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.     

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.     

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.     

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.     

ARIH2抑制LUBAC的线性泛素化连接酶活性

线性泛素化修饰在肿瘤及免疫系统中均发挥着重要作用。线性泛素链组装复合体(linear ubiquitin chain assembly complex,LUBAC)是目前已知的唯一能够催化合成线性泛素链的泛素连接酶。本研究发现,泛素连接酶2(ariadne homolog 2,ARIH2)作为LUBAC新的相互作用蛋白质,能够抑制LUBAC对底物的线性泛素化修饰水平。通过免疫共沉淀实验发现,ARIH2与HOIP存在相互作用,且GST pull-down结果说明,HOIP通过ZF-NZF结构域与ARIH2发生相互作用。进一步的免疫沉淀结果证明,LUBAC并不能线性泛素化修饰ARIH2。反之,ARIH2能够抑制LUBAC对底物的线性泛素化修饰水平,其机制可能是ARIH2影响了SHARPIN的泛素化水平,从而影响LUBAC酶活性,进而导致LUBAC对底物的线性泛素化水平减弱。     

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.     

A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7

Linear polyubiquitination of proteins has recently been implicated in NF-κB signalling and is mediated by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP and Sharpin. However, the mechanisms that regulate linear ubiquitination are still unknown. Here, we show that A20 is rapidly recruited to NEMO and LUBAC upon TNF stimulation and that A20 inhibits LUBAC-induced NF-κB activation via its C-terminal zinc-finger 7 (ZF7) domain. Expression of a polypeptide corresponding to only ZF7 was sufficient to inhibit TNF-induced NF-κB activation. Both A20 and ZF7 can form a complex with NEMO and LUBAC, and are able to prevent the TNF-induced binding of NEMO to LUBAC. Finally, we show that ZF7 preferentially binds linear polyubiquitin chains in vitro, indicating A20–ZF7 as a novel linear ubiquitin-binding domain (LUBID). We thus propose a model in which A20 inhibits TNF- and LUBAC-induced NF-κB signalling by binding to linear polyubiquitin chains via its seventh zinc finger, which prevents the TNF-induced interaction between LUBAC and NEMO.     

RBR E3‐ligases at work

The RING‐in‐between‐RING (RBR) E3s are a curious family of ubiquitin E3‐ligases, whose mechanism of action is unusual in several ways. Their activities are auto‐inhibited, causing a requirement for activation by protein‐protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT‐like mechanism in which the RING1 domain facilitates E2‐discharge to directly form a thioester intermediate with a cysteine in RING2. This short‐lived, HECT‐like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino‐terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.     

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.     

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.     

The linear ubiquitin chain assembly complex regulates TRAIL-induced gene activation and cell death

The linear ubiquitin chain assembly complex (LUBAC) is the only known E3 ubiquitin ligase which catalyses the generation of linear ubiquitin linkages de novo. LUBAC is a crucial component of various immune receptor signalling pathways. Here, we show that LUBAC forms part of the TRAIL-R-associated complex I as well as of the cytoplasmic TRAIL-induced complex II. In both of these complexes, HOIP limits caspase-8 activity and, consequently, apoptosis whilst being itself cleaved in a caspase-8-dependent manner. Yet, by limiting the formation of a RIPK1/RIPK3/MLKL-containing complex, LUBAC also restricts TRAIL-induced necroptosis. We identify RIPK1 and caspase-8 as linearly ubiquitinated targets of LUBAC following TRAIL stimulation. Contrary to its role in preventing TRAIL-induced RIPK1-independent apoptosis, HOIP presence, but not its activity, is required for preventing necroptosis. By promoting recruitment of the IKK complex to complex I, LUBAC also promotes TRAIL-induced activation of NF-κB and, consequently, the production of cytokines, downstream of FADD, caspase-8 and cIAP1/2. Hence, LUBAC controls the TRAIL signalling outcome from complex I and II, two platforms which both trigger cell death and gene activation.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

Site‐specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site‐specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)‐induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF‐κB reporter relative to wild‐type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated Hoip^K778R/K778R knock‐in mice, which show no overt developmental phenotypes; however, in response to TNF, Hoip^K778R/K778R mouse embryonic fibroblasts display mildly suppressed NF‐κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF‐induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in Hoip^K778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site‐specific ubiquitination of HOIP regulates a LUBAC‐dependent switch between survival and apoptosis in TNF signaling.     

Structural analysis of SHARPIN, a subunit of a large multi-protein E3 ubiquitin ligase, reveals a novel dimerization function for the pleckstrin homology superfold

SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.     

TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin‐RING ligase complexes

RING (Really Interesting New Gene)‐in‐between‐RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc‐binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto‐inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin‐RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto‐ubiquitylation, their ability to stimulate dissociation of a thioester‐linked UBCH7～ubiquitin intermediate, and reactivity with ubiquitin‐vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL‐induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.     

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase

RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.     

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.     

Linear polyubiquitin chains: A new modifier involved in NFκB activation and chronic inflammation including dermatitis

The ubiquitin conjugation system regulates a wide variety of biological phenomena, including protein degradation and signal transduction, by regulating protein function via polyubiquitin conjugation in most cases. Several types of polyubiquitin chains exist in cells, and the type of polyubiquitin chain conjugated to a protein seems to determine how that protein is regulated. We identified a novel linear polyubiquitin chain and the ubiquitin-protein ligase complex that assembles it, designated LUBAC. Both were shown to have crucial roles in the canonical NFκB activation pathway. This year, three groups, including our laboratory, identified SHARPIN as a new subunit of LUBAC. Of great interest, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm), which is characterized by numerous inflammatory symptoms including chronic dermatitis, arthritis and immune disorders. Deletion of SHARPIN drastically reduces the amount of LUBAC and attenuates signal-induced NFκB activation. The pleomorphic symptoms of cpdm mice suggest that LUBAC-mediated NFκB activation may play critical roles in mammals and be involved in various disorders. A forward look into the linear polyubiquitin research is also discussed.Key words: ubiquitin, linear ubiquitination, NFκB, LUBAC, SHARPIN, cpdm, chronic dermatitis, TNFα     

Linear ubiquitination by LUBEL has a role in Drosophila heat stress response

The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL‐1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro. We detect endogenous linear ubiquitin chain‐derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination‐defective flies by mutating residues essential for the catalytic activity of LUBEL. Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.     

Identification of Clusterin Domain Involved in NF-��B Pathway Regulation

Clusterin (CLU) is a ubiquitous protein that has been implicated in tumorigenesis, apoptosis, inflammation, and cell proliferation. We and others have previously shown that CLU is an inhibitor of the NF-κB pathway. However, the exact form of CLU and the region(s) of CLU involved in this effect were unknown. Using newly generated molecular constructs encoding for CLU and various regions of the molecule, we demonstrated that the presecretory form of CLU (psCLU) form bears the NF-κB regulatory activity. Sequence comparison analysis showed sequence motif identity between CLU and β-transducin repeat-containing protein (β-TrCP), a main E3 ubiquitin ligase involved in IκB-α degradation. These homologies were localized in the disulfide constraint region of CLU. We generated a specific molecular construct of this region, named ΔCLU, and showed that it has the same NF-κB regulatory activity as CLU. Neither the α-chain nor the β-chain of CLU had any NF-κB regulatory activity. Furthermore, we showed that following tumor necrosis factor-α stimulation of transfected cells, we could co-immunoprecipitate phospho-IκB-α with ΔCLU. Moreover, we showed that ΔCLU could localize both in the cytoplasm and in the nucleus. These results demonstrate the identification of a new CLU activity site involved in NF-κB pathway regulation.