MOLECULAR MECHANISMS OF LYMPHOCYTE ACTIVATION AND LYMPHOMA DEVELOPMENT
Lymphocytes play a crucial role in the defense against pathogens and tumor cells. One focus of our research is to understand the molecular mechanisms that control the activation of T-lymphocytes, initiated upon triggering of the T-cell antigen receptor by MHC-bound antigen. This leads to the initiation of multiple signaling pathways that regulate changes in cell shape and gene expression that are critical for efficient T-cell activation, proliferation and survival. Another focus of our research is to understand the molecular mechanisms underlying aberrant lymphocyte proliferation and survival that occurs in certain lymphoid tumors (lymphomas).
By uncovering new molecular players and enzymatic activities relevant to these pathways, we aim at identifying possible targets for therapeutic immuno-modulation or treatment of lymphomas.
Signaling pathways relevant for lymphocyte activation and survival
The initiation of the adaptive immune response depends on the recognition of pathogenic substances or tumor-specific molecules (microbial or tumor antigens) by antigen receptors on the lymphocyte cell surface. Antigen recognition leads to formation of a complex of signaling proteins comprising CARMA1, BCL10 and MALT1 (CBM proteins). An important role of CBM proteins is the activation of the transcription factor NF-κB, which controls genes that are essential for lymphocyte proliferation and survival (for recent reviews, see: Thome, 2008; Hailfinger et al., 2009b; Thome et al., 2010). Mutations of the genes encoding CARMA1, BCL10 or MALT1 or their upstream regulators are associated with constitutive CBM-dependent signaling, and the development of human B-cell lymphomas, such as lymphomas of the mucosa-associated lymphoid tissue (MALT lymphomas) and diffuse large B-cell lymphomas (DLBCL).
The main focus of our recent work has been the identification and characterization of a protease activity of MALT1 (Rebeaud et al., 2008), and the demonstration of constitutive MALT1 activity in cells derived from ABC-DLBCL, which critically depend on MALT1 protease activity for growth and survival (Hailfinger et al. 2009). More recently, we have identified RelB as a new substrate for MALT1 (Hailfinger et al., 2011) and identified a new mechanism of MALT1 activation that requires its C-terminal mono-ubiquitination (Pelzer et al., 2013).
MALT1 controls NF-κB activation via its scaffold and protease functions
The transcription factor NF-κB is pivotal to the expression of genes that control lymphocyte activation and the generation of the immune response. In resting lymphocytes, NF-κB family members are present in the cytoplasm in an inactive form, bound to inhibitory κ-B (IκB) proteins. Triggering of the T-cell antigen receptor leads to activation of the IκB kinase (IKK) complex that induces phosphorylation and subsequent degradation of IκB proteins. This allows NF-κB to translocate into the nucleus and to initiate the transcription of genes that control lymphocyte proliferation and survival.
MALT1 contributes to NF-κB activation as a scaffold protein, by binding to the ubiquitin ligase TRAF6, which in turn controls the recruitment and activation of the IKK complex. MALT1 also contributes to NF-κB activation by its protease domain, which has Arg-directed substrate specificity (Rebeaud et al., 2008; Thome et al., 2010). Interestingly, the development of a peptide-based inhibitor of MALT1 has allowed us to show that inhibition of its protease activity impairs NF-κB activation in an IKK-independent manner. Based on these findings, we had predicted the existence of a MALT1 cleavage substrate that affects NF-κB activity independently of the IKK complex.
MALT1-dependent cleavage of RelB controls NF-κB activation in an IKK-independent manner
In a recent study, we have now identified RelB as the MALT1 substrate responsible for the protease-dependent, IKK-independent control of the NF-κB pathway by MALT1 (Hailfinger et al., 2011). We showed that Malt1 cleaves the NF-κB family member RelB after Arg 85, at a conserved LVSR sequence that acts as an optimal MALT1 substrate in vitro. RelB cleavage induced its proteasomal degradation, which promoted DNA binding of RelA- or c-Rel-containing NF-κB complexes. In contrast, overexpression of RelB inhibited expression of canonical NF-κB target genes. Interestingly, RelB was constitutively cleaved in cell lines derived from ABC-DLBCL, and overexpression of RelB led to impaired survival of these diffuse large B-cell lymphoma cell lines, which are characterized by constitutive MALT1 activity. Collectively, these findings support the idea that MALT1 controls NF-κB activation in lymphocytes by both, its scaffold and its protease function (Figure 1). Moreover, our findings provide a rationale for the targeting of MALT1 in immuno-modulation and cancer treatment.
Fig. 1: MALT1 controls antigen receptor-mediated NF-kB activation by its scaffold and protease functions.
(A) Antigen receptor triggering leads to formation of the CARMA1-BCL10-MALT1 complex. MALT1 recruits the ubiquitin ligase TRAF6, which promotes IKK activation via its physical recruitment and activation of the Ser/Thr kinase TAK1. MALT1 also contributes to T-cell activation via its protease function by cleavage of RelB, which acts as an inhibitor of canonical NF-kB activation in lymphocytes. (B) Sequence alignment of the cleavage sites of known MALT1 substrates. (C) Comparison of the in vitro cleavage efficiency of fluorogenic tetrapeptide substrates derived from Bcl10 and RelB.
The MALT1 protease activity is controlled by inducible mono-ubiquitination
Our previous work had demonstrated that the protease activity of the paracaspase MALT1 is central to lymphocyte activation and lymphomagenesis, but how the catalytic activity was controlled remained unknown. In a recent study, we have now identified a monoubiquitination of Malt1 on lysine 644 (K644), which activates Malt1 protease function (Pelzer et al., 2013). Monoubiquitinated Malt1 showed increased protease activity, while a ubiquitination-deficient lysine to arginine mutant (K644R) had reduced protease activity correlating with impaired TCR-induced IL-2 induction in activated T cells. Expression of the K644R mutant diminished survival of cells derived from diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL), which require constitutive Malt1 protease activity for survival. Monoubiquitinated MALT1 preferentially formed dimers in vitro, suggesting that monoubiquitination promotes or stabilizes the formation of catalytically active dimers. Thus, monoubiquitination of Malt1 is essential for its catalytic activation (Figure 2) and the responsible ubiquitin ligase should be an interesting target for immuno-modulation and treatment of ABC DLBCL. Further studies are now targeted at the identification of the enzyme responsible for MALT1 monoubiquitination.
Fig. 2: MALT1 activity is controlled by monoubiquitination.
(A) Purified primary human T cells were activated using PMA and ionomycin, and lysates analyzed by Western blot. Open arrow indicates monoubiquitinated MALT1. (B) Size exclusion chromatography of purified recombinant MALT1 proteins. A purified MALT1-ubiquitin fusion protein that mimics monoubiquitination preferentially elutes as a dimer, while unmodified MALT1 preferentially elutes as a monomer. (C) Hypothetical model of MALT1 activation by monoubiquitination. MALT1 activity is negatively regulated by its C-terminal region. Monoubiquitination within this region most likely promotes MALT1 activation by overcoming this inhibition and by favoring dimerization of the protease domain. Original data are from Pelzer et al., 2013.
Parts of this work were done in collaboration with the laboratories of Georg Lenz (Charité, Berlin, Germany) and Louis Staudt (National Cancer Institute, Bethesda, MD, USA).