RESUMO
CARD-BCL10-MALT1 (CBM) signalosomes connect distal signaling of innate and adaptive immune receptors to proximal signaling pathways and immune activation. Four CARD scaffold proteins (CARD9, 10, 11, 14) can form seeds that nucleate the assembly of BCL10-MALT1 filaments in a cell- and stimulus-specific manner. MALT1 (also known as PCASP1) serves a dual function within the assembled CBM complexes. By recruiting TRAF6, MALT1 acts as a molecular scaffold that initiates IκB kinase (IKK)/NF-κB and c-Jun N-terminal kinase (JNK)/AP-1 signaling. In parallel, proximity-induced dimerization of the paracaspase domain activates the MALT1 protease which exerts its function by cleaving a set of specific substrates. While complete MALT1 ablation leads to immune deficiency, selective destruction of either scaffolding or protease function provokes autoimmune inflammation. Thus, balanced MALT1-TRAF6 recruitment and MALT1 substrate cleavage are critical to maintain immune homeostasis and to promote optimal immune activation. Further, MALT1 protease activity drives the survival of aggressive lymphomas and other non-hematologic solid cancers. However, little is known about the relevance of the cleavage of individual substrates for the pathophysiological functions of MALT1. Unbiased serendipity, screening and computational predictions have identified and validated ~20 substrates, indicating that MALT1 targets a quite distinct set of proteins. Known substrates are involved in CBM auto-regulation (MALT1, BCL10 and CARD10), regulation of signaling and adhesion (A20, CYLD, HOIL-1 and Tensin-3), or transcription (RelB) and mRNA stability/translation (Regnase-1, Roquin-1/2 and N4BP1), indicating that MALT1 often targets multiple proteins involved in similar cellular processes. Here, we will summarize what is known about the fate and functions of individual MALT1 substrates and how their cleavage contributes to the biological functions of the MALT1 protease. We will outline what is needed to better connect critical pathophysiological roles of the MALT1 protease with the cleavage of distinct substrates.
Assuntos
Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Transdução de Sinais , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Humanos , Animais , Especificidade por Substrato , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteína 10 de Linfoma CCL de Células B/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteólise , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
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.
Assuntos
Proteína 10 de Linfoma CCL de Células B , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Domínios Proteicos , Multimerização Proteica , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteína 10 de Linfoma CCL de Células B/química , Proteína 10 de Linfoma CCL de Células B/genética , Humanos , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Caspases/metabolismo , Caspases/químicaRESUMO
B-cell lymphoma 10 (Bcl10) is a scaffolding protein that functions as an upstream regulator of NF-κB signaling by forming a complex with Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (Malt1) and CARD-coiled coil protein family. This study showed that Bcl10 was involved in type I interferon (IFN) expression in response to DNA virus infection and that Bcl10-deficient mice were more susceptible to Herpes simplex virus 1 (HSV-1) infection than control mice. Mechanistically, DNA virus infection can trigger Bcl10 recruitment to the STING-TBK1 complex, leading to Bcl10 phosphorylation by TBK1. The phosphorylated Bcl10 undergoes droplet-like condensation and forms oligomers, which induce TBK1 phosphorylation and translocation to the perinuclear region. The activated TBK1 phosphorylates IRF3, which induces the expression of type I IFNs. This study elucidates that Bcl10 induces an innate immune response by undergoing droplet-like condensation and participating in signalosome formation downstream of the cGAS-STING pathway.
