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.

Inhibition of the MALT1-LPCAT3 axis protects cartilage degeneration and osteoarthritis.

The proinflammatory cytokines and arachidonic acid (AA)-derived eicosanoids play a key role in cartilage degeneration in osteoarthritis (OA). The lysophosphatidylcholine acyltransferase 3 (LPCAT3) preferentially incorporates AA into the membranes. Our recent studies showed that MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that LPCAT3 expression was up-regulated in both human and mice articular cartilage of OA, and correlated with severity of OA. The IL-1ß-induces cell death via upregulation of LPCAT3, MMP3, ADAMTS5, and eicosanoids via MALT1. Gene silencing or pharmacological inhibition of LPCAT3 or MALT1 in chondrocytes and human cartilage explants notably suppressed the IL-1ß-induced cartilage catabolism through inhibition of expression of MMP3, ADAMTS5, and also secretion of cytokines and eicosanoids. Mechanistically, overexpression of MALT1 in chondrocytes significantly upregulated the expression of LPCAT3 along with MMP3 and ADAMTS5 via c-Myc. Inhibition of c-Myc suppressed the IL-1ß-MALT1-dependent upregulation of LPCAT3, MMP3 and ADAMTS5. Consistent with the in vitro data, pharmacological inhibition of MALT1 or gene silencing of LPCAT3 using siRNA-lipid nanoparticles suppressed the synovial articular cartilage erosion, pro-inflammatory cytokines, and eicosanoids such as PGE2, LTB4, and attenuated osteoarthritis induced by the destabilization of the medial meniscus in mice. Overall, our data reveal a previously unrecognized role of the MALT1-LPCAT3 axis in osteoarthritis. Targeting the MALT1-LPCAT3 pathway with MALT1 inhibitors or siRNA-liposomes of LPCAT3 may become an effective strategy to treat OA by suppressing eicosanoids, matrix-degrading enzymes, and proinflammatory cytokines.

The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.

Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.

Chimeric and mutant CARD9 constructs enable analyses of conserved and diverged autoinhibition mechanisms in the CARD-CC protein family.

Caspase recruitment domain-containing protein (CARD)9, CARD10, CARD11, and CARD14 all belong to the CARD-coiled coil (CC) protein family and originated from a single common ancestral protein early in vertebrate evolution. All four proteins form CARD-CC/BCL10/MALT1 (CBM) complexes leading to nuclear factor-kappa-B (NF-κB) activation after upstream phosphorylation by various protein kinase C (PKC) isoforms. CBM complex signaling is critical for innate and adaptive immunity, but aberrant activation can cause autoimmune or autoinflammatory diseases, or be oncogenic. CARD9 shows a superior auto-inhibition compared with other CARD-CC family proteins, with very low spontaneous activity when overexpressed in HEK293T cells. In contrast, the poor auto-inhibition of other CARD-CC family proteins, especially CARD10 (CARMA3) and CARD14 (CARMA2), is hampering characterization of upstream activators or activating mutations in overexpression studies. We grafted different domains from CARD10, 11, and 14 on CARD9 to generate chimeric CARD9 backbones for functional characterization of activating mutants using NF-κB reporter gene activation in HEK293T cells as readout. CARD11 (CARMA1) activity was not further reduced by grafting on CARD9 backbones. The chimeric CARD9 approach was subsequently validated by using several known disease-associated mutations in CARD10 and CARD14, and additional screening allowed us to identify several previously unknown activating natural variants in human CARD9 and CARD10. Using Genebass as a resource of exome-based disease association statistics, we found that activated alleles of CARD9 correlate with irritable bowel syndrome (IBS), constipation, osteoarthritis, fibromyalgia, insomnia, anxiety, and depression, which can occur as comorbidities.

