Research updateLinear ubiquitination in NF-κB signaling and inflammation: What we do understand and what we do not
Graphical abstract
Introduction
Ubiquitin is a small protein of 76 amino acids that is encoded as a precursor protein in mammals by 4 different genes and is used by all eukaryotic cells to modify proteins posttranslationally. This ubiquitin label will appoint a new destiny to the ubiquitinated proteins as it influences their activity, interactions and/or stability. As a consequence, the ubiquitin system drives numerous cellular responses including DNA repair, cell cycle progression, signal transduction and membrane protein transport [1]. All these processes can be accomplished by covalent attachment of ubiquitin as a monomer (monoubiquitination) or as a polymer (polyubiquitination) to a target substrate [2]. Ubiquitination is achieved by the concerted action of a ubiquitin activating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin ligase (E3) [3]. E1 activates the ubiquitin residue by forming an E1–ubiquitin thioester in an ATP-dependent manner [3], [4], allowing the transfer of activated ubiquitin to E2. The final step of ubiquitination requires the activity of an E3, which functions as the substrate recognition module of the system and can interact with both E2 and substrate. While only 2 E1s and 38 E2s are known, more than 600 E3 ubiquitin ligases are predicted in the human genome [5]. Ubiquitination can be reversed by deubiquitinases (DUBs) that cleave protein–ubiquitin bonds [6]. The human genome encodes nearly 100 DUBs that can be classified in 4 cysteine protease families and 1 metalloprotease family. Besides reversing the ubiquitination of target proteins, DUBs are also responsible for the processing of ubiquitin precursor proteins and the recycling of ubiquitin from polyubiquitin chains.
Most commonly, the C-terminal Gly76 of ubiquitin is connected via an isopeptide bond to the ɛ-amino group of a Lys in the substrate [7]. During the last years it has become clear that also non-Lys residues can accept ubiquitin. For example, ubiquitin can be linked to the N-terminal amino acid of a protein [8]. In addition, even non-amino groups such as the sulfhydryl group of Cys [9], or the hydroxyl group of Thr or Ser residues can bind ubiquitin [10], [11]. Proteins cannot only be modified by single ubiquitin molecules, but also by polyubiquitin chains. In order to generate these linkages, the C-terminal Gly of the first ubiquitin molecule is linked to the ɛ-amino group of one of the 7 internal Lys residues of the second ubiquitin moiety (Lys6, Lys11, Lys27, Lys33, Lys48 or Lys63). More recently, binding between Gly76 of one ubiquitin and Met1 of another ubiquitin molecule was described [12]. In this way, consecutive ubiquitin molecules are assembled to each other by their N- and C-termini, which is known as linear or head-to-tail ubiquitination. Linear ubiquitin chains are already known for years as ubiquitin genes encode linear polyubiquitin precursors that are hydrolyzed to monomers by DUBs [13]. However, it still came as a surprise that a cell would assemble linear polyubiquitin chains from single ubiquitin molecules by means of specific E3 ligases, and use them to modify specific substrates.
The type of linkage and the resulting conformation of the polyubiquitin chain determine the outcome of ubiquitination for a cell. For example, linear, Lys48- and Lys6-linked polyubiquitin chains can target their substrates for proteasomal degradation [14], [15], [16]; Lys63- and Lys6-linked polyubiquitin chains contribute to DNA repair [17], [18] and innate immunity [19], while Lys11-ubiquitination has mainly been described in cell cycle events in mammals [20], [21]. Each type of ubiquitination is recognized by specific ubiquitin-binding domains (UBDs) in other proteins that decode and transmit information conferred by protein ubiquitination to control various cellular events [22].
The prototypic example of a signaling pathway that integrates multiple types of ubiquitination is the NF-κB pathway [23]. Lys63-ubiquitination of signaling intermediates leads to the formation of multi-protein signaling complexes, which ultimately results in activation of the inhibitor of κB (IκB) kinase (IKK) complex. Lys48-ubiquitination of IκBα on the other hand, leads to its proteasomal degradation and sets NF-κB free to translocate to the nucleus. Recently, modification of signaling proteins by linear ubiquitination has been added as a novel regulatory mechanism in NF-κB signaling.
