RESUMO
Thromboembolic events are common in patients with essential thrombocythemia (ET). However, the pathophysiological mechanisms underlying the increased thrombotic risk remain to be determined. Here, we perform the first phenotypical characterization of platelet expression using single-cell mass cytometry in six ET patients and six age- and sex-matched healthy individuals. A large panel of 18 transmembrane regulators of platelet function and activation were analyzed, at baseline and after ex-vivo stimulation with thrombin receptor-activating peptide (TRAP). We detected a significant overexpression of the activation marker CD62P (p-Selectin) (p = .049) and the collagen receptor GPVI (p = .044) in non-stimulated ET platelets. In contrast, ET platelets had a lower expression of the integrin subunits of the fibrinogen receptor GPIIb/IIIa CD41 (p = .036) and CD61 (p = .044) and of the von Willebrand factor receptor CD42b (p = .044). Using the FlowSOM algorithm, we identified 2 subclusters of ET platelets with a prothrombotic expression profile, one of them (cluster 3) significantly overrepresented in ET (22.13% of the total platelets in ET, 2.94% in controls, p = .035). Platelet counts were significantly increased in ET compared to controls (p = .0123). In ET, MPV inversely correlated with platelet count (r=-0.96). These data highlight the prothrombotic phenotype of ET and postulate GPVI as a potential target to prevent thrombosis in these patients.
Essential thrombocythemia (ET) is a rare disease characterized by an increased number of platelets in the blood. As a complication, many of these patients develop a blood clot, which can be life-threatening. So far, the reason behind the higher risk of blood clots is unclear. In this study, we analyzed platelet surface markers that play a critical role in platelet function and platelet activation using a modern technology called mass cytometry. For this purpose, blood samples from 6 patients with ET and 6 healthy control individuals were analyzed. We found significant differences between ET platelets and healthy platelets. ET platelets had higher expression levels of p-Selectin (CD62P), a key marker of platelet activation, and of the collagen receptor GPVI, which is important for clot formation. These results may be driven by a specific platelet subcluster overrepresented in ET. Other surface markers, such as the fibrinogen receptor GPIIb/IIIa CD41, CD61, and the von Willebrand factor receptor CD42b, were lower expressed in ET platelets. When ET platelets were treated with the clotting factor thrombin (thrombin receptor-activating peptide, TRAP), we found a differential response in platelet activation compared to healthy platelets. In conclusion, our results show an increased activation and clotting potential of ET platelets. The platelet surface protein GPVI may be a potential drug target to prevent abnormal blood clotting in ET patients.
Assuntos
Plaquetas , Trombocitemia Essencial , Trombose , Humanos , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/complicações , Plaquetas/metabolismo , Masculino , Feminino , Trombose/metabolismo , Trombose/etiologia , Pessoa de Meia-Idade , Idoso , Citometria de Fluxo/métodos , Ativação Plaquetária , Estudos de Casos e Controles , AdultoRESUMO
OBJECTIVE: Mutations in the glucocerebrosidase (GBA1) gene and subthalamic nucleus deep brain stimulation (STN-DBS) are independently associated with cognitive dysfunction in persons with Parkinson's disease (PwP). We hypothesized that PwP with both GBA1 mutations and STN-DBS are at greater risk of cognitive dysfunction than PwP with only GBA1 mutations or STN-DBS, or neither. In this study, we determined the pattern of cognitive dysfunction in PwP based on GBA1 mutation status and STN-DBS treatment. METHODS: PwP who are GBA1 mutation carriers with or without DBS (GBA1+DBS+, GBA1+DBS-), and noncarriers with or without DBS (GBA1-DBS+, GBA1-DBS-) were included. Using the NIH Toolbox, cross-sectional differences in response inhibition, processing speed, and episodic memory were compared using analysis of variance with adjustment for relevant covariates. RESULTS: Data were available for 9 GBA1+DBS+, 14 GBA1+DBS-, 17 GBA1-DBS+, and 26 GBA1-DBS- PwP. In this cross-sectional study, after adjusting for covariates, we found that performance on the Flanker test (measure of response inhibition) was lower in GBA1+DBS+ PwP compared with GBA1-DBS+ PwP (P = 0.030). INTERPRETATION: PwP who carry GBA1 mutations and have STN-DBS have greater impaired response inhibition compared with PwP with STN-DBS but without GBA1 mutations. Longitudinal data, including preoperative scores, are required to definitively determine whether GBA1 mutation carriers respond differently to STN-DBS, particularly in the domain of response inhibition.
Assuntos
Estimulação Encefálica Profunda , Doença de Parkinson , Núcleo Subtalâmico , Humanos , Doença de Parkinson/complicações , Doença de Parkinson/genética , Doença de Parkinson/terapia , Estudos Transversais , Glucosilceramidase/genéticaRESUMO
VAV1-MYO1F is a recently identified gain-of-function fusion protein of the proto-oncogene Vav guanine nucleotide exchange factor 1 (VAV1) that is recurrently detected in T-cell non-Hodgkin's lymphoma (T-NHL) patients. However, the pathophysiological functions of VAV1-MYO1F in lymphomagenesis are insufficiently defined. Therefore, we generated transgenic mouse models to conditionally express VAV1-MYO1F in T-cells in vivo. We demonstrate that VAV1-MYO1F triggers cell autonomous activation of T-cell signaling with an activation of the ERK, JNK, and AKT pathways. VAV1-MYO1F expression induces a T-cell activation phenotype with high surface expression of CD25, ICOS, CD44, PD-1, and decreased CD62L as well as aberrant T-cell differentiation, proliferation, and neoplastic transformation. Consequently, the VAV1-MYO1F expressing T-cells induce a malignant T lymphoproliferative disease with 100% penetrance in vivo that mimics key aspects of human peripheral T-cell lymphoma. These results demonstrate that the human T-cell oncogene VAV1-MYO1F is sufficient to trigger oncogenic T-cell signaling and neoplastic transformation, and moreover, it provides a new clinically relevant mouse model to explore the pathogenesis of and treatment concepts for human T-cell lymphoma.
Assuntos
Linfoma de Células T Periférico , Proteínas Proto-Oncogênicas c-vav , Camundongos , Humanos , Animais , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Linfoma de Células T Periférico/genética , Transdução de Sinais , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos Transgênicos , Oncogenes , Miosina Tipo I/genética , Miosina Tipo I/metabolismoRESUMO
Genetic counselors (GCs) and healthcare interpreters (HIs) are key members of the healthcare team when providing genetic counseling services to patients with Limited English Proficiency (LEP); however, the working relationship between GCs and HIs and the role each member plays within a genetic counseling session is unclear. Previous studies assessing this relationship have been qualitative and limited in sample size (Agather et al., 2018, Journal of Genetic Counseling, 26, 1388; Krieger et al., 2018, Journal of Genetic Counseling, 26, 1388; Lara-Otero et al., 2019, Health Communication, 34, 1608; Rosenbaum et al., 2020, Journal of Genetic Counseling, 29, 352). This study utilized a quantitative approach to allow for sampling of larger populations and to simultaneously understand current perspectives of GCs and HIs regarding each other's and their own roles within a genetic counseling session. GC and HI participants from the United States were recruited via email to complete an online survey with questions regarding interactions prior to a session, roles during a session, and opportunities for collaboration and constraints in the working relationship. Descriptive and inferential statistics were utilized to analyze responses of GCs and HIs. 130 GC and 40 HI participants were included in this study. There were statistically significant differences (p < .001) in responses between GC and HI participants on the expected distribution of roles during a session in advocacy, psychosocial and cultural domains. Additionally, this study identified that HI desired resources and training regarding genetics and genetic counseling are currently not being met. To our knowledge, this is the largest study to simultaneously survey GC and HI perspectives on these topics. Our findings suggest the need for greater communication and collaboration between GCs and HIs to ensure high-quality care for patients with LEP. Integrating a pre-session meeting between the GC and HI for sessions with patients with LEP and increasing education for GCs and HIs on the roles each group brings into a session is warranted to optimize this collaborative relationship and patient care.