Assuntos
Proteína 10 de Linfoma CCL de Células B , Imunidade Inata , Animais , Camundongos , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Imunidade Inata/fisiologia , NF-kappa B/metabolismo , FosforilaçãoRESUMO
The caspase activation and recruitment domain-coiled-coil (CARD-CC) family of proteins-CARD9, CARD10, CARD11, and CARD14-is collectively expressed across nearly all tissues of the body and is a crucial mediator of immunologic signaling as part of the CARD-B-cell lymphoma/leukemia 10-mucosa-associated lymphoid tissue lymphoma translocation protein 1 (CBM) complex. Dysfunction or dysregulation of CBM proteins has been linked to numerous clinical manifestations known as "CBM-opathies." The CBM-opathy spectrum encompasses diseases ranging from mucocutaneous fungal infections and psoriasis to combined immunodeficiency and lymphoproliferative diseases; however, there is accumulating evidence that the CARD-CC family members also contribute to the pathogenesis and progression of allergic inflammation and allergic diseases. Here, we review the 4 CARD-CC paralogs, as well as B-cell lymphoma/leukemia 10 and mucosa-associated lymphoid tissue lymphoma translocation protein 1, and their individual and collective roles in the pathogenesis and progression of allergic inflammation and 4 major allergic diseases (allergic asthma, atopic dermatitis, food allergy, and allergic rhinitis).
Assuntos
Leucemia , Linfoma de Zona Marginal Tipo Células B , Humanos , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Guanilato Ciclase , Transdução de Sinais , Inflamação , Proteínas Reguladoras de Apoptose/metabolismo , NF-kappa B/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Deep vein thrombosis (DVT) is the third most common cause of cardiovascular mortality. Several studies suggest that DVT occurs at the intersection of dysregulated inflammation and coagulation upon activation of inflammasome and secretion of interleukin 1ß (IL-1ß) in restricted venous flow conditions. Our recent studies showed a signaling adapter protein, Gab2 (Grb2-associated binder 2), plays a crucial role in propagating inflammatory signaling triggered by IL-1ß and other inflammatory mediators in endothelial cells. The present study shows that Gab2 facilitates the assembly of the CBM (CARMA3 [CARD recruited membrane-associated guanylate kinase protein 3]-BCL-10 [B-cell lymphoma 10]-MALT1 [mucosa-associated lymphoid tissue lymphoma translocation protein 1]) signalosome, which mediates the activation of Rho and NF-κB in endothelial cells. Gene silencing of Gab2 or MALT1, the effector signaling molecule in the CBM signalosome, or pharmacological inhibition of MALT1 with a specific inhibitor, mepazine, significantly reduced IL-1ß-induced Rho-dependent exocytosis of P-selectin and von Willebrand factor (VWF) and the subsequent adhesion of neutrophils to endothelial cells. MALT1 inhibition also reduced IL-1ß-induced NF-κB-dependent expression of tissue factor and vascular cell adhesion molecule 1. Consistent with the in vitro data, Gab2 deficiency or pharmacological inhibition of MALT1 suppressed the accumulation of monocytes and neutrophils at the injury site and attenuated venous thrombosis induced by the inferior vena cava ligation-induced stenosis or stasis in mice. Overall, our data reveal a previously unrecognized role of the Gab2-MALT1 axis in thromboinflammation. Targeting the Gab2-MALT1 axis with MALT1 inhibitors may become an effective strategy to treat DVT by suppressing thromboinflammation without inducing bleeding complications.
