RESUMO
Ligation of the B cell antigen receptor (BCR) initiates humoral immunity. However, BCR signaling without appropriate co-stimulation commits B cells to death rather than to differentiation into immune effector cells. How BCR activation depletes potentially autoreactive B cells while simultaneously primes for receiving rescue and differentiation signals from cognate T lymphocytes remains unknown. Here, we use a mass spectrometry-based proteomic approach to identify cytosolic/nuclear shuttling elements and uncover transcription factor EB (TFEB) as a central BCR-controlled rheostat that drives activation-induced apoptosis, and concurrently promotes the reception of co-stimulatory rescue signals by supporting B cell migration and antigen presentation. CD40 co-stimulation prevents TFEB-driven cell death, while enhancing and prolonging TFEB's nuclear residency, which hallmarks antigenic experience also of memory B cells. In mice, TFEB shapes the transcriptional landscape of germinal center B cells. Within the germinal center, TFEB facilitates the dark zone entry of light-zone-residing centrocytes through regulation of chemokine receptors and, by balancing the expression of Bcl-2/BH3-only family members, integrates antigen-induced apoptosis with T cell-provided CD40 survival signals. Thus, TFEB reprograms antigen-primed germinal center B cells for cell fate decisions.
Assuntos
Apoptose , Linfócitos B , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Antígenos CD40 , Centro Germinativo , Receptores de Antígenos de Linfócitos B , Animais , Centro Germinativo/imunologia , Centro Germinativo/citologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Camundongos , Antígenos CD40/metabolismo , Antígenos CD40/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Camundongos Endogâmicos C57BL , Ativação Linfocitária/imunologia , Diferenciação Celular/imunologia , Transdução de Sinais , Apresentação de Antígeno/imunologiaRESUMO
Biomolecular condensates play a major role in cell compartmentalization, besides membrane-enclosed organelles. The multivalent SLP65 and CIN85 proteins are proximal B-cell antigen receptor (BCR) signal effectors and critical for proper immune responses. In association with intracellular vesicles, the two effector proteins form phase separated condensates prior to antigen stimulation, thereby preparing B lymphocytes for rapid and effective activation upon BCR ligation. Within this tripartite system, 6 proline-rich motifs (PRMs) of SLP65 interact promiscuously with 3 SH3 domains of the CIN85 monomer, establishing 18 individual SH3-PRM interactions whose individual dissociation constants we determined. Based on these 18 dissociation constants, we measured the phase-separation properties of the natural SLP65/CIN85 system as well as designer constructs that emphasize the strongest SH3/PRM interactions. By modeling these various SLP65/CIN85 constructs with the program LASSI (LAttice simulation engine for Sticker and Spacer Interactions), we reproduced the observed phase-separation properties. In addition, LASSI revealed a deviation in the experimental measurement, which was independently identified as a previously unknown intramolecular interaction. Thus, thermodynamic properties of the individual PRM/SH3 interactions allow us to model the phase-separation behavior of the SLP65/CIN85 system faithfully.
