MALT1 proteolytic activity suppresses autoimmunity in a T cell intrinsic manner

MALT1 is a central signaling component in innate and adaptive immunity by regulating NF-kappa B and other key signaling pathways in different cell types. Activities of MALT1 are mediated by its scaffold and protease functions. Because of its role in lymphocyte activation and proliferation, inhibition of MALT1 proteolytic activity is of high interest for therapeutic targeting in autoimmunity and certain lymphomas. However, recent studies showing that Mak1 protease-dead knock-in (Malt1-PD) mice suffer from autoimmune disease have somewhat tempered the initial enthusiasm. Although it has been proposed that an imbalance between immune suppressive regulatory T cells (Tregs) and activated effector CD4(+) T cells plays a key role in the autoimmune phenotype of Malt1-PD mice, the specific contribution of MALT1 proteolytic activity in T cells remains unclear. Using T cell-conditional Malt1 protease-dead knock-in (Malt1-PDT) mice, we here demonstrate that MALT1 has a T cell-intrinsic role in regulating the homeostasis and function of thymic and peripheral T cells. T cell-specific ablation of MALT1 proteolytic activity phenocopies mice in which MALT1 proteolytic activity has been genetically inactivated in all cell types. The Malt1-PDT mice have a reduced number of Tregs in the thymus and periphery, although the effect in the periphery is less pronounced compared to full-body Malt1-PD mice, indicating that also other cell types may promote Treg induction in a MALT1 protease-dependent manner. Despite the difference in peripheral Treg number, both T cell-specific and full-body Malt1-PD mice develop ataxia and multi-organ inflammation to a similar extent. Furthermore, reconstitution of the full-body Malt1-PD mice with T cell-specific expression of wild-type human MALT1 eliminated all signs of autoimmunity. Together, these findings establish an important T cell-intrinsic role of MALT1 proteolytic activity in the suppression of autoimmune responses

Mepazine inhibits RANK-induced osteoclastogenesis independent of its MALT1 inhibitory function

Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is an intracellular cysteine protease (paracaspase) that plays an integral role in innate and adaptive immunity. The phenothiazine mepazine has been shown to inhibit the proteolytic activity of MALT1 and is frequently used to study its biological role. MALT1 has recently been suggested as a therapeutic target in rheumatoid arthritis. Here, we analyzed the effect of mepazine on the receptor activator of nuclear factor κ-B (RANK)-induced osteoclastogenesis. The treatment of mouse bone marrow precursor cells with mepazine strongly inhibited the RANK ligand (RANKL)-induced formation of osteoclasts, as well as the expression of several osteoclast markers, such as TRAP, cathepsin K, and calcitonin. However, RANKL induced osteoclastogenesis equally well in bone marrow cells derived from wild-type and Malt1 knock-out mice. Furthermore, the protective effect of mepazine was not affected by MALT1 deficiency. Additionally, the absence of MALT1 did not affect RANK-induced nuclear factor κB (NF-κB) and activator protein 1 (AP-1) activation. Overall, these studies demonstrate that MALT1 is not essential for RANK-induced osteoclastogenesis, and implicate a MALT1-independent mechanism of action of mepazine that should be taken into account in future studies using this compound

Long-term MALT1 inhibition in adult mice without severe systemic autoimmunity

The protease MALT1 is a key regulator of NF-kappa B signaling and a novel therapeutic target in autoimmunity and cancer. Initial enthusiasm supported by preclinical results with MALT1 inhibitors was tempered by studies showing that germline MALT1 protease inactivation in mice results in reduced regulatory T cells and lethal multi-organ inflammation due to expansion of IFN-gamma-producing T cells. However, we show that long-term MALT1 inactivation, starting in adulthood, is not associated with severe systemic inflammation, despite reduced regulatory T cells. In contrast, IL-2-, TNF-, and IFN-gamma-producing CD4(+) T cells were strongly reduced. Limited formation of tertiary lymphoid structures was detectable in lungs and stomach, which did not affect overall health. Our data illustrate that MALT1 inhibition in prenatal or adult life has a different outcome and that long-term MALT1 inhibition in adulthood is not associated with severe side effects

MALT1-deficient mice develop atopic-like dermatitis upon aging

MALT1 plays an important role in innate and adaptive immune signaling by acting as a scaffold protein that mediates NF-kappa B signaling. In addition, MALT1 is a cysteine protease that further fine tunes proinflammatory signaling by cleaving specific substrates. Deregulated MALT1 activity has been associated with immunodeficiency, autoimmunity, and cancer in mice and humans. Genetically engineered mice expressing catalytically inactive MALT1, still exerting its scaffold function, were previously shown to spontaneously develop autoimmunity due to a decrease in Tregs associated with increased effector T cell activation. In contrast, complete absence of MALT1 does not lead to autoimmunity, which has been explained by the impaired effector T cell activation due to the absence of MALT1-mediated signaling. However, here we report that MALT1-deficient mice develop atopic-like dermatitis upon aging, which is preceded by Th2 skewing, an increase in serum IgE, and a decrease in Treg frequency and surface expression of the Treg functionality marker CTLA-4

Biomarkers of aging in Drosophila

Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of aging-related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of aging-related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of aging-related damage. Using this approach, we assessed several commonly used biomarkers of aging-related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of aging-related damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of aging-related damage. Our approach provides a powerful method for identification of biomarkers of aging.This work was supported by the Wellcome Trust and in part by I+D grants from the Spanish Ministry of Education and Science (BFU2006-14495 ⁄ BFI), the Spanish Ministry of Health (ISCIII, Red de Envejecimiento y Fragilidad, RD06 ⁄ 0013 ⁄ 0012), and the Generalitat of Catalunya (2005SGR00101) to R.P; the Spanish Ministry of Health (FIS PI081843), Spanish Ministry of Education and Science (AGL2006-12433), and ‘‘La Caixa’’ Foundation to M.P.O. Also supported by the Max Planck Society (J.J. and L.P), COST B-35 Action; Research into Ageing (A.J.L.) and the Medical Research Council and National Institutes of Health (P01 AG025901, PL1 AG032118 and P30 AG025708) (M.D.B.)