Assuntos
Trombose , Trombose Venosa , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Endoteliais/metabolismo , Guanilato Quinases/metabolismo , Inflamassomos/metabolismo , Inflamação , Mediadores da Inflamação , Interleucina-1beta/metabolismo , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , NF-kappa B/metabolismo , Selectina-P/metabolismo , Tromboinflamação , Tromboplastina/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Trombose Venosa/genética , Fator de von Willebrand/metabolismoRESUMO
CARD11 acts as a gatekeeper for adaptive immune responses after T cell or B cell antigen receptor (TCR/BCR) ligation on lymphocytes. PKCθ/ß-catalyzed phosphorylation of CARD11 promotes the assembly of the CARD11-BCL10-MALT1 (CBM) complex and lymphocyte activation. Here, we demonstrated that PKCθ/ß-dependent CARD11 phosphorylation also suppressed CARD11 functions in T or B cells. Through mass spectrometry-based proteomics analysis, we identified multiple constitutive and inducible CARD11 phosphorylation sites in T cells. We demonstrated that a single TCR- or BCR-inducible phosphorylation on Ser893 in the carboxyl terminus of CARD11 prevented the activation of the transcription factor NF-κB, the kinase JNK, and the protease MALT1. Moreover, CARD11 Ser893 phosphorylation sensitized BCR-addicted lymphoma cells to toxicity induced by Bruton's tyrosine kinase (BTK) inhibitors. Phosphorylation of Ser893 in CARD11 by PKCθ controlled the strength of CARD11 scaffolding by impairing the formation of the CBM complex. Thus, PKCθ simultaneously catalyzes both stimulatory and inhibitory CARD11 phosphorylation events, which shape the strength of CARD11 signaling in lymphocytes.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Serina , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Linfócitos B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , FosforilaçãoRESUMO
Regulatory T cells (Tregs) are a critical subset of CD4 T cells that modulate the immune response to prevent autoimmunity and chronic inflammation. CARD11, a signaling hub and scaffold protein that links antigen receptor engagement to activation of NF-κB and other downstream signaling pathways, is essential for the development and function of thymic Tregs. Mouse models with deficiencies in CARD11 and CARD11-associated signaling components generally have Treg defects, but some mouse models develop overt autoimmunity and inflammatory disease whereas others do not. Inhibition of CARD11 signaling in Tregs within the tumor microenvironment can potentially promote anti-tumor immunity. In this review, we summarize evidence for the involvement of CARD11 signaling in Treg development and function and discuss key unanswered questions and future research opportunities.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Linfócitos T Reguladores , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Camundongos , NF-kappa B/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is a multifunctional protein. MALT1 functions as an adaptor protein to assemble and recruit proteins such as B-cell lymphoma 10 (BCL10) and caspase-recruitment domain (CARD)-containing coiled-coil protein 11 (CARD11). Conversely it also acts as a paracaspase to cleave specified substrates. Because of its involvement in immunity, inflammation and cancer through its dual functions of scaffolding and catalytic activity, MALT1 is becoming a promising therapeutic target in B cell- and T cell-related diseases. There is growing evidence that the function of MALT1 is subtly modulated via post-translational modifications. This review summarized recent progress in relevant studies regarding the physiological and pathophysiological functions of MALT1, post-translational modifications of MALT1 and its role in B cell- and T cell- related diseases. In addition, the current available MALT1 inhibitors were also discussed.
Assuntos
Proteínas Adaptadoras de Sinalização CARD , Guanilato Ciclase , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Caspases/genética , Caspases/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Processamento de Proteína Pós-Traducional , Linfócitos T/metabolismoRESUMO