RESUMO
Signal transduction by the ligated B cell antigen receptor (BCR) depends on the preorganization of its intracellular components, such as the effector proteins SLP65 and CIN85 within phase-separated condensates. These liquid-like condensates are based on the interaction between three Src homology 3 (SH3) domains and the corresponding proline-rich recognition motifs (PRM) in CIN85 and SLP65, respectively. However, detailed information on the protein conformation and how it impacts the capability of SLP65/CIN85 condensates to orchestrate BCR signal transduction is still lacking. This study identifies a hitherto unknown intramolecular SH3:PRM interaction between the C-terminal SH3 domain (SH3C) of CIN85 and an adjacent PRM. We used high-resolution nuclear magnetic resonance (NMR) experiments to study the flexible linker region containing the PRM and determined the extent of the interaction in multidomain constructs of the protein. Moreover, we observed that the phosphorylation of a serine residue located in the immediate vicinity of the PRM regulates this intramolecular interaction. This allows for a dynamic modulation of CIN85's valency toward SLP65. B cell culture experiments further revealed that the PRM/SH3C interaction is crucial for maintaining the physiological level of SLP65/CIN85 condensate formation, activation-induced membrane recruitment of CIN85, and subsequent mobilization of Ca2+. Our findings therefore suggest that the intramolecular interaction with the adjacent disordered linker is effective in modulating CIN85's valency both in vitro and in vivo. This therefore constitutes a powerful way for the modulation of SLP65/CIN85 condensate formation and subsequent B cell signaling processes within the cell.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/química , Transdução de Sinais/fisiologia , Domínios de Homologia de src , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Ligação ProteicaRESUMO
B cell maturation in germinal centers (GCs) depends on cognate interactions between the T and B cells. Upon interaction with CD40 ligand (CD40L) on T cells, CD40 delivers co-stimulatory signals alongside B cell antigen receptor (BCR) signaling to regulate affinity maturation and antibody class-switch during GC reaction. Mutations in CD40L disrupt interactions with CD40, which lead to abnormal antibody responses in immune deficiencies known as X-linked Hyper IgM syndrome (X-HIgM). Assuming that physical interactions between highly mobile T and B cells generate mechanical forces on CD40-CD40L bonds, we set out to study the B cell mechanobiology mediated by CD40-CD40L interaction. Using a suite of biophysical assays we find that CD40 forms catch bond with CD40L where the bond lasts longer at larger forces, B cells exert tension on CD40-CD40L bonds, and force enhances CD40 signaling and antibody class-switch. Significantly, X-HIgM CD40L mutations impair catch bond formation, suppress endogenous tension, and reduce force-enhanced CD40 signaling, leading to deficiencies in antibody class switch. Our findings highlight the critical role of mechanotransduction in CD40 function and provide insights into the molecular mechanisms underlying X-HIgM syndrome.
RESUMO
IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.
Assuntos
Receptores de Antígenos de Linfócitos B , Receptores Fc , Animais , Camundongos , Imunoglobulina M , Receptores Fc/metabolismo , Isoformas de ProteínasRESUMO
The altered wiring of signaling pathways downstream of antigen receptors of T and B cells contributes to the dysregulation of the adaptive immune system, potentially causing immunodeficiency and autoimmunity. In humans, the investigation of such complex systems benefits from nature's experiments in patients with genetically defined primary immunodeficiencies. Disturbed B-cell receptor (BCR) signaling in a subgroup of common variable immunodeficiency (CVID) patients with immune dysregulation and expanded T-bethighCD21low B cells in peripheral blood has been previously reported. Here, we investigate PI3K signaling and its targets as crucial regulators of survival, proliferation and metabolism by intracellular flow cytometry, imaging flow cytometry and RNAseq. We observed increased basal but disturbed BCR-induced PI3K signaling, especially in T-bethighCD21low B cells from CVID patients, translating into impaired activation of crucial downstream molecules and affecting proliferation, survival and the metabolic profile. In contrast to CVID, increased basal activity of PI3K in patients with a gain-of-function mutation in PIK3CD and activated PI3K delta syndrome (APDS) did not result in impaired BCR-induced AKT-mTOR-S6 phosphorylation, highlighting that signaling defects in B cells in CVID and APDS patients are fundamentally different and that assessing responses to BCR stimulation is an appropriate confirmative diagnostic test for APDS. The active PI3K signaling in vivo may render autoreactive T-bethighCD21low B cells in CVID at the same time to be more sensitive to mTOR or PI3K inhibition.
Assuntos
Imunodeficiência de Variável Comum , Fosfatidilinositol 3-Quinases , Linfócitos B , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Doenças da Imunodeficiência Primária , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.
