expression of muscle-specific E3 Ub ligases. Genetic suppression of poly-Ub conjugation prevented inflammation-induced diaphragm muscle atrophy, as did muscle-specific inhibition of NF-κB signaling. In conclusion, the present study is the first to demonstrate that diaphragm muscle atrophy, resulting from acute pulmonary inflammation, requires NF-κB activation and UPS-mediated protein degradation.     

LUBAC synthesizes linear ubiquitin chains via a thioester intermediate

The linear ubiquitin chain assembly complex (LUBAC) is a RING E3 ligase that regulates immune and inflammatory signalling pathways. Unlike classical RING E3 ligases, LUBAC determines the type of ubiquitin chain being formed, an activity normally associated with the E2 enzyme. We show that the RING-in-between-RING (RBR)-containing region of HOIP-the catalytic subunit of LUBAC-is sufficient to generate linear ubiquitin chains. However, this activity is inhibited by the N-terminal portion of the molecule, an inhibition that is released upon complex formation with HOIL-1L or SHARPIN. Furthermore, we demonstrate that HOIP transfers ubiquitin to the substrate through a thioester intermediate formed by a conserved cysteine in the RING2 domain, supporting the notion that RBR ligases act as RING/HECT hybrids.     

Suppression of NF-κB signaling by KEAP1 regulation of IKKβ activity through autophagic degradation and inhibition of phosphorylation

IκB kinase β (IKKβ) plays a crucial role in biological processes, including immune response, stress response, and tumor development by mediating the activation of various signaling molecules such as NF-κB. Extensive studies on the mechanisms underlying IKK activation have led to the identification of new activators and have facilitated an understanding of the cellular responses related to NF-κB and other target molecules. However, the molecular processes that modulate IKK activity are still unknown. In this study, we show that KEAP1 is a new IKK binding partner, which is responsible for the down-regulation of TNFα-stimulated NF-κB activation. The E(T/S)GE motif, which is found only in the IKKβ subunit of the IKK complex, is essential for interaction with the C-terminal Kelch domain of KEAP1. Reduction of KEAP1 expression by small interfering RNA enhanced NF-κB activity, and up-regulated the expression of NF-κB target genes. Ectopic expression of KEAP1 decreased the expression of IKKβ, which was restored by an autophagy inhibitor. IKK phosphorylation stimulated by TNFα was blocked by KEAP1. Our data demonstrate that KEAP1 is involved in the negative regulation of NF-κB signaling through the inhibition of IKKβ phosphorylation and the mediation of autophagy-dependent IKKβ degradation.     

LUBAC, a novel ubiquitin ligase for linear ubiquitination, is crucial for inflammation and immune responses

LUBAC (linear ubiquitin chain assembly complex) is a ubiquitin ligase complex composed of SHARPIN, HOIL-IL and HOIP that generates linear polyubiquitin chains and regulates the NF-κB pathway, which is pivotal in inflammatory and immune responses. Recent findings on the regulation of NF-κB by LUBAC and the diseases associated with this process are the focus of this review.