API2-MALT1 Fusion Protein Research Articles

Study of individual domains contributing to MALT1 dimerization in BCL10-independent and dependent assembly

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.

Abstract IA49: MALT1 targeting for B-cell lymphomas

Abstract IA49: MALT1 targeting for B-cell lymphomas

T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

Microarray ANALYSIS of t(11;18)(Q21;Q21)-positive and -negative gastrointestinal MALT lymphomas.

e14610 Background: Among the genetic abnormalities that occur in MALT lymphomas, the translocation t(11;18) is of particular interest because it is predominantly documented in MALT lymphomas of the gut. It results in the creation of a fusion protein API2-MALT1 that activates the transcription factor NF-kB via enhanced IKKγ polyubiquitination. Here, we apply microarray analysis to identify differentially expressed genes related to the pathogenesis of gastrointestinal (GI) MALT lymphomas. Methods: 8 t(11;18)-positive and 6 t(11;18)-negative GI MALT lymphomas were compared with normal lymphoid by microarray (Affymetrix Plus 2.0 Gene Chip). 6 splenectomy specimens (with a prominent marginal zone but without obvious morphologic anomalies) of healthy individuals suffering from traumatic splenic rupture were selected as reference tissue, as the marginal zone is assumed to be the non-tumoural counterpart of MALT lymphomas. For this particular study, the number of representative cases documented with both surgical resection specimens and frozen tissue (as required for gene expression profiling) is limited, due to the current conservative treatment of GI MALT lymphomas (the majority of gastric lymphomas regresses after Helicobacter pylori eradication therapy). Results: By comparing the 8 t(11;18)-positive MALT lymphomas as well as their 6 t(11;18)-negative counterparts to normal lymphoid tissue (e.g. spleen with prominent marginal zones), we could define a common GI MALT lymphoma signature, most likely comprising the genes with significance in the development of type of cancer. Moreover, unsupervised analysis of the gene expression profiles of these fourteen GI MALT lymphomas clustered in 2 groups, with the clustering being determined by presence or absence of the API2-MALT1 fusion protein. The latter finding suggests different pathways for both t(11;18)-positive and -negative GI MALT lymphomas. Interestingly, within the group of t(11;18)-positive GI MALT lymphomas, there was a tendency that the gene expression profiles of these tumours tended to cluster according to the location of the MALT1 breakpoint. Conclusions: Our study reveals several genes that play an important rol in MALT lymphomagenesis. No significant financial relationships to disclose.

Auto-Ubiquitination-Induced Degradation of MALT1-API2 Prevents BCL10 Destabilization in t(11;18)(q21;q21)-Positive MALT Lymphoma

BackgroundThe translocation t(11;18)(q21;q21) is the most frequent chromosomal aberration associated with MALT lymphoma and results in constitutive NF-κB activity via the expression of an API2-MALT1 fusion protein. The properties of the reciprocal MALT1-API2 were never investigated as it was reported to be rarely transcribed.Principal FindingsOur data indicate the presence of MALT1-API2 transcripts in the majority of t(11;18)(q21;q21)-positive MALT lymphomas. Based on the breakpoints in the MALT1 and API2 gene, the MALT1-API2 protein contains the death domain and one or both immunoglobulin-like domains of MALT1 (∼90% of cases) - mediating the possible interaction with BCL10 - fused to the RING domain of API2. Here we show that this RING domain enables MALT1-API2 to function as an E3 ubiquitin ligase for BCL10, inducing its ubiquitination and proteasomal degradation in vitro. Expression of MALT1-API2 transcripts in t(11;18)(q21;q21)-positive MALT lymphomas was however not associated with a reduction of BCL10 protein levels.ConclusionAs we observed MALT1-API2 to be an efficient target of its own E3 ubiquitin ligase activity, our data suggest that this inherent instability of MALT1-API2 prevents its accumulation and renders a potential effect on MALT lymphoma development via destabilization of BCL10 unlikely.