T cell activation initiates protective adaptive immunity, but counterbalancing mechanisms are critical to prevent overshooting responses and to maintain immune homeostasis. The CARD11-BCL10-MALT1 (CBM) complex bridges T cell receptor engagement to NF-κB signaling and MALT1 protease activation. Here, we show that ABIN-1 is modulating the suppressive function of A20 in T cells. Using quantitative mass spectrometry, we identified ABIN-1 as an interactor of the CBM signalosome in activated T cells. A20 and ABIN-1 counteract inducible activation of human primary CD4 and Jurkat T cells. While A20 overexpression is able to silence CBM complex-triggered NF-κB and MALT1 protease activation independent of ABIN-1, the negative regulatory function of ABIN-1 depends on A20. The suppressive function of A20 in T cells relies on ubiquitin binding through the C-terminal zinc finger (ZnF)4/7 motifs, but does not involve the deubiquitinating activity of the OTU domain. Our mechanistic studies reveal that the A20/ABIN-1 module is recruited to the CBM complex via A20 ZnF4/7 and that proteasomal degradation of A20 and ABIN-1 releases the CBM complex from the negative impact of both regulators. Ubiquitin binding to A20 ZnF4/7 promotes destructive K48-polyubiquitination to itself and to ABIN-1. Further, after prolonged T cell stimulation, ABIN-1 antagonizes MALT1-catalyzed cleavage of re-synthesized A20 and thereby diminishes sustained CBM complex signaling. Taken together, interdependent post-translational mechanisms are tightly controlling expression and activity of the A20/ABIN-1 silencing module and the cooperative action of both negative regulators is critical to balance CBM complex signaling and T cell activation.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Linfócitos T/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/fisiologia , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Células Cultivadas , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Células Jurkat , Ativação Linfocitária/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Ligação Proteica , Interferência de RNA/imunologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologiaRESUMO
Caspase recruitment domain and membrane-associated guanylate kinase-like protein 3 (CARMA3) is a scaffold protein widely expressed in non-hematopoietic cells. It is encoded by the caspase recruitment domain protein 10 (CARD10) gene. CARMA3 can form a CARMA3-BCL10-MALT1 complex by recruiting B cell lymphoma 10 (BCL10) and mucosa-âassociated lymphoid tissue lymphoma translocation protein 1 (MALT1), thereby activating nuclear factor-âκB (NF-κB), a key transcription factor that involves in various biological responses. CARMA3 mediates different receptors-dependent signaling pathways, including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). Inappropriate expression and activation of GPCRs and/or RTKs/CARMA3 signaling lead to the pathogenesis of human diseases. Emerging studies have reported that CARMA3 mediates the development of various types of cancers. Moreover, CARMA3 and its partners participate in human non-cancer diseases, including atherogenesis, abdominal aortic aneurysm, asthma, pulmonary fibrosis, liver fibrosis, insulin resistance, inflammatory bowel disease, and psoriasis. Here we provide a review on its structure, regulation, and molecular function, and further highlight recent findings in human non-cancerous diseases, which will provide a novel therapeutic target.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Sinalização CARD , Neoplasias , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/terapia , Neoplasias/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
B cell lymphoma/leukemia 10 (BCL10) is an important member of the caspase recruitment domain-containing (CARD) protein family, which plays crucial roles in mediating the host inflammatory response. In the present study, a BCL10 homologue was identified from Pacific oyster Crassostrea gigas (designed as CgBCL10). The full length cDNA of CgBCL10 was of 897 bp with an open reading frame of 522 bp encoding a polypeptide of 174 amino acids containing a classical CARD domain. The deduced amino acid sequence of CgBCL10 shared low similarity with the previously identified BCL10s from other species. In the phylogenetic tree, CgBCL10 was firstly clustered with CvBCL10 from Crassostrea virginica and then assigned into the branch of invertebrate BCL10s. The mRNA transcripts of CgBCL10 were highly expressed in gonad, gill, adductor muscle, and haemocytes. After Vibrio splendidus stimulation, the mRNA expression level of CgBCL10 in haemocytes increased significantly (p < 0.01) at 24, 72 and 96 h. In CgBCL10-RNAi oysters, the phosphorylation level of mitogen-activated protein kinases (MAPKs), nuclear translocation of NF-κB/Rel and activator protein-1 (AP-1) in haemocytes were inhibited, and the mRNA expressions of inflammatory cytokines including CgIL17-1, CgIL17-2, CgIL17-3, CgIL17-6 and CgTNF all decreased significantly (p < 0.01) at 12 h after V. splendidus stimulation. These results suggested that CgBCL10 regulated the expression of inflammatory cytokines by activating MAPK kinase, and nuclear translocation of NF-κB/Rel and AP-1 to defense pathogen.