Assuntos
Conselheiros , Proficiência Limitada em Inglês , Pessoal Técnico de Saúde , Conselheiros/psicologia , Atenção à Saúde , Aconselhamento Genético/psicologia , Humanos , Estados UnidosRESUMO
OBJECTIVE: This study was undertaken to compare the rate of change in cognition between glucocerebrosidase (GBA) mutation carriers and noncarriers with and without subthalamic nucleus deep brain stimulation (STN-DBS) in Parkinson disease. METHODS: Clinical and genetic data from 12 datasets were examined. Global cognition was assessed using the Mattis Dementia Rating Scale (MDRS). Subjects were examined for mutations in GBA and categorized as GBA carriers with or without DBS (GBA+DBS+, GBA+DBS-), and noncarriers with or without DBS (GBA-DBS+, GBA-DBS-). GBA mutation carriers were subcategorized according to mutation severity (risk variant, mild, severe). Linear mixed modeling was used to compare rate of change in MDRS scores over time among the groups according to GBA and DBS status and then according to GBA severity and DBS status. RESULTS: Data were available for 366 subjects (58 GBA+DBS+, 82 GBA+DBS-, 98 GBA-DBS+, and 128 GBA-DBS- subjects), who were longitudinally followed (range = 36-60 months after surgery). Using the MDRS, GBA+DBS+ subjects declined on average 2.02 points/yr more than GBA-DBS- subjects (95% confidence interval [CI] = -2.35 to -1.69), 1.71 points/yr more than GBA+DBS- subjects (95% CI = -2.14 to -1.28), and 1.49 points/yr more than GBA-DBS+ subjects (95% CI = -1.80 to -1.18). INTERPRETATION: Although not randomized, this composite analysis suggests that the combined effects of GBA mutations and STN-DBS negatively impact cognition. We advise that DBS candidates be screened for GBA mutations as part of the presurgical decision-making process. We advise that GBA mutation carriers be counseled regarding potential risks associated with STN-DBS so that alternative options may be considered. ANN NEUROL 2022;91:424-435.
Assuntos
Cognição/fisiologia , Estimulação Encefálica Profunda/métodos , Glucosilceramidase/genética , Heterozigoto , Doença de Parkinson/terapia , Núcleo Subtalâmico/fisiopatologia , Idoso , Bases de Dados Factuais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Testes Neuropsicológicos , Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Doença de Parkinson/psicologiaRESUMO
Mass cytometry (CyTOF) is a new technology that allows the investigation of protein expression at single cell level with high resolution. While several protocols are available to investigate leukocyte expression, platelet staining and analysis with CyTOF have been described only from whole blood. Moreover, available protocols do not allow sample storage but require fresh samples to be obtained, processed, and measured immediately. We provide a structured and reproducible method to stain platelets from platelet-rich plasma to study thrombocyte protein expression and reactivity using mass cytometry. With our method, it is possible to acquire a large number of events allowing deep bioinformatic investigation of platelet expression heterogeneity. Integrated in our protocol is also a previously established freezing protocol that allows the storage of stained samples and to delay their measurement. Finally, we provide a structured workflow using different platelet stimulators and a freely available bioinformatic pipeline to analyze platelet expression. Our protocol unlocks the potential of CyTOF analysis for studying platelet biology in health and disease.