Identification of an Ortholog of MALT1 from Shrimp That Induces NF-κB-Mediated Antiviral Immunity.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) serves as a pivotal mediator for NF-κB activation in response to a wide spectrum of transmembrane receptor stimuli. In the present study, a homolog of MALT1, named LvMALT1, is cloned from the Pacific white shrimp (Litopenaeus vannamei) and its potential function in shrimp innate immunity is explored. The open reading frame of LvMALT1 is 2364 bp that encodes 787 amino acids. The predicted LvMALT1 protein structure comprises a death domain, three immunoglobulin domains, and a caspase-like domain, exhibiting remarkable similarity to other homologs. LvMALT1 is a cytoplasmic-localized protein and could interact with LvTRAF6. Overexpression of LvMALT1 induces the activation of promoter elements governing the expression of several key antimicrobial peptides (AMPs), including penaeidins (PENs) and crustins (CRUs). Conversely, silencing of LvMALT1 leads to a reduction in the phosphorylation levels of Dorsal and Relish, along with a concomitant decline in the in vivo expression levels of multiple AMPs. Furthermore, LvMALT1 is prominently upregulated in response to a challenge by the white spot syndrome virus (WSSV), facilitating the NF-κB-mediated expression of AMPs as a defense against viral infection. Taken together, we identified a MALT1 homolog from the shrimp L. vannamei, which plays a positive role in the TRAF6/NF-κB/AMPs axis-mediated innate immunity.

In silico study of polyphenols as potential inhibitors of MALT1 protein in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common cancer types. Deregulated signaling pathways can trigger certain NHL subtypes, including Diffuse Large B-cell lymphoma. NF-ĸB signaling pathway, which is responsible for the proliferation, growth, and survival of cells, has an essential role in lymphoma development. Although different signals control NF-ĸB activation in various lymphoid malignancies, the characteristic one is the CARD11-BCL10-MALT1 (CBM) complex. The CBM complex is responsible for the initiation of adaptive immune response. Our study is focused on the molecular docking of ten polyphenols as potential CARD11-BCL10-MALT1 complex inhibitors, essentially through MALT1 inhibition. Molecular docking was performed by Auto Dock Tools and AutoDock Vina tool, while SwissADME was used for drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of the ligands. Out of 66 ligands that were used in this study, we selected and visualized five. Selection criteria were based on the binding energy score and position of the ligands on the used protein. 2D and 3D visualizations showed interactions of ligands with the protein. Five ligands are considered potential inhibitors of MALT1, thus affecting NF-ĸB signaling pathway. However, additional in vivo and in vitro studies are required to confirm their mechanism of inhibition.

Identification of Tensin-3 as a MALT1 substrate that controls B cell adhesion and lymphoma dissemination.

The protease MALT1 promotes lymphocyte activation and lymphomagenesis by cleaving a limited set of cellular substrates, most of which control gene expression. Here, we identified the integrin-binding scaffold protein Tensin-3 as a MALT1 substrate in activated human B cells. Activated B cells lacking Tensin-3 showed decreased integrin-dependent adhesion but exhibited comparable NF-κB1 and Jun N-terminal kinase transcriptional responses. Cells expressing a noncleavable form of Tensin-3, on the other hand, showed increased adhesion. To test the role of Tensin-3 cleavage in vivo, mice expressing a noncleavable version of Tensin-3 were generated, which showed a partial reduction in the T cell-dependent B cell response. Interestingly, human diffuse large B cell lymphomas and mantle cell lymphomas with constitutive MALT1 activity showed strong constitutive Tensin-3 cleavage and a decrease in uncleaved Tensin-3 levels. Moreover, silencing of Tensin-3 expression in MALT1-driven lymphoma promoted dissemination of xenografted lymphoma cells to the bone marrow and spleen. Thus, MALT1-dependent Tensin-3 cleavage reveals a unique aspect of the function of MALT1, which negatively regulates integrin-dependent B cell adhesion and facilitates metastatic spread of B cell lymphomas.

MALT1 Positively Relates to T Helper 1 and T Helper 17 cells, and Serves as a Potential Biomarker for Predicting 30-Day Mortality in Stanford Type A Aortic Dissection Patients.

