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

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

Structure-function analysis of the A20-binding inhibitor of NF-kappa B activation, ABIN-1

AbstractNuclear factor κB (NF-κB)-dependent gene expression plays an important role in numerous cellular processes including stress responses, inflammation and cell proliferation. Therefore, the activity of this transcription factor needs to be tightly regulated. Among others, the NF-κB-dependent zinc finger protein A20 is involved in the negative feedback regulation of NF-κB activation in response to tumor necrosis factor (TNF). We previously demonstrated that A20 can interact with A20-binding inhibitors of NF-κB activation (ABINs), which have the potential to inhibit TNF-induced activation of NF-κB upon overexpression. The ABIN proteins were therefore proposed to mediate the NF-κB inhibiting function of A20. Here we demonstrate the presence of a short homologous region in ABINs and IκB kinase γ, the regulatory subunit of the IκB kinase complex. Site-specific mutagenesis of this region abolished the NF-κB inhibiting function of ABIN-1, without affecting the interaction with A20. Furthermore, coexpression of these ABIN-1 mutants interfered in a dominant negative manner with the NF-κB inhibiting function of ABIN-1, whereas the A20-mediated inhibition was unaffected. These results suggest that A20 and ABIN-1 probably act independently of their mutual interaction

Importance of Validating Antibodies and Small Compound Inhibitors Using Genetic Knockout Studies—T Cell Receptor-Induced CYLD Phosphorylation by IKKε/TBK1 as a Case Study

CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it could still be prevented by MRT67307, indicating that the initially observed inhibitory effect of MRT67307 on TCR-induced CYLD phosphorylation is IKKε/TBK1-independent. Most surprisingly, the phospho(Ser418)-CYLD immunoreactive band was still detectable upon immunoblotting of cell extracts obtained from CYLD deficient cells. These data demonstrate the non-specificity of MRT67307 and phospho(Ser418)-CYLD specific antibodies, implying that previously published results based on these tools may also have led to wrong conclusions. We therefore advise to use genetic knockout studies or alternative approaches for a better validation of antibodies and small compound inhibitors. Interestingly, immunoprecipitation with the phospho(Ser418)-CYLD antibody, followed by immunoblotting with anti-CYLD, revealed that CYLD is phosphorylated by IKKε/TBK1 at Ser418 upon T cell stimulation, but that its direct detection with the phospho(Ser418)-CYLD-specific antibody in a western blot is masked by another inducible protein of the same size that is recognized by the same antibody

Yeast two-hybrid screening for proteins interacting with the anti-apoptotic protein A20

he yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors. Therefore, the protein of interest (bait) is fused to a DNA-binding domain, and complimentary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined and transcription from a reporter gene is started. Here, we will give a comprehensive guide for the GAL4-based two-hybrid system, exemplified by the detection of binding partners for the zinc finger protein A20. The latter is an inducible cellular inhibitor of tumor necrosis factor (TNF)-induced apoptosis and nuclear factor (NF)-kappaB-dependent gene expression. Yeast two-hybrid screening with A20 as bait revealed several A20-binding proteins, including A20 itself, members of the 14-3-3 family, as well as three novel proteins ABIN-1, ABIN-2, and TXBP151. The latter protein was subsequently shown to mediate at least part of the anti-apoptotic activities of A20, whereas ABIN-1 and -2 are more likely to be involved in the NF-kappaB inhibitory effects of A20

Yeast two-hybrid screening for proteins interacting with the anti-apoptotic protein A20

he yeast two-hybrid system is a powerful technique for identifying proteins that interact with a specific protein of interest. The rationale of the yeast two-hybrid system relies on the physical separation of the DNA-binding domain from the transcriptional activation domain of several transcription factors. Therefore, the protein of interest (bait) is fused to a DNA-binding domain, and complimentary DNA (cDNA) library-encoded proteins are fused to a transcriptional activation domain. When a protein encoded by the cDNA library binds to the bait, both activities of the transcription factor are rejoined and transcription from a reporter gene is started. Here, we will give a comprehensive guide for the GAL4-based two-hybrid system, exemplified by the detection of binding partners for the zinc finger protein A20. The latter is an inducible cellular inhibitor of tumor necrosis factor (TNF)-induced apoptosis and nuclear factor (NF)-kappaB-dependent gene expression. Yeast two-hybrid screening with A20 as bait revealed several A20-binding proteins, including A20 itself, members of the 14-3-3 family, as well as three novel proteins ABIN-1, ABIN-2, and TXBP151. The latter protein was subsequently shown to mediate at least part of the anti-apoptotic activities of A20, whereas ABIN-1 and -2 are more likely to be involved in the NF-kappaB inhibitory effects of A20

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<p>CYLD is a deubiquitinating enzyme that plays a crucial role in immunity and inflammation as a negative regulator of NF-κB transcription factor and JNK kinase signaling. Defects in either of these pathways contribute to the progression of numerous inflammatory and autoimmune disorders. Therefore, we set out to unravel molecular mechanisms that control CYLD activity in the context of T cell receptor (TCR) signaling. More specifically, we focused on CYLD phosphorylation at Ser418, which can be detected upon immunoblotting of cell extracts with phospho(Ser418)-CYLD specific antibodies. Jurkat T cells stimulated with either anti-CD3/anti-CD28 or PMA/Ionomycin (to mimic TCR signaling) were used as a model system. The role of specific kinases was analyzed using pharmacological as well as genetic approaches. Our initial data indicated that CYLD is directly phosphorylated by the noncanonical IκB kinases (IKKs) IKKε and TANK Binding Kinase 1 (TBK1) at Ser418 upon TCR stimulation. Treatment with MRT67307, a small compound inhibitor for IKKε and TBK1, inhibited TCR-induced CYLD phosphorylation. However, the phospho(Ser418)-CYLD immunoreactive band was still present in CRISPR/Cas9 generated IKKε/TBK1 double knockout cell lines, where it could still be prevented by MRT67307, indicating that the initially observed inhibitory effect of MRT67307 on TCR-induced CYLD phosphorylation is IKKε/TBK1-independent. Most surprisingly, the phospho(Ser418)-CYLD immunoreactive band was still detectable upon immunoblotting of cell extracts obtained from CYLD deficient cells. These data demonstrate the non-specificity of MRT67307 and phospho(Ser418)-CYLD specific antibodies, implying that previously published results based on these tools may also have led to wrong conclusions. We therefore advise to use genetic knockout studies or alternative approaches for a better validation of antibodies and small compound inhibitors. Interestingly, immunoprecipitation with the phospho(Ser418)-CYLD antibody, followed by immunoblotting with anti-CYLD, revealed that CYLD is phosphorylated by IKKε/TBK1 at Ser418 upon T cell stimulation, but that its direct detection with the phospho(Ser418)-CYLD-specific antibody in a western blot is masked by another inducible protein of the same size that is recognized by the same antibody.</p