Assuntos
Plaquetas , Plasma Rico em Plaquetas , Plaquetas/metabolismo , Citometria de Fluxo/métodos , Humanos , Leucócitos , Plasma Rico em Plaquetas/metabolismoRESUMO
Regulatory T cells (Tregs) are essential for the inhibition of immunity and the maintenance of tissue homeostasis. Signals from the T-cell antigen receptor (TCR) are critical for early Treg development, their expansion, and inhibitory activity. Although TCR-engaged activation of the paracaspase MALT1 is important for these Treg activities, the MALT1 effector pathways in Tregs remain ill-defined. Here, we demonstrate that MALT1 protease activity controls the TCR-induced upregulation of the transcription factor MYC and the subsequent expression of MYC target genes in Tregs. These mechanisms are important for Treg-intrinsic mitochondrial function, optimal respiratory capacity, and homeostatic Treg proliferation. Consistently, conditional deletion of Myc in Tregs results similar to MALT1 inactivation in a lethal autoimmune inflammatory syndrome. Together, these results identify a MALT1 protease-mediated link between TCR signaling in Tregs and MYC control that coordinates metabolism and Treg expansion for the maintenance of immune homeostasis.
Assuntos
Proliferação de Células , Ativação Linfocitária , Mitocôndrias/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Proteínas Proto-Oncogênicas c-myc/imunologia , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Transgênicos , Mitocôndrias/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.
Assuntos
Vacina BNT162 , Plaquetas , COVID-19 , Anticorpos Antivirais , Vacina BNT162/efeitos adversos , Plaquetas/metabolismo , Ligante de CD40 , COVID-19/prevenção & controle , Vacinas contra COVID-19/efeitos adversos , Humanos , Proteínas de Membrana/metabolismo , Selectina-P/metabolismo , Receptores de Trombina/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
Balanced control of T cell signaling is critical for adaptive immunity and protection from autoimmunity. By combining genetically engineered mouse models, biochemical analyses and pharmacological interventions, we describe an unexpected dual role of the tumor necrosis factor receptorassociated factor 6 (TRAF6) E3 ligase as both a positive and negative regulator of mucosa-associated lymphoid tissue 1 (MALT1) paracaspase. Although MALT1-TRAF6 recruitment is indispensable for nuclear factor κB signaling in activated T cells, TRAF6 counteracts basal MALT1 protease activity in resting T cells. In mice, loss of TRAF6-mediated homeostatic suppression of MALT1 protease leads to severe autoimmune inflammation, which is completely reverted by genetic or therapeutic inactivation of MALT1 protease function. Thus, TRAF6 functions as a molecular brake for MALT1 protease in resting T cells and a signaling accelerator for MALT1 scaffolding in activated T cells, revealing that TRAF6 controls T cell activation in a switch-like manner. Our findings have important implications for development and treatment of autoimmune diseases.
Assuntos
Homeostase/imunologia , Inflamação/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Fator 6 Associado a Receptor de TNF/imunologia , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
INTRODUCTION: Myoclonus-dystonia is an inherited disorder characterized by a combination of myoclonic jerks and dystonia. Mutations in the epsilon-sarcoglycan gene (SGCE) represent the main known genetic cause. In the last few years, deep brain stimulation (DBS) has shown significant promise in treating these patients. There is only one report in the literature of a patient with positive SGCE mutation and isolated myoclonus phenotype who has been successfully treated with DBS. CASE PRESENTATION: We present a case of a 16-year-old young man with a history of quick jerks since childhood. They progressed gradually over the years involving the entire body and interfering with most of his daily activities. He had no dystonia. Genetic testing identified a single base deletion in exon 3 of the SGCE gene, considered very likely pathogenic. After unsuccessfully trying several oral medications, he underwent DBS of the globus pallidus internus (GPi). His Unified Myoclonus Rating Scale score during rest and with action improved by 92.8% and 82.6%, respectively. DISCUSSION: The striking effect of DBS on myoclonic jerks confirms the superior benefit of DBS over oral medications. Further study is needed to determine the role of mutation status in predicting DBS response, especially considering that myoclonus-dystonia is genetically heterogeneous. CONCLUSION: Our case confirms the poor response to oral medications and supports the use of GPi DBS for patients with genetically confirmed myoclonus-dystonia and isolated-myoclonus phenotype. In addition, our case represents familial myoclonus-dystonia due to a novel SGCE mutation.