RESUMO
Circulating autoantibodies (AB) of different immunoglobulin classes (IgM, IgA, and IgG), directed against the obligatory N-methyl-D-aspartate-receptor subunit NR1 (NMDAR1-AB), belong to the mammalian autoimmune repertoire, and appear with age-dependently high seroprevalence across health and disease. Upon access to the brain, they can exert NMDAR-antagonistic/ketamine-like actions. Still unanswered key questions, addressed here, are conditions of NMDAR1-AB formation/boosting, intraindividual persistence/course in serum over time, and (patho)physiological significance of NMDAR1-AB in modulating neuropsychiatric phenotypes. We demonstrate in a translational fashion from mouse to human that (1) serum NMDAR1-AB fluctuate upon long-term observation, independent of blood-brain barrier (BBB) perturbation; (2) a standardized small brain lesion in juvenile mice leads to increased NMDAR1-AB seroprevalence (IgM + IgG), together with enhanced Ig-class diversity; (3) CTLA4 (immune-checkpoint) genotypes, previously found associated with autoimmune disease, predispose to serum NMDAR1-AB in humans; (4) finally, pursuing our prior findings of an early increase in NMDAR1-AB seroprevalence in human migrants, which implicated chronic life stress as inducer, we independently replicate these results with prospectively recruited refugee minors. Most importantly, we here provide the first experimental evidence in mice of chronic life stress promoting serum NMDAR1-AB (IgA). Strikingly, stress-induced depressive-like behavior in mice and depression/anxiety in humans are reduced in NMDAR1-AB carriers with compromised BBB where NMDAR1-AB can readily reach the brain. To conclude, NMDAR1-AB may have a role as endogenous NMDAR antagonists, formed or boosted under various circumstances, ranging from genetic predisposition to, e.g., tumors, infection, brain injury, and stress, altogether increasing over lifetime, and exerting a spectrum of possible effects, also including beneficial functions.
Assuntos
Autoanticorpos , Lesões Encefálicas , Animais , Barreira Hematoencefálica , Camundongos , Receptores de N-Metil-D-Aspartato , Estudos Soroepidemiológicos , Estresse PsicológicoRESUMO
BACKGROUND: Allergic inflammation is driven by IgE-producing plasma cells (PCs), which are required for IgE-mediated activation of mast cells and basophils. Repeated antigen encounter elicits a memory IgE response with elevated serum IgE titers and accumulation of IgE-producing PCs. However, the cellular compartment and molecular signals that underlie the immunologic memory of IgE responses remain unclear. OBJECTIVE: With this study we aimed at clarifying whether inactivation of the cytoplasmic immunoglobulin tail tyrosine (ITT) motif in transmembrane IgE (mIgE) impairs the memory IgE response in mice. METHODS: We generated mice with an inactivated mIgE-ITT motif and analyzed serum IgE levels as well as the generation of IgE-producing germinal center B cells and PCs subsequent to primary and secondary infection with helminths. In vitro cultures were used to study the mIgE-ITT-controlled expression of mIgE on the surface of PCs. Systemic mast cell activation was determined by serum Mcpt1 ELISA in response to ovalbumin challenge. RESULTS: mIgE-ITT-mutant mice showed an impaired memory IgE response subsequent to helminth infection. Furthermore, sensitization and challenge of mIgE-ITT-mutant mice with ovalbumin resulted in diminished serum IgE titers and reduced mast cell activation. The mIgE-ITT motif was required for optimal cell surface expression of mIgE B-cell antigen receptors but not for intracellular IgE expression in PCs. CONCLUSION: These results indicate that the mIgE B-cell antigen receptor plays a critical role in establishing or maintaining the population of IgE-producing PCs during memory IgE responses.
Assuntos
Imunoglobulina E/imunologia , Memória Imunológica , Plasmócitos/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Tricostrongiloidíase/imunologia , Alérgenos/imunologia , Animais , Quimases/imunologia , Feminino , Masculino , Mastócitos/imunologia , Camundongos Transgênicos , Ovalbumina/imunologia , TrichostrongyloideaRESUMO
Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Microscopia Crioeletrônica , Proteínas Intrinsicamente Desordenadas , Espectroscopia de Ressonância Magnética , Ligação Proteica , Domínios Proteicos , Transdução de Sinais/fisiologia , Domínios de Homologia de srcRESUMO
In contrast to other B-cell antigen receptor (BCR) classes, the function of IgD BCR on mature B cells remains largely elusive as mature B cells co-express IgM, which is sufficient for development, survival, and activation of B cells. Here, we show that IgD expression is regulated by the forkhead box transcription factor FoxO1, thereby shifting the responsiveness of mature B cells towards recognition of multivalent antigen. FoxO1 is repressed by phosphoinositide 3-kinase (PI3K) signaling and requires the lipid phosphatase Pten for its activation. Consequently, Pten-deficient B cells expressing knock-ins for BCR heavy and light chain genes are unable to upregulate IgD. Furthermore, in the presence of autoantigen, Pten-deficient B cells cannot eliminate the autoreactive BCR specificity by secondary light chain gene recombination. Instead, Pten-deficient B cells downregulate BCR expression and become unresponsive to further BCR-mediated stimulation. Notably, we observed a delayed germinal center (GC) reaction by IgD-deficient B cells after immunization with trinitrophenyl-ovalbumin (TNP-Ova), a commonly used antigen for T-cell-dependent antibody responses. Together, our data suggest that the activation of IgD expression by Pten/FoxO1 results in mature B cells that are selectively responsive to multivalent antigen and are capable of initiating rapid GC reactions and T-cell-dependent antibody responses.