Mucosa-associated lymphoid tissue 1 (MALT1) regulates inflammation and T helper (Th) cell differentiation, which may participate in the progression of Stanford type A aortic dissection (TAAD). This study intended to assess the association of MALT1 expression with prognosis in TAAD patients. In this prospective study, MALT1 expression was measured by reverse transcription-quantitative polymerase chain reaction assay from peripheral blood samples in 100 TAAD patients and 100 non-AD controls (non-AD patients with chest pain) before treatment. Besides, Th1, Th2, and Th17 cells of TAAD patients before treatment were measured by flow cytometry assay, and their 30-day mortality was recorded. MALT1 expression was ascended in TAAD patients vs. non-AD controls (P < 0.001). In TAAD patients, elevated MALT1 expression was linked with hypertension complication (P = 0.009), increased systolic blood pressure (r = 0.291, P = 0.003), C-reactive protein (CRP) (r = 0.286, P = 0.004), and D-dimer (r = 0.359, P < 0.001). Additionally, MALT1 expression was positively correlated with Th1 cells (r = 0.312, P = 0.002) and Th17 cells (r = 0.397, P < 0.001), but not linked with Th2 cells (r = -0.166, P = 0.098). Notably, the 30-day mortality of TAAD patients was 28.0%. MALT1 expression [odds ratio (OR) = 1.936, P = 0.004], CRP (OR = 1.108, P = 0.002), D-dimer (OR = 1.094, P = 0.003), and surgery timing (emergency vs. selective) (OR = 8.721, P = 0.024) independently predicted increased risk of death within 30 days in TAAD patients. Furthermore, the combination of the above-mentioned independent factors had an excellent ability in predicting 30-day mortality with the area under curve of 0.949 (95% confidence interval: 0.909-0.989). MALT1 expression relates to increased Th1 cells, Th17 cells, and 30-day mortality risk in TAAD patients.

Function and targeting of MALT1 paracaspase in cancer.

The paracaspase MALT1 has emerged as a key regulator of immune signaling, which also promotes tumor development by both cancer cell-intrinsic and -extrinsic mechanisms. As an integral subunit of the CARD11-BCL10-MALT1 (CBM) signaling complex, MALT1 has an intriguing dual function in lymphocytes. MALT1 acts as a scaffolding protein to drive activation of NF-κB transcription factors and as a protease to modulate signaling and immune activation by cleavage of distinct substrates. Aberrant MALT1 activity is critical for NF-κB-dependent survival and proliferation of malignant cancer cells, which is fostered by paracaspase-catalyzed inactivation of negative regulators of the canonical NF-κB pathway like A20, CYLD and RelB. Specifically, B cell receptor-addicted lymphomas rely strongly on this cancer cell-intrinsic MALT1 protease function, but also survival, proliferation and metastasis of certain solid cancers is sensitive to MALT1 inhibition. Beyond this, MALT1 protease exercises a cancer cell-extrinsic role by maintaining the immune-suppressive function of regulatory T (Treg) cells in the tumor microenvironment (TME). MALT1 inhibition is able to convert immune-suppressive to pro-inflammatory Treg cells in the TME of solid cancers, thereby eliciting a robust anti-tumor immunity that can augment the effects of checkpoint inhibitors. Therefore, the cancer cell-intrinsic and -extrinsic tumor promoting MALT1 protease functions offer unique therapeutic opportunities, which has motivated the development of potent and selective MALT1 inhibitors currently under pre-clinical and clinical evaluation.

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.

MALT1 paracaspase is overexpressed in hepatocellular carcinoma and promotes cancer cell survival and growth.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Despite recent advances in treatment options, therapeutic management of HCC remains a challenge, emphasizing the importance of exploring novel targets. MALT1 paracaspase is a druggable signaling molecule whose dysregulation has been linked to hematological and solid tumors. However, the role of MALT1 in HCC remains poorly understood, leaving its molecular functions and oncogenic implications unclear. Here we provide evidence that MALT1 expression is elevated in human HCC tumors and cell lines, and that correlates with tumor grade and differentiation state, respectively. Our results indicate that ectopic expression of MALT1 confers increased cell proliferation, 2D clonogenic growth, and 3D spheroid formation in well differentiated HCC cell lines with relatively low MALT1 levels. In contrast, stable silencing of endogenous MALT1 through RNA interference attenuates these aggressive cancer cell phenotypes, as well as migration, invasion, and tumor-forming ability, in poorly differentiated HCC cell lines with higher paracaspase expression. Consistently, we find that pharmacological inhibition of MALT1 proteolytic activity with MI-2 recapitulates MALT1 depletion phenotypes. Finally, we show that MALT1 expression is positively correlated with NF-kB activation in human HCC tissues and cell lines, suggesting that its tumor promoting functions may involve functional interaction with the NF-kB signaling pathway. This work unveils new insights into the molecular implications of MALT1 in hepatocarcinogenesis and places this paracaspase as a potential marker and druggable liability in HCC.