RESUMO
Culture bump theory provides a practical and goal-oriented framework for addressing cultural differences that can impact communication and patient care. Differences in language and culture, coupled with a lack of knowledge or competency regarding these differences, often contribute to 'culture bumps' between healthcare providers and patients. Interpreters serve the unique role of 'cultural brokers', going beyond bridging the linguistic divide to close cultural gaps. Research from the perspective of interpreters focused on culture bumps and cultural competency within genetic counseling sessions is lacking. We aimed to assess interpreters' experiences with significant 'culture bumps' in genetic counseling sessions, obtain interpreters' perspectives regarding genetic counselors' gaps in cultural competency, and explore interpreters' perceptions of the impact of cultural competency on the genetic counseling sessions. Spanish and Polish interpreters experienced in working in person with genetic counselors were recruited through interpreter supervisors at medical centers, hospitals, and interpreter training and service agencies in the Chicagoland area. Using a semi-structured interview guide, phone interviews were conducted with eligible participants and transcribed verbatim. A codebook was developed between two coders, and inter-rater reliability was assessed (κ = 0.82). Grounded theory was used as a guiding principle to code data. The results of this study revealed significant culture bumps identified by interpreters in genetic counseling sessions in the areas of exchange of information, gender and family dynamics, and incorporation of religious and faith beliefs. Interpreters identified the impact on rapport, both negative and positive, due to gaps and strengths in cultural competency, respectively. These responses offer useful insight for training and providing practicing genetic counselors with tools to promote cultural competency, in order to provide optimal care for patients with limited English proficiency (LEP). Further research is necessary to explore these concepts within other languages and cultures, as well as to determine the most appropriate methods for implementing these suggestions for improvement.
Assuntos
Competência Cultural , Aconselhamento Genético/psicologia , Tradução , Adulto , Barreiras de Comunicação , Feminino , Humanos , MasculinoRESUMO
Regulatory T cells (Tregs) have crucial functions in the inhibition of immune responses. Their development and suppressive functions are controlled by the T cell receptor (TCR), but the TCR signaling mechanisms that mediate these effects remain ill-defined. Here we show that CARD11-BCL10-MALT1 (CBM) signaling mediates TCR-induced NF-κB activation in Tregs and controls the conversion of resting Tregs to effector Tregs under homeostatic conditions. However, in inflammatory milieus, cytokines can bypass the CBM requirement for this differentiation step. By contrast, CBM signaling, in a MALT1 protease-dependent manner, is essential for mediating the suppressive function of Tregs. In malignant melanoma models, acute genetic blockade of BCL10 signaling selectively in Tregs or pharmacological MALT1 inhibition enhances anti-tumor immune responses. Together, our data uncover a segregation of Treg differentiation and suppressive function at the CBM complex level, and provide a rationale to explore MALT1 inhibitors for cancer immunotherapy.