Assuntos
Proteína 10 de Linfoma CCL de Células B , Crassostrea , Citocinas , NF-kappa B , Transdução de Sinais , Animais , Proteína 10 de Linfoma CCL de Células B/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Crassostrea/genética , Crassostrea/imunologia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Hemócitos/metabolismo , Imunidade Inata , Proteínas Quinases Ativadas por Mitógeno , NF-kappa B/genética , NF-kappa B/metabolismo , Filogenia , RNA Mensageiro , Fator de Transcrição AP-1Assuntos
Carcinoma de Células Acinares/patologia , Neoplasias Gástricas/patologia , Adenocarcinoma de Pulmão/patologia , Idoso , Proteína 10 de Linfoma CCL de Células B/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Masculino , Segunda Neoplasia Primária/patologiaRESUMO
Transmissible gastroenteritis (TGE) is an acute viral disease and characterized as severe acute inflammation response that leads to diarrhea, vomiting, and high lethality of piglets. Transmissible gastroenteritis virus (TGEV), a member of coronavirus, is the pathogen of TGE. We previously found NF-κB pathway was activated and 65 miRNAs were changed in response to inflammation caused by TGEV in cell line porcine intestinal epithelial cells-jejunum 2 (IPEC-J2). Bioinformatics results showed that these altered miRNAs were relevant to inflammation. In this study, the candidate targets of differentially expressed (DE) miRNAs were predicted and analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Based on the results of KEGG analysis, miR-885-3p might participate in regulating activation of NF-κB pathway and TNF pathway. To study the function of miR-885-3p, miR-885-3p mimics and inhibitors were artificially synthesized and respectively used for overexpression and silence of miR-885-3p in cells. Our results showed that miR-885-3p inhibited NF-κB signaling pathway and tumor necrosis factor-α (TNF-α) production. B-cell CLL/lymphoma 10 (Bcl-10) was identified as the target of miR-885-3p, and promoted NF-κB pathway activation and TNF-α production. It was found that TGEV open reading frame 3b (TGEV-ORF3b) suppressed Bcl-10 expression, activation of NF-κB pathway, and TNF-α production by uniquely up-regulated miR-885-3p expression. Overall, the results indicated that TGEV-ORF3b counteracted NF-κB pathway and TNF-α via regulating miR-885-3p and Bcl-10.
Assuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Gastroenterite Suína Transmissível/virologia , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Vírus da Gastroenterite Transmissível/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Proteína 10 de Linfoma CCL de Células B/genética , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação da Expressão Gênica , Mucosa Intestinal/citologia , MicroRNAs/genética , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Suínos , Regulação para Cima , Proteínas ViraisRESUMO
The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes.
Assuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Algoritmos , Animais , Autofagossomos/imunologia , Autofagossomos/metabolismo , Proteína 10 de Linfoma CCL de Células B/química , Proteína 10 de Linfoma CCL de Células B/genética , Linhagem Celular , Biologia Computacional , Simulação por Computador , Camundongos , Modelos Biológicos , Método de Monte Carlo , Polimerização , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
Deubiquitinases (DUBs) regulate diverse biological processes and represent a novel class of drug targets. However, the biological function of only a small fraction of DUBs, especially in adaptive immune response regulation, is well-defined. In this study, we identified DUB ubiquitin-specific peptidase 12 (USP12) as a critical regulator of CD4+ T cell activation. USP12 plays an intrinsic role in promoting the CD4+ T cell phenotype, including differentiation, activation, and proliferation. Although USP12-deficient CD4+ T cells protected mice from autoimmune diseases, the immune response against bacterial infection was subdued. USP12 stabilized B cell lymphoma/leukemia 10 (BCL10) by deubiquitinating, and thereby activated the NF-κB signaling pathway. Interestingly, this USP12 regulatory mechanism was identified in CD4+ T cells, but not in CD8+ T cells. Our study results showed that USP12 activated CD4+ T cell signaling, and targeting USP12 might help develop therapeutic interventions for treating inflammatory diseases or pathogen infections.