Selective Expansion of Marginal Zone B Cells in Eμ-API2-MALT1 Mice Is Linked to Enhanced IκB Kinase γ Polyubiquitination

The translocation t(11;18)(q21;q21) that generates an API2-MALT1 fusion protein is the most common structural abnormality among the genetic defects reported in mucosa-associated lymphoid tissue (MALT)-type lymphomas, and its presence correlates with the apparent lack of further genetic instability or chromosomal imbalances. Hence, constitutive nuclear factor-kappaB (NF-kappaB) activation induced by the API2-MALT1 fusion protein is considered essential for B-cell transformation. To examine its role in B-cell development and lymphomagenesis, Emu-API2-MALT1 transgenic mice were produced. Our data show that expression of the API2-MALT1 fusion protein alone is not sufficient for the development of lymphoma masses within 50 weeks. Nevertheless, API2-MALT1 expression affected B-cell maturation in the bone marrow and triggered the specific expansion of splenic marginal zone B cells. Polyubiquitination of IkappaB kinase gamma (IKKgamma), indicative for enhanced NF-kappaB activation, was increased in splenic lymphocytes and promoted the survival of B cells ex vivo. In addition, we show that the API2-MALT1 fusion resided in the cholesterol- and sphingolipid-enriched membrane microdomains, termed lipid rafts. We provide evidence that association of the MALT1 COOH terminal with the lipid rafts, which is mediated by the API2 portion, is sufficient to trigger NF-kappaB activation via enhanced polyubiquitination of IKKgamma. Taken together, these data support the hypothesis that the API2-MALT1 fusion protein can contribute to MALT lymphoma formation via increased NF-kappaB activation.

Molecular pathogenesis of MALT lymphoma: two signaling pathways underlying the antiapoptotic effect of API2-MALT1 fusion protein

At least three recurrent chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32), t(14;18)(q32;q21), involving the API2-MALT1 fusion protein, BCL10 and MALT1, have been implicated in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphoma. Several lines of evidence indicated that both BCL10 and MALT1 are required for nuclear factor kappa B (NF-kappaB) activation by antigen receptor stimulation in lymphocytes, and API2-MALT1 can bypass this BCL10/MALT1 signaling pathway. Nuclear factor kappa B activation may contribute to antiapoptotic effect through NF-kappaB-mediated upregulation of apoptotic inhibitor genes. We recently demonstrated that API2-MALT1 can induce transactivation of the API2 gene through NF-kappaB activation, thus highlighting a positive feedback-loop mechanism of self-activation by upregulating its own expression in t(11;18) MALT lymphomas. We also demonstrated that API2-MALT1 possesses an antiapoptotic effect, in part, through its direct interaction with apoptotic regulators. These findings therefore led us to hypothesize that the antiapoptotic effect by API2-MALT1 may be mediated by its interaction with apoptotic regulators, on the one hand, and by NF-kappaB-mediated upregulation of apoptotic inhibitor genes on the other. We also found that BCL10 and MALT1 are shuttling between nucleus and cytoplasm, and that MALT1 can regulate the subcellular location of BCL10.

API2-MALT1 fusion protein induces transcriptional activation of the API2 gene through NF-κB binding elements: Evidence for a positive feed-back loop pathway resulting in unremitting NF-κB activation

t(11;18)(q21;q21) is a characteristic as well as the most frequent chromosomal translocation in mucosa-associated lymphoid tissue (MALT) type lymphoma, and this translocation results in a fusion transcript, API2-MALT1. Although API2-MALT1 has been shown to enforce activation of NF-κB signaling, the transcriptional target genes of this fusion protein remains to be identified. Our analyses of the API2-MALT transfectants suggested that one of the target genes may be the apoptotic inhibitor API2 gene. Luciferase reporter assays with deletion and mutational constructs of the API2 promoter and electrophoretic mobility shift assays established that API2-MALT1 induces transcriptional activation of the API2 gene through two NF-κB binding elements. Moreover, supershift experiments indicated that these elements are recognized by the NF-κB p50/p65 heterodimer. Taken together, our results strongly indicated that API2-MALT1 possesses a novel mechanism of self-activation by up-regulating its own expression in t(11;18)(q21;q21)-carrying MALT lymphomas, highlighting a positive feedback-loop pathway resulting in unremitting NF-κB activation.

