The paracaspase MALT1 dampens NF‐κB signalling by cleaving the LUBAC subunit HOIL‐1
2015-12-30

The mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a key player in the activation of the transcription factor nuclear factor κB (NF-κB) upon antigen receptor stimulation. MALT1 promotes NF-κB activity and lymphocyte activation via two distinct pathways. In the first such pathway, MALT1, together with its binding partners BCL10 and CARMA1/CARD11, physically recruits and activates the inhibitor of κB kinase (IKK) complex, which in turn leads to the phosphorylation and subsequent degradation of the inhibitor of κB (IκB). This scaffold function of MALT1 results in the translocation of NF-κB proteins to the nucleus and induction of target gene expression. The second pathway involves the caspase-like protease domain of MALT1, which is activated during antigen receptor stimulation. The protease domain cleaves substrates such as RelB and A20, which are negative regulators of classical NF-κB signalling in lymphocytes [1-3]. Cleavage of other MALT1 substrates, including BCL10, Regnase-1, Roquin-1 and CYLD, also promotes the activation of lymphocytes by regulating cell adhesion, mRNA stability and JNK activity [1]. Two papers now report a potential negative regulatory role for MALT1 in these processes, via cleaving heme-oxidized iron-responsive element-binding protein 2 ubiquitin ligase-1 (HOIL-1) [4, 5]. HOIL-1, together with its interaction partners HOIP and SHARPIN, forms the so-called linear ubiquitin chain assembly complex (LUBAC). MALT1-mediated cleavage of HOIL-1 dampens NF-κB and lymphocyte activation, suggesting that MALT1 has pleiotropic functions that are not restricted to the initiation of NF-κB responses (Fig. 1). Ubiqutination of several signalling molecules represents a key step in NF-κB activation in response to antigen receptor triggering. K48-linked polyubiquitination of IκB targets it to the proteasome and thus activates NF-κB. In contrast, MALT1 and BCL10 become transiently modified with K63-linked polyubiquitin, which is recognized by the regulatory subunit IKKγ and thus recruits the IKK complex [1]. IKKγ itself is activated by the conjugation of linear ubiquitin, which is characterized by an intermolecular bond between the amino-terminal methionine (Met1) of one ubiquitin and the carboxyl group of the C-terminal glycine (Gly76) of another ubiquitin [6]. The only complex known to mediate de novo linear di-ubiquitin linkage is the LUBAC complex. The ubiquitin ligase activity of the LUBAC complex, which is crucial for the attachment of linear ubiquitin, is contained within the HOIP protein, whereas HOIL-1 and SHARPIN regulate the localization, stability and activity of the complex [7, 8]. Even though the detailed molecular mechanism remains unclear, there is strong evidence that the LUBAC complex is required for NF-κB activation upon antigen receptor triggering. In stimulated T cells, the LUBAC components are transiently recruited to the CARMA1-BCL10-MALT1 (CBM) complex. Silencing of HOIP weakens the interaction of the CBM complex with the IKKγ subunit and this correlates with a decrease in IKK activity, reduced phosphorylation of IκB, lower NF-κB activity and strongly diminished secretion of the NF-κB target gene IL-2 [9]. Additionally, the identification of two single-nucleotide polymorphisms in the HOIP gene (Q584H and Q622L) in an aggressive subtype of diffuse large B cell lymphoma (DLBCL) supports a central role for the LUBAC complex in the B cell receptor (BCR) signalling pathway. The activated B cell-like (ABC) DLBCL subtype is characterized by chronic activation of antigen receptor signalling, leading to deregulated CBM complex assembly, hyperactivation of the MALT1 protease and, consequently, increased NF-κB signalling. This pathway is partially driven by activating mutations of the BCR co-receptor CD79 and CARMA1 that lead to recognition of self-antigens [10]. The expression of the two HOIP single-nucleotide polymorphisms in ABC-DLBCL cells leads to elevated levels of IKKγ modified with linear ubiquitin and thus promotes NF-κB activity. Conversely, silencing of the LUBAC complex decreases IKKγ ubiquitination, phosphorylation of IκB and NF-κB activity and thus reduces survival of ABC-DLBCL lymphoma cells [11]. Christopher Overall and Rudi Beyaert and their colleagues report in Nature Communications and The FEBS Journal, respectively, that MALT1 cleaves the LUBAC member HOIL-1 