Assuntos
Linfócitos B/fisiologia , Centro Germinativo/fisiologia , Imunoglobulina D/genética , PTEN Fosfo-Hidrolase/fisiologia , Receptores de Antígenos de Linfócitos B/genética , Animais , Células Cultivadas , Proteína Forkhead Box O1/fisiologia , Regulação da Expressão Gênica/imunologia , Centro Germinativo/metabolismo , Imunoglobulina D/imunologia , Imunoglobulina D/metabolismo , Camundongos , Camundongos Transgênicos , PTEN Fosfo-Hidrolase/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
The generation of memory B cells (MBCs) that have undergone immunoglobulin class switching from IgM, which dominates primary antibody responses, to other immunoglobulin isoforms is a hallmark of immune memory. Hence, humoral immunological memory is characterized by the presence of serum immunoglobulins of IgG subtypes known as the γ-globulin fraction of blood plasma proteins. These antibodies reflect the antigen experience of B lymphocytes and their repeated triggering. In fact, efficient protection against a previously encountered pathogen is critically linked to the production of pathogen-specific IgG molecules even in those cases where the primary immune response required cellular immunity, for example, T cell-mediated clearance of intracellular pathogens such as viruses. Besides IgG, also IgA and IgE can provide humoral immunity depending on the microbe's nature and infection route. The molecular mechanisms underlying the preponderance of switched immunoglobulin isotypes during memory antibody responses are a matter of active and controversial debate. Here, we summarize the phenotypic characteristics of distinct MBC subpopulations and discuss the decisive roles of different B cell antigen receptor isotypes for the functional traits of class-switched B cell populations.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Imunidade Humoral , Memória Imunológica , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Formação de Anticorpos/imunologia , Antígenos/imunologia , Antígenos/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Humanos , Imunidade Humoral/genética , Imunidade nas Mucosas/genética , Imunidade nas Mucosas/imunologia , Imunoglobulina A/genética , Imunoglobulina A/imunologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Memória Imunológica/genética , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos B/genética , Transdução de SinaisRESUMO
Ubiquitously expressed Cbl-interacting protein of 85 kD (CIN85) is a multifunctional adapter molecule supposed to regulate numerous cellular processes that are critical for housekeeping as well as cell type-specific functions. However, limited information exists about the in vivo roles of CIN85, because only conditional mouse mutants with cell type-specific ablation of distinct CIN85 isoforms in brain and B lymphocytes have been generated so far. No information is available about the roles of CIN85 in humans. Here, we report on primary antibody deficiency in patients harboring a germline deletion within the CIN85 gene on the X chromosome. In the absence of CIN85, all immune cell compartments developed normally, but B lymphocytes showed intrinsic defects in distinct effector pathways of the B cell antigen receptor, most notably NF-κB activation and up-regulation of CD86 expression on the cell surface. These results reveal nonredundant functions of CIN85 for humoral immune responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Anticorpos/metabolismo , Cromossomos Humanos X/genética , Deleção de Genes , Células Germinativas/metabolismo , Síndromes de Imunodeficiência/genética , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Linfócitos B/imunologia , Cálcio/metabolismo , Humanos , Imunoglobulina G/sangue , Síndromes de Imunodeficiência/sangue , Imunofenotipagem , Ativação Linfocitária/imunologia , Masculino , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Irmãos , Transdução de SinaisRESUMO
Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH i-1, NH i) or flanking a proline at position i-1 (NH i-2, NH i). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of â2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Amidas/química , Ressonância Magnética Nuclear Biomolecular , Prolina/química , alfa-Sinucleína/química , HumanosRESUMO
The B cell antigen receptor (BCR) employs enzymatically inactive adaptor proteins to facilitate activation of intracellular signaling pathways. In animal model systems, adaptor proteins of the growth factor receptor-bound 2 (Grb2) family have been shown to serve critical functions in lymphocytes. However, the roles of Grb2 and the Grb2-related adaptor protein (GRAP) in human B lymphocytes remain unclear. Using TALEN-mediated gene targeting, we show that in human B cells Grb2 and GRAP amplify signaling by the immunoglobulin tail tyrosine (ITT) motif of mIgE-containing BCRs and furthermore connect immunoreceptor tyrosine-based activation motif (ITAM) signaling to activation of the Ras-controlled Erk MAP kinase pathway. In contrast to mouse B cells, BCR-induced activation of Erk in human B cells is largely independent of phospholipase C-É£ activity and diacylglycerol-responsive members of Ras guanine nucleotide releasing proteins. Together, our results demonstrate that Grb2 family adaptors are critical regulators of ITAM and ITT signaling in naïve and IgE-switched human B cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Proteína Adaptadora GRB2/metabolismo , Sistema de Sinalização das MAP Quinases , Receptores de Antígenos de Linfócitos B/metabolismo , Linhagem Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Fosfolipases Tipo C/metabolismoRESUMO
Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species.