Anti-apoptotic action of API2-MALT1 fusion protein involved in t(11;18)(q21;q21) MALT lymphoma

At least three distinct chromosomal translocations, t(11;18)(q21;q21), t(1;14)(p22;q32) and t(14;18)(q32;q21) involving the API2 (also known as c-IAP2)-MALT1 fusion protein, BCL10, and MALT1, respectively, have been implicated in the molecular pathogenesis of mucosa associated lymphoid tissue (MALT) lymphoma. Our findings showed that several variants of the API2-MALT1 fusion protein can occur in patients with t(11;18)(q21;q21), and that API2-MALT1 can potently enfance activation of nuclear factor (NF)-kappaB signaling, which may be relevant to the pathogenesis of MALT lymphomas. We also found that MALT1 is rapidly degraded via the ubiquitin-proteasome pathway, as is the case with API2, but upon the synthesis of fusion, API2-MALT1 becomes stable against this pathway. This stability of API2-MALT1 may thus result in inappropriate nuclear factor (NF)-kappaB activation, thereby contributing to the pathogenesis of MALT lymphoma. Recent biochemical and genetic studies have clearly shown that BCL10 and MALT1 form a physical and functional complex and are both required for NF-kappaB activation by antigen receptor stimulation in T and B lymphocytes. It has also been shown that CARMA1, a newly discovered member of the membrane-associated guanylate kinase (MAGUK) families, is critical for antigen receptor-stimulated NF-kappaB activation. It can be assumed that API2-MALT1 can bypass this normal BCL10/MALT1 cellular signaling pathway linked to NF-kappaB activation, thereby inducing antigen receptor-independent proliferation of lymphocytes. Furthermore, BCL10/MALT1- and API2-MALT1-induced NF-kappaB activation may contribute to anti-apoptotic action probably through NF-kappaB-mediated upregulation of apoptotic inhibitor genes. We recently provided direct evidence that API2-MALT1 indeed exerts anti-apoptotic action, in part, through its direct interaction with apoptotic regulators including Smac. Taken together, these findings prompt us to hypothesize that the anti-apoptotic action of API2-MALT1 may be mediated partly by the direct interaction with apoptotic regulators as well as partly by upregulation of apoptotic inhibitor genes. Further studies can be expected to stimulate research into the development of therapeutic drugs that specifically inhibit the antigen receptor signaling-stimulated NF-kappaB activation pathway: such molecule targeting drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders.

Nuclear Factor 3B Activation and Antiapoptosis in Mucosa-Associated Lymphoid Tissue Lymphoma

Three distinct chromosomal translocations, t(11;18), t(1;14), and t(14;18), involving the API2-MALT1 fusion protein, BCL10, and MALT1 have been convincingly implicated in the pathogenesis of mucosa-associated lymphoid tissue (MALT) lymphomas. Recent genetic and biochemical studies have indicated that BCL10 and MALT1 form a physical and functional complex and are both essential for nuclear factor kappaB (NF-kappaB) activation by antigen receptor stimulation in lymphocytes. API2-MALT1 can bypass the BCL10/MALT1 pathway linking to NF-kappaB activation, thereby inducing antigen receptor-independent events of lymphocytes. BCL10/MALT1- and API2-MALT1-induced NF-kappaB activation can be assumed to be able to contribute to antiapoptosis, probably through NF-kappaB-mediated up-regulation of several apoptotic inhibitor genes. We also have provided direct evidence that API2-MALT1 can exert an antiapoptotic effect, in part through its direct interaction with apoptotic regulators. We therefore hypothesize that the antiapoptotic effect of API2-MALT1 may be mediated by the interaction with apoptotic regulators as well as by the up-regulation of apoptotic inhibitor genes. Finally, we hope that further studies will stimulate research leading to the development of therapeutic drugs that specifically inhibit the antigen receptor-stimulated NF-kappaB activation pathway. Such drugs should be useful for interfering with inappropriate proliferation of lymphocytes associated with inflammatory and neoplastic disorders, including MALT lymphomas.