after T and B cell receptor stimulation [4, 5]. HOIL-1 cleavage in stimulated lymphocytes is blocked by MALT1 silencing and treatment with MALT1 inhibitors. Intriguingly, T and B cells from a patient with a homozygous MALT1 Trp580Ser mutation exhibit strongly reduced HOIL-1 cleavage after stimulation [4]. Both groups map the cleavage site to Arg165 after an LQPR (murine LQSR) sequence, which is in accordance with the consensus site of previously described MALT1 substrates (LXS/PR). MALT1 cleaves HOIL-1 between its N-terminal ubiquitin-like (UBL) and the ubiquitin-binding Npl4-type zinc finger (NZF) domain. In addition to the NZF domain, the C-terminal fragment of HOIL-1 also contains a catalytic RING-IBR-RING (RBR) domain, which is dispensable for linear ubiquitination activity. As the UBL domain of HOIL-1 binds the ubiquitin-associated domain of HOIP, MALT1-mediated HOIL-1 cleavage releases the NZF and the RBR of HOIL-1 from the LUBAC complex. This has two effects: (a) rapid degradation of HOIP and destabilization of the LUBAC complex (indeed, HOIP is destabilized upon stimulation in B cells from a healthy donor, but not in patient-derived B cells with the homozygous MALT1 Trp580Ser mutation [4]); and (b) a reduction of linear ubiquitin. HOIL-1 derepresses the auto-inhibitory effect of the HOIP N-terminus on linear ubiquitination activity. Whereas overexpression of full-length HOIL-1 promotes the production of linear ubiquitin chains, neither the N-terminal nor the C-terminal HOIL-1 fragments are sufficient for this activity. MALT1-mediated HOIL-1 cleavage thus decreases levels of ubiquitin-modified IKKγ and impairs the NF-κB response [4, 5]. Beyaert and colleagues also propose an inhibitory function or LUBAC-destabilizing effect of the newly generated C-terminal HOIL-1 fragment, although the molecular mechanism remains to be elucidated [5]. These results, proposing a role of MALT1 in dampening the NF-κB response, are quite surprising, because MALT1 has so far been considered solely as an activator of NF-κB signalling. Lymphocytes from MALT1-knockout mice display impaired NF-κB activity upon antigen receptor triggering. How is it possible that MALT1 is essential for NF-κB activation but at the same time limits NF-κB signalling via HOIL-1 cleavage? One explanation could be the chronological sequence of events. In the initiation phase of lymphocyte activation, the scaffold function of MALT1 and the preferential cleavage of NF-κB inhibitory factors predominate. HOIL-1 cleavage, in contrast, might be important for the termination of CBM/IKK-mediated NF-κB activation (Fig. 1). In line with this model, the initial activation of NF-κB, which is characterized by the rapid phosphorylation of IκB and RelA, is not affected by MALT1 inhibitor treatment [4]. Indeed, both studies suggest an important role of MALT1 protease activity in the termination of NF-κB signalling in lymphocytes. Overall and colleagues correlate a lack of MALT1 activity with the autoinflammatory phenotype observed in a patient expressing a homozygous MALT1 Trp580Ser mutation, which mainly affects the skin and gastrointestinal tract [4]. Interestingly, knock-in mice expressing a protease-inactive mutant of MALT1 (MALT1 KI) also suffer from spontaneous autoimmune gastritis, exhibit an accumulation of activated T cells and have high IgE/IgG1 serum levels [12]. The autoinflammatory phenotype in the MALT1 KI mice was explained by reduced numbers of regulatory T cells (Tregs); accordingly, the observed phenotype could be counteracted by reconstitution with wild-type Tregs. Furthermore, because the LUBAC complex is essential for the survival of ABC-DLBCL cells, but those cells are at the same time characterized by constitutive MALT1 protease activity, it will be interesting to understand why MALT1-mediated cleavage of HOIL-1 is unable to terminate NF-κB signalling in these cells. The molecular role of the deubiquitinases CYLD and A20 in the remodelling of linear ubiquitin also remains vague, especially as both proteins are known to be recruited to the CBM complex and cleaved by MALT1. These findings reveal that MALT1 has a central role not only in the onset of NF-κB activation but also potentially in the termination of the signal, uncovering the complexity of NF-κB activation in response to antigen receptor triggering, and provides potential avenues for therapeutic intervention. All authors wrote this manuscript and prepared the artwork.