MALT1-dependent cleavage of CYLD promotes NF-κB signaling and growth of aggressive B-cell receptor-dependent lymphomas.

The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is a protease and scaffold protein essential in propagating B-cell receptor (BCR) signaling to NF-κB. The deubiquitinating enzyme cylindromatosis (CYLD) is a recently discovered MALT1 target that can negatively regulate NF-κB activation. Here, we show that low expression of CYLD is associated with inferior prognosis of diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) patients, and that chronic BCR signaling propagates MALT1-mediated cleavage and, consequently, inactivation and rapid proteasomal degradation of CYLD. Ectopic overexpression of WT CYLD or a MALT1-cleavage resistant mutant of CYLD reduced phosphorylation of IκBα, repressed transcription of canonical NF-κB target genes and impaired growth of BCR-dependent lymphoma cell lines. Furthermore, silencing of CYLD expression rendered BCR-dependent lymphoma cell lines less sensitive to inhibition of NF-κΒ signaling and cell proliferation by BCR pathway inhibitors, e.g., the BTK inhibitor ibrutinib, indicating that these effects are partially mediated by CYLD. Taken together, our findings identify an important role for MALT1-mediated CYLD cleavage in BCR signaling, NF-κB activation and cell proliferation, which provides novel insights into the underlying molecular mechanisms and clinical potential of inhibitors of MALT1 and ubiquitination enzymes as promising therapeutics for DLBCL, MCL and potentially other B-cell malignancies.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

TRAF6 controls T cell homeostasis by maintaining the equilibrium of MALT1 scaffolding and protease functions.

MALT1 is a core component of the CARD11-BCL10-MALT1 (CBM) signalosome, in which it acts as a scaffold and a protease to bridge T cell receptor (TCR) ligation to immune activation. As a scaffold, MALT1 binds to TRAF6, and T cell-specific TRAF6 ablation or destruction of MALT1-TRAF6 interaction provokes activation of conventional T (Tconv) effector cells. In contrast, MALT1 protease activity controls the development and suppressive function of regulatory T (Treg) cells in a T cell-intrinsic manner. Thus, complete loss of TRAF6 or selective inactivation of MALT1 catalytic function in mice skews the immune system towards autoimmune inflammation, but distinct mechanisms are responsible for these immune disorders. Here we demonstrate that TRAF6 deletion or MALT1 paracaspase inactivation are highly interdependent in causing the distinct immune pathologies. We crossed mice with T cell-specific TRAF6 ablation (Traf6-ΔT) and mice with a mutation rendering the MALT1 paracaspase dead in T cells (Malt1 PD-T) to yield Traf6-ΔT;Malt1 PD-T double mutant mice. These mice reveal that the autoimmune inflammation caused by TRAF6-ablation relies strictly on the function of the MALT1 protease to drive the activation of Tconv cells. Vice versa, despite the complete loss of Treg cells in Traf6-ΔT;Malt1 PD-T double mutant mice, inactivation of the MALT1 protease is unable to cause autoinflammation, because the Tconv effector cells are not activated in the absence of TRAF6. Consequentially, combined MALT1 paracaspase inactivation and TRAF6 deficiency in T cells mirrors the immunodeficiency seen upon T cell-specific MALT1 ablation.

Targeting tumor exosomal circular RNA cSERPINE2 suppresses breast cancer progression by modulating MALT1-NF-𝜠B-IL-6 axis of tumor-associated macrophages.