No Influence of Indy on Lifespan in Drosophila after Correction for Genetic and Cytoplasmic Background Effects

To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, we studied lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls we included the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. We report that only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, we saw no effects on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans

Ancient origin of the CARD-coiled coil/Bcl10/MALT1-like paracaspase signaling complex indicates unknown critical functions

The CARD-coiled coil (CC)/Bcl10/MALT1-like paracaspase (CBM) signaling complexes composed of a CARD-CC family member (CARD-9, -10, -11, or -14), Bcl10, and the type 1 paracaspase MALT1 (PCASP1) play a pivotal role in immunity, inflammation, and cancer. Targeting MALT1 proteolytic activity is of potential therapeutic interest. However, little is known about the evolutionary origin and the original functions of the CBM complex. Type 1 paracaspases originated before the last common ancestor of planulozoa (bilaterians and cnidarians). Notably in bilaterians, Ecdysozoa (e.g., nematodes and insects) lacks Bcl10, whereas other lineages have a Bcl10 homolog. A survey of invertebrate CARD-CC homologs revealed such homologs only in species with Bcl10, indicating an ancient common origin of the entire CBM complex. Furthermore, vertebrate-like Syk/Zap70 tyrosine kinase homologs with the ITAM-binding SH2 domain were only found in invertebrate organisms with CARD-CC/Bcl10, indicating that this pathway might be related to the original function of the CBM complex. Moreover, the type 1 paracaspase sequences from invertebrate organisms that have CARD-CC/Bcl10 are more similar to vertebrate paracaspases. Functional analysis of protein-protein interactions, NF-kappa B signaling, and CYLD cleavage for selected invertebrate type 1 paracaspase and Bcl10 homologs supports this scenario and indicates an ancient origin of the CARD-CC/Bcl10/paracaspase signaling complex. By contrast, many of the known MALT1-associated activities evolved fairly recently, indicating that unknown functions are at the basis of the protein conservation. As a proof-of-concept, we provide initial evidence for a CBM- and NF-kappa B-independent neuronal function of the Caenorhabditis elegans type 1 paracaspase malt-1. In conclusion, this study shows how evolutionary insights may point at alternative functions of MALT1

MALT1 is not alone after all : identification of novel paracaspases

Paracaspases and metacaspases are two families of caspase-like proteins identified in 2000. Up until now paracaspases were considered a single gene family with one known non-metazoan paracaspase in the slime mold Dictyostelium and a single animal paracaspase called MALT1. Human MALT1 is a critical signaling component in many innate and adaptive immunity pathways that drive inflammation, and when it is overly active, it can also cause certain forms of cancer. Here, we report the identification and functional analysis of two new vertebrate paracaspases, PCASP2 and PCASP3. Functional characterization indicates that both scaffold and protease functions are conserved across the three vertebrate paralogs. This redundancy might explain the loss of two of the paralogs in mammals and one in Xenopus. Several of the vertebrate paracaspases currently have incorrect or ambiguous annotations. We propose to annotate them accordingly as PCASP1, PCASP2, and PCASP3 similar to the caspase gene nomenclature. A comprehensive search in other metazoans and in non-metazoan species identified additional new paracaspases. We also discovered the first animal metacaspase in the sponge Amphimedon. Comparative analysis of the active site suggests that paracaspases constitute one of the several subclasses of metacaspases that have evolved several times independently

Normal lymphocyte homeostasis and function in MALT1 protease-resistant HOIL-1 knock-in mice

The uniqueness of MALT1 protease activity in controlling several aspects of immunity in humans has made it a very attractive therapeutic target for multiple autoimmune diseases and lymphoid malignancies. Despite several encouraging preclinical studies with MALT1 inhibitors, severe reduction in regulatory T cells and immune-mediated pathology seen in MALT1 protease-dead (MALT1-PD) mice and some, but not all, studies analysing the effect of prolonged pharmacological MALT1 protease inhibition, indicates the need to further unravel the mechanism of MALT1 protease function. Notably, the contribution of individual MALT1 substrates to the immune defects seen in MALT1-PD mice is still unclear. Previous in vitro studies indicated a role for MALT1-mediated cleavage of the E3 ubiquitin ligase HOIL-1 in the modulation of nuclear factor-kappa B (NF-kappa B) signalling and inflammatory gene expression in lymphocytes. Here, we addressed the immunological consequences of inhibition of HOIL-1 cleavage by generating and immunophenotyping MALT1 cleavage-resistant HOIL-1 knock-in (KI) mice. HOIL-1 KI mice appear healthy and have no overt phenotype. NF-kappa B activation in T or B cells, as well as IL-2 production and in vitro T-cell proliferation, is comparable between control and HOIL-1 KI cells. Inhibition of HOIL-1 cleavage in mice has no effect on thymic T-cell development and conventional T-cell homeostasis. Likewise, B-cell development and humoral immune responses are not affected. Together, these data exclude an important role of MALT1-mediated HOIL-1 cleavage in T- and B-cell development and function in mice