Section snippets
The linear ubiquitination machinery
The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase complex known to catalyze the generation of linear polyubiquitin chains in cells [12]. LUBAC was initially identified as a 600 kDa complex containing two related multidomain proteins: HOIL-1 (heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1; also known as RBCK1) and HOIP (HOIL-1 interacting protein; also known as RNF31), which have masses of 58 and 120 kDa, respectively [12]. Both HOIP and HOIL-1 contain
Linear ubiquitination in NF-κB signaling
Ubiquitination plays a key role in NF-κB signaling. From the early days of NF-κB signaling research it is known that SCF-βTrCP-mediated Lys48 polyubiquitination of the NF-κB inhibitor protein IκBα results in its proteasomal degradation, allowing NF-κB to translocate into the nucleus (Fig. 2). In the year 2000, a new regulatory function for ubiquitin was revealed by the finding that IKK is activated through the assembly of Lys63-linked polyubiquitin chains [32]. In the meantime, members of the
Role of linear ubiquitination in other signaling pathways
Besides NF-κB activation, TNF-R1 triggering also leads to the activation of MAP kinases such as JNK and ERK. A role for linear ubiquitination in their activation has been suggested but current evidence is rather controversial. Two independent studies showed reduced TNF-induced JNK activation in SHARPIN mutant (cpdm) MEFs [24], [25], whereas Tokunaga and colleagues demonstrated higher JNK and ERK activity in these cells and in HOIL-1-deficient MEFs [26], [27]. Studies with B cells or peritoneal
Sensing and modulation of linear ubiquitination
The recognition of different types of polyubiquitin chains by chain specific UBDs that are present in other proteins determines the outcome of ubiquitination [22]. At least three different UBDs: UBAN, NZF and UBA were identified as linear polyubiquitin sensors [24], [34], [47], [48]. As already mentioned, the UBAN domain of NEMO recognizes linear ubiquitin chains attached to RIP1 and NEMO in the TNF-R1 signaling complex, which enhances its recruitment and stabilizes the TNF-R1 signaling
Functional role of linear ubiquitination in immunity and inflammation
Information on the physiological role of linear ubiquitination is still very limited. Mice harboring a mutation in the SHARPIN gene develop a chronic proliferative dermatitis (mutation symbol, cpdm) phenotype. The skin of these mice displays epidermal hyperplasia with infiltration of inflammatory cells and keratinocyte cell death [25], [60]. Inflammatory lesions are also found in other epithelial layers like those of the mouth, oesophagus and forestomach, as well as in the liver, lungs and
Targeting linear ubiquitination for therapeutic purposes
Persistent NF-κB activation is central to the pathogenesis of many inflammatory diseases and cancer [65]. Therefore, targeting NF-κB activation is of high therapeutic interest and several inhibitors have been described, including antioxidants, peptides, decoy oligodeoxynucleotides, dominant-negative or constitutive active proteins, natural products, and small synthetic compounds [66]. Several of these molecules target specific steps such as IKK activity, nuclear translocation or DNA binding.
Outlook
Although the use of cells deficient in one of the LUBAC components clearly illustrates the contribution of LUBAC and linear ubiquitin in NF-κB activation and in resistance towards TNF-induced apoptosis, further studies are required to unravel the role of this modification in other pathways or processes. As the existence of linear ubiquitination and LUBAC is a rather recent finding, still much has to be (re)investigated. Are NEMO, RIP1 and TRIM25 the only substrates for LUBAC in the NF-κB and
Acknowledgements
Research in the authors lab is supported by grants from the ‘Interuniversity Attraction Poles’ (IAP6/18), the Fund for Scientific Research (FWO)-Flanders (grants G.0619.10, G.0089.10, 3G023611), the ‘Foundation Against Cancer’, the ‘Strategic Basic Research’ of the IWT, the ‘Queen Elisabeth Medical Foundation’, and the ‘Hercules’ and ‘Group-ID MRP’ initiatives of Ghent University. K.V. was supported by a predoctoral fellowship from the IWT and L.V. holds a postdoctoral fellowship of the FWO.
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2011, Cytokine and Growth Factor ReviewsCitation Excerpt :It should be mentioned that the essential role of RIP1 in TNF-induced NF-κB activation has recently been questioned based on the finding that multiple cell types from E18 RIP1 knockout embryos can still activate NF-κB in response to TNF [61], which contradicts previous findings [47] and is still a matter of intense debate. Recent studies suggest that linear ubiquitination of NEMO, and possibly also RIP1, by the tripartite linear ubiquitin chain assembly complex (LUBAC) play an important role in TNF-induced NF-κB activation [62]. Linear polyubiquitin chains are not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin.