MALT1 protease function – from ubiquitin binding to substrate cleavage

The protease MALT1 has a key function in the activation of lymphocytes and the regulation of the immune response, by promoting the activation of pro-inflammatory transcription factors such as NF-B. Dysregulation of MALT1 is implicated in immunodeficiency, autoimmune diseases, lymphomagenesis and non-lymphoid malignancies. Therefore, MALT1 proteolytic activity has appeared as a possible pharmaceutical target, however, the precise mechanism of MALT1 protease activation and the role of individual substrate cleavage events remain incompletely defined. Thus, the aims of this study are to firstly elucidate the molecular mechanism of MALT1 activation and second, to explore the role of the MALT1-dependent cleavage of one particular substrate namely, A20. The protease activity of MALT1 is tightly controlled by conjugation of monoubiquitin to its third auto-inhibitory Ig-like domain, but the mechanism governing the release of the protease domain by a single ubiquitin moiety remains unknown. Here, we identify the Ig3 domain of MALT1 as a novel ubiquitin-binding domain, responsible for MALT1 monoubiquitination, which is essential for MALT1 proteolytic activity and lymphocyte activation. Furthermore, we reveal an allosteric communication from the monoubiquitination site through the protease-Ig3 interaction surface to the catalytic active site of the protease domain. One of the first substrates of MALT1 that has been identified is A20, a potent anti-inflammatory protein. A20 is a well-described negative regulator of the NF-B signaling pathway downstream of different pro-inflammatory stimuli and a regulator of cell death. Although MALT1-dependent A20 cleavage is generally thought to promote NF-B activity, the functional role of A20 cleavage remains controversial and not well defined. This is due to the fact that the originally described single cleavage site in A20 is not conserved among species and only a small proportion of cellular A20 undergoes cleavage. Here, we demonstrate that MALT1 cleaves A20 at a total of four distinct sites in B and T lymphocytes, including three novel cleavage sites with unusual sequence properties, which are conserved in the mouse and other species. The cleavage fragments lost their capacity to regulate the NF-B pathway, but are stable within the cell, suggesting that they retain an unknown physiological function in lymphocytes. Collectively, our findings provide fundamentally new insights into the mechanism of MALT1 protease activation and its cleavage site specificity, and suggest that MALT1-dependent A20 cleavage has roles that go beyond the enhancement of NF-B activity. -- La protéase MALT1 a une fonction clé dans l’activation des lymphocytes et la régulation de la réponse immunitaire, en favorisant l’activation de facteurs de transcription pro- inflammatoires comme le NF-B. La dérégulation de MALT1 est impliquée dans l’immunodéficience, les maladies auto-immunes, la lymphomagénèse et les tumeurs malignes non-lymphoides. Par conséquent, l’activité protéolytique de MALT1 est apparue comme une cible thérapeutique possible, cependant le mécanisme précis de l’activation de la protéase MALT1 et le rôle du clivage de chacun de ses substrats restent en partie incompris. Ainsi les objectifs de cette étude sont d’abord d’élucider le mécanisme moléculaire d’activation de MALT1 puis d’explorer le rôle du clivage dépendant de MALT1 d’un substrat en particulier nommé A20. L’activité protéase de MALT1 est étroitement contrôlée par la conjugaison d’une monoubiquitine à son troisième domaine auto-inhibiteur de type Ig, mais le mécanisme régissant la libération du domaine protéase par un seul fragment d'ubiquitine reste inconnu. Ici, nous avons identifié le domaine Ig3 de MALT1 comme un nouveau domaine de liaison à l’ubiquitine, responsable de la monoubiquitination de MALT1, qui est essentielle pour son activité protéolytique et l’activation lymphocytaire. De plus, nous avons révélé une transmission allostérique du site de monoubiquitination à travers la surface d’interaction protéase-Ig3 au site catalytique actif du domaine protéase. L’un des premiers substrats de MALT1 qui a été identifié est A20, une protéine anti-inflammatoire puissante. A20 est un régulateur négatif bien décrit de la voie de signalisation NF-B en aval de différents stimuli pro-inflammatoires et un régulateur de la mort cellulaire. Bien que le clivage de A20 dépendant de MALT1 soit généralement vu comme un promoteur de l’activité de NF-B, le rôle fonctionnel du clivage de A20 reste controversé et mal défini. Cela est dû au fait que le site de clivage originellement découvert de A20 n’est pas conservé chez les autres espèces et que seulement une petite partie de la protéine cellulaire est clivée. Ici, nous démontrons que MALT1 clive A20 sur un total de quatre sites distincts dans les lymphocytes B et T. Nous avons également identifié trois nouveaux sites de clivage avec des motifs inhabituels, sites qui sont conservés chez la souris et d’autres espèces. Les fragments issus du clivage ont perdu leur capacité à réguler la voie NF-B, mais sont stables dans la cellule, suggérant qu’ils gardent une fonction physiologique inconnue dans les lymphocytes. Ensemble, nos résultats apportent de nouvelles informations fondamentales sur le mécanisme d’activation de la protéase MALT1 et sur la spécificité de ses sites de clivage, et suggèrent que le clivage de A20 dépendant de MALT1 a un rôle qui va au- delà d’une simple activation de NF-B