BACKGROUND: Circular RNAs (circRNAs) have important regulatory functions in cancer, but the role of circRNAs in the tumor microenvironment (TME) remains unclear. Moreover, we also explore the effects of si-circRNAs loaded in nanoparticles as therapeutic agent for anti-tumor in vivo. METHODS: We conducted bioinformatics analysis, qRT-PCR, EdU assays, Transwell assays, co-culture system and multiple orthotopic xenograft models to investigate the expression and function of circRNAs. Additionally, PLGA-based nanoparticles loaded with si-circRNAs were used to evaluate the potential of nanotherapeutic strategy in anti-tumor response. RESULTS: We identified oncogene SERPINE2 derived circRNA, named as cSERPINE2, which was notably elevated in breast cancer and was closely related to poor clinical outcome. Functionally, tumor exosomal cSERPINE2 was shuttled to tumor associated macrophages (TAMs) and enhanced the secretion of Interleukin-6 (IL-6), leading to increased proliferation and invasion of breast cancer cells. Furthermore, IL-6 in turn increased the EIF4A3 and CCL2 levels within tumor cells in a positive feedback mechanism, further enhancing tumor cSERPINE2 biogenesis and promoting the recruitment of TAMs. More importantly, we developed a PLGA-based nanoparticle loaded with si-cSERPINE2, which effectively attenuated breast cancer progression in vivo. CONCLUSIONS: Our study illustrates a novel mechanism that tumor exosomal cSERPINE2 mediates a positive feedback loop between tumor cells and TAMs to promote cancer progression, which may serve as a promising nanotherapeutic strategy for the treatment of breast cancer.

MALT1 promotes necroptosis in stroke rat brain via targeting the A20/RIPK3 pathway.

Necroptosis has been demonstrated to contribute to brain injury in ischemic stroke, whereas A20 can exert anti-necroptosis effect via deubiquitinating receptor-interacting protein kinase (RIPK3) at k63 and it can be cleaved by MALT1. This study aims to explore whether MALT1 is upregulated in the brain during ischemic stroke and promotes brain cell necroptosis through enhancing the degradation of A20. Ischemic stroke model was established in Sprague Dawley rats by occlusion of the middle cerebral artery (MCA) for 2 h, followed by 24 h reperfusion, which showed brain injury (increase in neurological deficit score and infarct volume) concomitant with an upregulation of MALT1, a decrease in A20 level, and increases in necroptosis-associated protein levels [RIPK3, mixed lineage kinase domain-like protein (MLKL) and p-MLKL] and k63-ubiquitination of RIPK3 in brain tissues. Administration of MALT1 inhibitor (Ml-2) at 8 or 15 mg/kg (i.p.) at 1 h after ischemia significantly improved neurological function and reduced infarct volume together with a downregulation of MALT1, an increase in A20 level and decreases in necroptosis-associated protein levels and k63-ubiquitination of RIPK3. Similarly, knockdown of MALT1 could also reduce oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in the cultured HT22 cells coincident with an increase in A20 level and decreases in necroptosis-associated protein levels and k63-ubiquitination of RIPK3. Based on these observations, we conclude that MALT1 promotes necroptosis in stroke rat brain via enhancing the degradation of A20, which leads to a decrease in the capability of A20 to deubiquitinate RIPK3 at k63 and a subsequent compromise in counteraction against the brain cell necroptosis.

The Weakened Interaction Between HECTD4 and GluN2B in Ischemic Stroke Promotes Calcium Overload and Brain Injury Through a Mechanism Involving the Decrease of GluN2B and MALT1 Ubiquitination.

Glutamate receptor ionotropic NMDA 2B (GluN2B) plays an essential role in calcium overload during excitotoxicity. Reverse-phase nano-liquid chromatography-tandem mass spectrometry has revealed an interaction between GluN2B and HECT domain E3 ubiquitin protein ligase 4 (HECTD4), an E3 ubiquitin ligase highly expressed in the brain. As a potential substrate for HECTD4, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) acts as a scaffold with hydrolysis activity. This study explores the relationship between HECTD4, GluN2B, and MALT1, focusing on their role in brain injury in ischemic stroke. Rats were subjected to 2 h-ischemia followed by 24-h reperfusion to establish an ischemic stroke model. We observed the downregulation of HECTD4 and the upregulation of MALT1. Additionally, an increased GluN2B phosphorylation was concomitant with weakened interactions between HECTD4 and GluN2B, followed by decreased striatal-enriched protein phosphatase (STEP61). Knockdown of HECTD4 exacerbated hypoxia- or NMDA-induced injury in nerve cells coincident with a decrease in GluN2B and MALT1 ubiquitination, and an increase in GluN2B phosphorylation as well as an increase in intracellular calcium level, which were counteracted by MALT1 siRNA. Blockage of MALT1 with its inhibitor or siRNA reduced STEP61 degradation, accompanied by a decrease in GluN2B phosphorylation, intracellular calcium concentration, and brain cell injury, which were reversed by overexpression of MALT1. Based on these observations, we conclude that the downregulation of HECTD4 in ischemic stroke rat brain accounts for calcium overload and brain injury due to activating GluN2B directly and indirectly through a mechanism involving the reduced ubiquitination of GluN2B and MALT1, respectively.