Assuntos
Proteína 10 de Linfoma CCL de Células B/imunologia , Proteínas Adaptadoras de Sinalização CARD/imunologia , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Diferenciação Celular , Citocinas/imunologia , Melanoma Experimental , Camundongos , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , NF-kappa B/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell-specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3- thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell-mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T Reguladores/fisiologia , Timo/citologia , Timo/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Diferenciação Celular , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais , Transplante de Células-Tronco , Timo/imunologia , Fator de Transcrição RelA/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiênciaRESUMO
Transcription factor EBF1 (early B-cell factor 1) regulates early B-cell differentiation by poising or activating lineage-specific genes and repressing genes associated with alternative cell fates. To identify proteins that regulate the diverse functions of EBF1, we used SILAC (stable isotope labeling by amino acids in cell culture)-based mass spectrometry of proteins associated with endogenous EBF1 in pro-B cells. This analysis identified most components of the multifunctional CCR4-NOT complex, which regulates transcription and mRNA degradation. CNOT3 interacts with EBF1, and we identified histidine 240 in EBF1 as a critical residue for this interaction. Complementation of Ebf1-/- progenitors with EBF1H240A revealed a partial block of pro-B-cell differentiation and altered expression of specific EBF1 target genes that show either reduced transcription or increased mRNA stability. Most deregulated EBF1 target genes show normal occupancy by EBF1H240A, but we also detected genes with altered occupancy, suggesting that the CCR4-NOT complex affects multiple activities of EBF1. Mice with conditional Cnot3 inactivation recapitulate the block of early B-cell differentiation, which we found to be associated with an impaired autoregulation of Ebf1 and reduced expression of pre-B-cell receptor components. Thus, the interaction of the CCR4-NOT complex with EBF1 diversifies the function of EBF1 in a context-dependent manner and may coordinate transcriptional and post-transcriptional gene regulation.
Assuntos
Linfócitos B/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Linfopoese/genética , Proteínas Nucleares/metabolismo , Estabilidade de RNA/genética , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Inativação Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Chaperonas Moleculares/genética , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Fatores de Transcrição/genética , TransgenesRESUMO
MZB1 (pERp1) is a B-cell-specific and endoplasmic reticulum (ER)-localized protein implicated in antibody secretion and integrin-mediated cell adhesion. Here, we examine the role of MZB1 in vivo by conditional gene inactivation in the mouse germline and at different stages of B lymphopoiesis. Deletion of MZB1 impairs humoral immune responses and antibody secretion in plasma cells that naturally undergo ER stress. In addition, we found that experimental induction of ER stress by tunicamycin injections in mice results in a block of pro-B-cell to pre-B-cell differentiation specifically in Mzb1(-/-) mice. A similar developmental block was observed in Mzb1(fl/fl)mb1(Cre) mice, whereby a Cre recombinase-induced genotoxic stress unmasks a role for MZB1 in the surface expression of immunoglobulin µ heavy chains (µHCs). MZB1 associates directly with the substrate-specific chaperone GRP94 (also called HSP90B1 or gp96) in an ATP-sensitive manner and is required for the interaction of GRP94 with µHCs upon ER stress. Thus, MZB1 seems to act as a substrate-specific cochaperone of GRP94 that enables proper biosynthesis of µHCs under conditions of ER stress.
Assuntos
Estresse do Retículo Endoplasmático/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Chaperonas Moleculares/metabolismo , Animais , Linfócitos B/metabolismo , Técnicas de Inativação de Genes , Imunidade Humoral/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares/genéticaRESUMO
Marginal zone (MZ) B cells of the spleen and B1 cells, termed innate-like B cells, differ from follicular B cells by their attenuated Ca(2+) mobilization, fast antibody secretion, and increased cell adhesion. We identified and characterized Mzb1 as an endoplasmic reticulum-localized and B cell-specific protein that was most abundantly expressed in MZ B and B1 cells. Knockdown of Mzb1 in MZ B cells increased Ca(2+) mobilization and nuclear NFAT transcription factor localization, but reduced lipopolysaccharide-induced antibody secretion and integrin-mediated cell adhesion. Conversely, ectopic expression of an Lck-Mzb1 transgene in peripheral T cells resulted in attenuated Ca(2+) mobilization and augmented integrin-mediated cell adhesion. In addition to its interaction with the substrate-specific chaperone Grp94, Mzb1 augmented the function of the oxidoreductase ERp57 in favoring the expression of integrins in their activated conformation. Thus, Mzb1 helps to diversify peripheral B cell functions by regulating Ca(2+) stores, antibody secretion, and integrin activation.