CARD14 gain-of-function mutation alone is sufficient to drive IL-23/IL-17-mediated psoriasiform skin inflammation in vivo

Rare autosomal dominant mutations in the gene encoding the keratinocyte signaling molecule, Caspase Recruitment Domain-Containing Protein 14 (CARD14), have been associated with an increased susceptibility to psoriasis but the physiological impact of CARD14 gain-of-function mutations remains to be fully determined in vivo. Here, we report that heterozygous mice harboring a CARD14 gain-of-function mutation (Card14ΔE138) spontaneously develop a chronic psoriatic phenotype with characteristic scaling skin lesions, epidermal thickening, keratinocyte hyperproliferation, hyperkeratosis and immune cell infiltration. Affected skin of these mice is characterized by elevated expression of anti-microbial peptides, chemokines and cytokines (including Th17 cell-signature cytokines), and an immune infiltrate rich in neutrophils, myeloid cells and T-cells, reminiscent of human psoriatic skin. Disease pathogenesis was driven by the IL-23/IL-17 axis and neutralization of IL-23p19, the key cytokine in maintaining Th17 cell polarization, significantly reduced skin lesions and the expression of antimicrobial peptides and pro-inflammatory cytokines. Therefore, hyperactivation of CARD14 alone is sufficient to orchestrate the complex immunopathogenesis that drives Th17-mediated psoriasis skin disease in vivo

The RORɣ/SREBP2 pathway is a master regulator of cholesterol metabolism and serves as potential therapeutic target in t(4;11) leukemia

Dysregulated cholesterol homeostasis promotes tumorigenesis and progression. Therefore, metabolic reprogramming constitutes a new hallmark of cancer. However, until today, only few therapeutic approaches exist to target this pathway due to the often-observed negative feedback induced by agents like statins leading to controversially increased cholesterol synthesis upon inhibition. Sterol regulatory element-binding proteins (SREBPs) are key transcription factors regulating the synthesis of cholesterol and fatty acids. Since SREBP2 is difficult to target, we performed pharmacological inhibition of retinoic acid receptor (RAR)-related orphan receptor gamma (RORγ), which acts upstream of SREBP2 and serves as master regulator of the cholesterol metabolism. This resulted in an inactivated cholesterol-related gene program with significant downregulation of cholesterol biosynthesis. Strikingly, these effects were more pronounced than the effects of fatostatin, a direct SREBP2 inhibitor. Upon RORγ inhibition, RNA sequencing showed strongly increased cholesterol efflux genes leading to leukemic cell death and cell cycle changes in a dose- and time-dependent manner. Combinatorial treatment of t(4;11) cells with the RORγ inhibitor showed additive effects with cytarabine and even strong anti-leukemia synergism with atorvastatin by circumventing the statin-induced feedback. Our results suggest a novel therapeutic strategy to inhibit tumor-specific cholesterol metabolism for the treatment of t(4;11) leukemia

Targeting MYC in combination with epigenetic regulators induces synergistic anti-leukemic effects in MLLr leukemia and simultaneously improves immunity

MLL rearranged (MLLr) leukemias are associated with a poor prognosis and a limited response to conventional therapies. Moreover, chemotherapies result in severe side effects with significant impairment of the immune system. Therefore, the identification of novel treatment strategies is mandatory.Recently, we developed a human MLLr leukemia model by inducing chromosomal rearrangements in CD34+ cells using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9. This MLLr model authentically mimics patient leukemic cells and can be used as a platform for novel treatment strategies. RNA sequencing of our model revealed MYC as one of the most important key drivers to promote oncogenesis. However, in clinical trials the BRD4 inhibitor JQ-1 leading to indirect blocking of the MYC pathway shows only modest activity. We and others previously reported that epigenetic drugs targeting MAT2A or PRMT5 promote cell death in MLLr cells. Therefore, we use these drugs in combination with JQ-1 leading to augmented anti-leukemic effects. Moreover, we found activation of T, NK and iNKT cells, release of immunomodulatory cytokines and downregulation of the PD-1/PD-L1 axis upon inhibitor treatment leading to improved cytotoxicity.In summary, the inhibition of MYC and MAT2A or PRMT5 drives robust synergistic anti-leukemic activity in MLLr leukemia. Moreover, the immune system is concomitantly activated upon combinatorial inhibitor treatment, hereby further augmenting the therapeutic efficiency