The combination of gemcitabine and ginsenoside Rh2 enhances the immune function of dendritic cells against pancreatic cancer via the CARD9-BCL10-MALT1 / NF-κB pathway.

Cold tumor immune microenvironment (TIME) of pancreatic cancer (PC) with minimal dendritic cell (DC) and T cell infiltration can result in insufficient immunotherapy and chemotherapy. While gemcitabine (GEM) is a first-line chemotherapeutic drug for PC, its efficacy is reduced by immunosuppression and drug resistance. Ginsenoside Rh2 (Rh2) is known to have anti-cancer and immunomodulatory properties. Combining GEM with Rh2 may thus overcome immunosuppression and induce lasting anti-tumor immunity in PC. Here, we showed that after GEM-Rh2 therapy, there was significantly greater tumor infiltration by DCs. Caspase recruitment domain-containing protein 9 (CARD9), a central adaptor protein, was strongly up-regulated DCs with GEM-Rh2 therapy and promoted anti-tumor immune responses by DCs. CARD9 was found to be a critical target for Rh2 to enhance DC function. However, GEM-Rh2 treatment did not achieve the substantial anti-PC efficacy in CARD9-/- mice as in WT mice. The adoptive transfer of WT DCs to DC-depleted PC mice treated with GEM-Rh2 elicited strong anti-tumor immune responses, although CARD9-/- DCs were less effective than WT DCs. Our results showed that GEM-Rh2 may reverse cold TIME by enhancing tumor immunogenicity and decreasing the levels of immunosuppressive factors, reactivating DCs via the CARD9-BCL10-MALT1/ NF-κB pathway. Our findings suggest a potentially feasible and safe treatment strategy for PC, with a unique mechanism of action. Thus, Rh2 activation of DCs may remodel the cold TIME and optimize GEM chemotherapy for future therapeutic use.

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-κB (NF-κB) 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-κB 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.

Regulation of gingival keratinocyte monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 expressions by periodontal bacteria, lipopolysaccharide, and interleukin-1ß.

BACKGROUND: The aim of this study was to evaluate oral bacteria- and interleukin (IL)-1ß-induced protein and mRNA expression profiles of monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 in human gingival keratinocyte monolayers and organotypic oral mucosal models. METHODS: Human gingival keratinocyte (HMK) monolayers were incubated with Porphyromonas gingivalis, Fusobacterium nucleatum, P. gingivalis lipopolysaccharide (LPS) and IL-1ß. The protein levels of MCPIP-1 and MALT-1 were examined by immunoblots and mRNA levels by qPCR. MCPIP-1 and MALT-1 protein expression levels were also analyzed immunohistochemically using an organotypic oral mucosal model. One-way analysis of variance followed by Tukey correction was used in statistical analyses. RESULTS: In keratinocyte monolayers, MCPIP-1 protein expression was suppressed by F. nucleatum and MALT-1 protein expression was suppressed by F. nucleatum, P. gingivalis LPS and IL-1ß. P. gingivalis seemed to degrade MCPIP-1 and MALT-1 at all tested time points and degradation was inhibited when P. gingivalis was heat-killed. MCPIP-1 mRNA levels were increased by P. gingivalis, F. nucleatum, and IL-1ß, however, no changes were observed in MALT-1 mRNA levels. CONCLUSION: Gingival keratinocyte MCPIP-1 and MALT-1 mRNA and protein expression responses are regulated by infection and inflammatory mediators. These findings suggest that periodontitis-associated bacteria-induced modifications in MCPIP-1 and MALT-1 responses can be a part of periodontal disease pathogenesis.