Assuntos
Proteína 10 de Linfoma CCL de Células B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Enzimas Desubiquitinantes/metabolismo , Linfócitos T/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Proliferação de Células , CamundongosRESUMO
BACKGROUND: Germline pathogenic variants impairing the caspase recruitment domain family member 11 (CARD11)-B cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) (CBM) complex are associated with diverse human diseases including combined immunodeficiency (CID), atopy, and lymphoproliferation. However, the impact of CARD11 deficiency on human B-cell development, signaling, and function is incompletely understood. OBJECTIVES: This study sought to determine the cellular, immunological, and biochemical basis of disease for 2 unrelated patients who presented with profound CID associated with viral and fungal respiratory infections, interstitial lung disease, and severe colitis. METHODS: Patients underwent next-generation sequencing, immunophenotyping by flow cytometry, signaling assays by immunoblot, and transcriptome profiling by RNA-sequencing. RESULTS: Both patients carried identical novel pathogenic biallelic loss-of-function variants in CARD11 (c.2509C>T; p.Arg837∗) leading to undetectable protein expression. This variant prevented CBM complex formation, severely impairing the activation of nuclear factor-κB, c-Jun N-terminal kinase, and MALT1 paracaspase activity in B and T cells. This functional defect resulted in a developmental block in B cells at the naive and type 1 transitional B-cell stage and impaired circulating T follicular helper cell (cTFH) development, which was associated with impaired antibody responses and absent germinal center structures on lymph node histology. Transcriptomics indicated that CARD11-dependent signaling is essential for immune signaling pathways involved in the development of these cells. Both patients underwent hematopoietic stem cell transplantations, which led to functional normalization. CONCLUSIONS: Complete human CARD11 deficiency causes profound CID by impairing naive/type 1 B-cell and cTFH cell development and abolishing activation of MALT1 paracaspase, NF-κB, and JNK activity. Hematopoietic stem cell transplantation functionally restores impaired signaling pathways.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Centro Germinativo/imunologia , Guanilato Ciclase/genética , Transplante de Células-Tronco Hematopoéticas , Mutação/genética , Células Precursoras de Linfócitos B/imunologia , Doenças da Imunodeficiência Primária/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Adolescente , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Criança , Perfilação da Expressão Gênica , Guanilato Ciclase/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Lactente , Masculino , NF-kappa B/metabolismo , Doenças da Imunodeficiência Primária/terapia , Transdução de SinaisRESUMO
Signal transduction typically displays a so-called bow-tie topology: Multiple receptors lead to multiple cellular responses but the signals all pass through a narrow waist of central signaling nodes. One such signaling node for several inflammatory and oncogenic signaling pathways is the CARD-CC/BCL10/MALT1 (CBM) complexes, which get activated by protein kinase C (PKC)-mediated phosphorylation of the caspase activation and recruitment domain (CARD)-coiled-coil domain (CC) component. In humans, there are four CARD-CC family proteins (CARD9, CARD10, CARD11, and CARD14) and 9 true PKC isozymes (α to ι). At this moment, less than a handful of PKC::CARD-CC relationships are known. In order to explore the biologically relevant combinatorial space out of all 36 potential permutations in this two-component signaling event, we made use of CARD10-deficient human embryonic kidney 293T cells for subsequent pairwise cotransfections of all CARD-CC family members and all activated PKCs. Upon analysis of NF-κB-dependent reporter gene expression, we could define specific PKC::CARD-CC relationships. Surprisingly, as many as 21 PKC::CARD-CC functional combinations were identified. CARD10 was responsive to most PKCs, while CARD14 was mainly activated by PKCδ. The CARD11 activation profile was most similar to that of CARD9. We also discovered the existence of mixed protein complexes between different CARD-CC proteins, which was shown to influence their PKC response profile. Finally, multiple PKCs were found to use a common phosphorylation site to activate CARD9, while additional phosphorylation sites contribute to CARD14 activation. Together, these data reveal the combinatorial space of PKC::CARD-CC signal transduction nodes, which will be valuable for future studies on the regulation of CBM signaling.