Assuntos
Linfócitos B/imunologia , Imunoglobulina E/química , Receptores de Antígenos de Linfócitos B/química , Processamento Alternativo , Sequência de Aminoácidos , Animais , Apoptose/imunologia , Linfócitos B/citologia , Linhagem Celular Tumoral , Retículo Endoplasmático/imunologia , Evolução Molecular , Espaço Extracelular/imunologia , Humanos , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Primatas/genética , Primatas/imunologia , Domínios Proteicos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The nonreceptor tyrosine kinase Syk, a central regulator of immune cell differentiation and activation, is a promising drug target for treatment of leukemia and allergic and inflammatory diseases. The clinical failure of Syk inhibitors underscores the importance of understanding the regulation of Syk function and activity. A series of previous studies emphasized the importance of three C-terminal tyrosines in Syk for kinase activity regulation, as docking sites for downstream effector molecules, and for Ca2+ mobilization. Here, we investigated the roles of these C-terminal tyrosines in the mouse. Surprisingly, expression of a triple tyrosine-to-phenylalanine human Syk mutant, SYK(Y3F), was not associated with discernible signaling defects either in reconstituted DT40 cells or in B or mast cells from mice expressing SYK(Y3F) instead of wild-type Syk. Remarkably, lymphocyte differentiation, calcium mobilization, and 2,4,6-trinitrophenyl (TNP)-specific immune responses were unperturbed in SYK(Y3F) mice. These results emphasize the capacity of immune cells to compensate for specific molecular defects, likely using redundant intermolecular interactions, and highlight the importance of in vivo analyses for understanding cellular signaling mechanisms.
Assuntos
Linfócitos B/metabolismo , Mastócitos/metabolismo , Mutação , Quinase Syk/genética , Quinase Syk/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular , Linhagem Celular , Técnicas de Introdução de Genes , Humanos , Camundongos , Fenilalanina/genética , Transdução de Sinais , Quinase Syk/química , Tirosina/genéticaRESUMO
Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.
Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas Nucleares/metabolismo , Linfócitos T/metabolismo , Humanos , Células Jurkat , Espectrometria de Massas , Fatores de Transcrição NFATC/genética , Proteínas Nucleares/genéticaRESUMO
The SH2 domain-containing inositol 5'-phosphatase (SHIP) plays a key role in preventing autoimmune phenomena by limiting antigen-mediated B cell activation. SHIP function is thought to require the dual engagement of the BCR and negative regulatory coreceptors as only the latter appear capable of recruiting SHIP from the cytosol to the plasma membrane by the virtue of phosphorylated immunoreceptor tyrosine-based inhibitory motifs. Here, we demonstrate a coreceptor-independent membrane recruitment and function of SHIP in B cells. In the absence of coreceptor ligation, SHIP translocates to sites of BCR activation through a concerted action of the protein adaptor unit Dok-3/Grb2 and phosphorylated BCR signaling components. Our data reveal auto-inhibitory SHIP activation by the activated BCR and suggest an unexpected negative-regulatory capacity of immunoreceptor tyrosine-based activation motifs in Igα and Igß.