Antiapoptotic function of apoptosis inhibitor 2-MALT1 fusion protein involved in t(11;18)(q21;q21) mucosa-associated lymphoid tissue lymphoma.

t(11;18)(q21;q21) is a characteristic chromosomal translocation in mucosa-associated lymphoid tissue (MALT) type lymphoma, and this translocation results in fusion transcript of apoptosis inhibitor 2 (API2), also known as c-IAP2, and MALT translocation gene 1 (MALT1). Although the API2-MALT1 fusion protein has been shown to enforce activation of nuclear factor kappaB signaling, its precise role in the apoptotic signaling pathway remains to be established. To identify proteins that bind the API2-MALT1 protein, we used coimmunoprecipitation and SDS-PAGE, followed by liquid chromatography-electrospray ionization tandem mass spectrometry. As a result, three important regulators of apoptosis, Smac, HtrA2, and TRAF2, and three other proteins were identified as potential API2-MALT1-binding proteins. Immunoprecipitation analyses verified that API2-MALT1 indeed binds to both exogeneous and endogeneous Smac proteins. It is especially noteworthy that stably transfected API2-MALT1 significantly suppressed both UV- and etoposide-induced apoptosis in HeLa cells, thus demonstrating for the first time that API2-MALT1 indeed possesses antiapoptotic function. Furthermore, API2-MALT1 significantly suppressed Smac-promoted apoptosis in UV-irradiated HeLa cells. Thus, our results provide direct experimental evidence that API2-MALT1 can confer resistance to apoptosis, at least in part, by neutralizing apoptosis promoted by Smac.

Genetic alterations underlying the pathogenesis of MALT lymphoma.

Extranodal marginal zone B-cell lymphoma is a discrete clinicopathologic entity arising in mucosaassociated lymphoid tissue (MALT), characterised by unique pathogenic, histologic and clinical features. MALT B-cell lymphoma occurs more frequently in organs, such as stomach, salivary glands, or thyroid, which acquire lymphoid tissue only after chronic phlogistic events. This occurs in response to either infectious conditions such as Helicobacter pyloriassociated chronic gastritis, or autoimmune processes like myo-epithelial sialadenitis and Hashimoto's thyroiditis. Within these prolonged lymphoid reactive proliferations, an abnormal B cell clone arises and replaces the normal population, giving rise to the MALT lymphoma. Clinical studies have shown that gastric MALT lymphoma can regress after anti-H. pylori antibiotic treatment, even if it is still not known whether antibiotics alone might induce de®nitive cure of the lymphoma. The molecular events underlying MALT lymphoma pathogenesis have become clearer during these last years and, at least for gastric lymphoma, a model can be drawn (Figure 1). Two distinct pathways seem to occur. MALT lymphoma might develop through a series of genetic lesions underlying the progression from a H. pylori-dependent lymphoma, with the presence of H. pylori strain-speci®c T cells necessary for the growth of lymphoma cells, to a H. pyloriindependent lesion. In contrast, the occurrence of the translocation t(11;18) would give rise to a MALT lymphoma, already H. pylori-independent. t(11;18)(q21;q21): API2-MALT1 fusion protein

Bcl10 and MALT1, Independent Targets of Chromosomal Translocation in MALT Lymphoma, Cooperate in a Novel NF-κB Signaling Pathway

At least two distinct recurrent chromosomal translocations have been implicated in the pathogenesis of MALT lymphoma. The first, t(1;14), results in the transfer of the entire Bcl10 gene to chromosome 14 wherein Bcl10 expression is inappropriately stimulated by the neighboring Ig enhancer. The second, t(11;18), results in the synthesis of a novel fusion protein, API2-MALT1. Until now, no common mechanism of action has been proposed to explain how the products of these seemingly unrelated translocations may contribute to the same malignant process. We show here that Bcl10 and MALT1 form a strong and specific complex within the cell, and that these proteins synergize in the activation of NF-kappaB. The data support a mechanism of action whereby Bcl10 mediates the oligomerization and activation of the MALT1 caspase-like domain. This subsequently activates the IKK complex through an unknown mechanism, setting in motion a cascade of events leading to NF-kappaB induction. Furthermore, the API2-MALT1 fusion protein also strongly activates NF-kappaB and shows dependence upon the same downstream signaling factors. We propose a model whereby both the Bcl10.MALT1 complex and the API2-MALT1 fusion protein activate a common downstream signaling pathway that originates with the oligomerization-dependent activation of the MALT1 caspase-like domain.