Assuntos
Proteína 10 de Linfoma CCL de Células B/genética , Proteínas Adaptadoras de Sinalização CARD/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , NF-kappa B/genética , Proteína Quinase C/genética , Sequência de Aminoácidos , Animais , Proteína 10 de Linfoma CCL de Células B/metabolismo , Sítios de Ligação , Proteínas Adaptadoras de Sinalização CARD/classificação , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/metabolismo , Fosforilação , Filogenia , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/classificação , Proteína Quinase C/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TransfecçãoRESUMO
The LUBAC ubiquitin ligase complex, composed of the HOIP, HOIL-1L, and SHARPIN subunits, stimulates the canonical nuclear factor-κB (NF-κB) activation pathways through its Met1-linked linear ubiquitination activity. Here we performed cellular and mathematical modeling analyses of the LUBAC involvement in the T cell receptor (TCR)-mediated NF-κB activation pathway, using the Jurkat human T cell line. LUBAC is indispensable for TCR-induced NF-κB and T cell activation, and transiently associates with and linearly ubiquitinates the CARMA1-BCL10-MALT1 (CBM) complex, through the catalytic HOIP subunit. In contrast, the linear ubiquitination of NEMO, a substrate of the TNF-α-induced canonical NF-κB activation pathway, was limited during the TCR pathway. Among deubiquitinases, OTULIN, but not CYLD, plays a major role in downregulating LUBAC-mediated TCR signaling. Mathematical modeling indicated that linear ubiquitination of the CBM complex accelerates the activation of IκB kinase (IKK), as compared with the activity induced by linear ubiquitination of NEMO alone. Moreover, simulations of the sequential linear ubiquitination of the CBM complex suggested that the allosteric regulation of linear (de)ubiquitination of CBM subunits is controlled by the ubiquitin-linkage lengths. These results indicated that, unlike the TNF-α-induced NF-κB activation pathway, the TCR-mediated NF-κB activation in T lymphocytes has a characteristic mechanism to induce LUBAC-mediated NF-κB activation.
Assuntos
Ativação Linfocitária , Modelos Imunológicos , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Anticorpos/farmacologia , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Simulação por Computador , Enzima Desubiquitinante CYLD/genética , Enzima Desubiquitinante CYLD/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Guanilato Ciclase/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Complexos Multienzimáticos , NF-kappa B/genética , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/genéticaRESUMO
Rationale: Constitutive activation of the NF-κB signalling pathway plays a pivotal role in the pathogenesis of activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCLs), the most aggressive and chemoresistant form of DLBCL. In ABC-DLBCLs, the CARMA1-BCL10 (CB) complex forms a filamentous structure and functions as a supramolecular organizing centre (CB-SMOC) that is required for constitutive NF-κB activation, making it an attractive drug target for ABC-DLBCL treatment. However, a pharmaceutical approach targeting CB-SMOC has been lacking. Here, we developed Bcl10 peptide inhibitors (BPIs) that specifically target the BCL10 filamentation process. Methods: Electron microscopy and immunofluorescence imaging were used to visualize the effect of the BPIs on the BCL10 filamentation process. The cytotoxicity of the tested BPIs was evaluated in DLBCL cell lines according to cell proliferation assays. Different in vitro experiments (pharmacokinetics, immunoprecipitation, western blotting, annexin V and PI staining) were conducted to determine the functional mechanisms of the BPIs. The in vivo therapeutic effect of the BPIs was examined in different xenograft DLBCL mouse models. Finally, Ki67 and TUNEL staining and histopathology analysis were used to evaluate the antineoplastic mechanisms and systemic toxicity of the BPIs. Results: We showed that these BPIs can effectively disrupt the BCL10 filamentation process, destabilize BCL10 and suppress NF-κB signalling in ABC-DLBCL cells. By examining a panel of DLBCL cell lines, we found that these BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL cells by inducing apoptosis and cell cycle arrest. Moreover, by converting the BPIs to acquire a D-retro inverso (DRI) configuration, we developed DRI-BPIs with significantly improved intracellular stability and unimpaired BPI activity. These DRI-BPIs selectively repressed the growth of CB-SMOC-dependent DLBCL tumors in mouse xenograft models without eliciting discernible adverse effects. Conclusion: We developed novel BPIs to target the BCL10 filamentation process and demonstrated that targeting BCL10 by BPIs is a potentially safe and effective pharmaceutical approach for the treatment of ABC-DLBCL and other CB-SMOC-dependent malignancies.