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1.
Mol Cell Proteomics ; 23(7): 100792, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38810695

RESUMO

Immune cells that infiltrate the tumor microenvironment (TME) play crucial roles in shaping cancer development and influencing clinical outcomes and therapeutic responses. However, obtaining a comprehensive proteomic snapshot of tumor-infiltrating immunity in clinical specimens is often hindered by small sample amounts and a low proportion of immune infiltrating cells in the TME. To enable in-depth and highly sensitive profiling of microscale tissues, we established an immune cell-enriched library-assisted strategy for data-independent acquisition mass spectrometry (DIA-MS). Firstly, six immune cell subtype-specific spectral libraries were established from sorted cluster of differentiation markers, CD8+, CD4+ T lymphocytes, B lymphocytes, natural killer cells, dendritic cells, and macrophages in murine mesenteric lymph nodes (MLNs), covering 7815 protein groups with surface markers and immune cell-enriched proteins. The feasibility of microscale immune proteomic profiling was demonstrated on 1 µg tissue protein from the tumor of murine colorectal cancer (CRC) models using single-shot DIA; the immune cell-enriched library increased coverage to quantify 7419 proteins compared to directDIA analysis (6978 proteins). The enhancement enabled the mapping of 841 immune function-related proteins and exclusive identification of many low-abundance immune proteins, such as CD1D1, and CD244, demonstrating high sensitivity for immune landscape profiling. This approach was used to characterize the MLNs in CRC models, aiming to elucidate the mechanism underlying their involvement in cancer development within the TME. Even with a low percentage of immune cell infiltration (0.25-3%) in the tumor, our results illuminate downregulation in the adaptive immune signaling pathways (such as C-type lectin receptor signaling, and chemokine signaling), T cell receptor signaling, and Th1/Th2/Th17 cell differentiation, suggesting an immunosuppressive status in MLNs of CRC model. The DIA approach using the immune cell-enriched libraries showcased deep coverage and high sensitivity that can facilitate illumination of the immune proteomic landscape for microscale samples.

2.
Nucleic Acids Res ; 51(19): 10768-10781, 2023 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-37739431

RESUMO

Translational readthrough of UGA stop codons by selenocysteine-specific tRNA (tRNASec) enables the synthesis of selenoproteins. Seryl-tRNA synthetase (SerRS) charges tRNASec with serine, which is modified into selenocysteine and delivered to the ribosome by a designated elongation factor (eEFSec in eukaryotes). Here we found that components of the human selenocysteine incorporation machinery (SerRS, tRNASec, and eEFSec) also increased translational readthrough of non-selenocysteine genes, including VEGFA, to create C-terminally extended isoforms. SerRS recognizes target mRNAs through a stem-loop structure that resembles the variable loop of its cognate tRNAs. This function of SerRS depends on both its enzymatic activity and a vertebrate-specific domain. Through eCLIP-seq, we identified additional SerRS-interacting mRNAs as potential readthrough genes. Moreover, SerRS overexpression was sufficient to reverse premature termination caused by a pathogenic nonsense mutation. Our findings expand the repertoire of selenoprotein biosynthesis machinery and suggest an avenue for therapeutic targeting of nonsense mutations using endogenous factors.


Assuntos
Biossíntese de Proteínas , Serina-tRNA Ligase , Humanos , Códon sem Sentido , Códon de Terminação , RNA Mensageiro/metabolismo , Selenocisteína/genética , Selenocisteína/metabolismo , Selenoproteínas/genética , Serina-tRNA Ligase/genética
3.
J Biomed Sci ; 31(1): 16, 2024 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-38280996

RESUMO

SUMOylation, which is a type of post-translational modification that involves covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to target substrates, regulates various important molecular and cellular processes, including transcription, the cell cycle, cell signaling, and DNA synthesis and repair. Newly synthesized SUMO is immature and cleaved by the SUMO-specific protease family, resulting in exposure of the C-terminal Gly-Gly motif to become the mature form. In the presence of ATP, mature SUMO is conjugated with the activating enzyme E1 through the cysteine residue of E1, followed by transfer to the cysteine residue of E2-conjugating enzyme Ubc9 in humans that recognizes and modifies the lysine residue of a substrate protein. E3 SUMO ligases promote SUMOylation. SUMOylation is a reversible modification and mediated by SUMO-specific proteases. Cumulative studies have indicated that SUMOylation affects the functions of protein substrates in various manners, including cellular localization and protein stability. Gene knockout studies in mice have revealed that several SUMO cycling machinery proteins are crucial for the development and differentiation of various cell lineages, including immune cells. Aberrant SUMOylation has been implicated in several types of diseases, including cancers, cardiovascular diseases, and autoimmune diseases. This review summarizes the biochemistry of SUMO modification and the general biological functions of proteins involved in SUMOylation. In particular, this review focuses on the molecular mechanisms by which SUMOylation regulates the development, maturation, and functions of immune cells, including T, B, dendritic, and myeloid cells. This review also discusses the underlying relevance of disruption of SUMO cycling and site-specific interruption of SUMOylation on target proteins in immune cells in diseases, including cancers and infectious diseases.


Assuntos
Neoplasias , Enzimas de Conjugação de Ubiquitina , Humanos , Animais , Camundongos , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Cisteína/genética , Ubiquitinas/metabolismo , Ubiquitina/metabolismo , Neoplasias/genética
4.
J Am Chem Soc ; 145(17): 9840-9849, 2023 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-37089019

RESUMO

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.


Assuntos
Vacinas Anticâncer , Neoplasias , Humanos , Vacinas Conjugadas , Apresentação de Antígeno , Anticorpos , Polissacarídeos , Oligossacarídeos
5.
PLoS Pathog ; 17(8): e1009724, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34352041

RESUMO

Hemagglutinin (HA) is the immunodominant protein of the influenza virus. We previously showed that mice injected with a monoglycosylated influenza A HA (HAmg) produced cross-strain-reactive antibodies and were better protected than mice injected with a fully glycosylated HA (HAfg) during lethal dose challenge. We employed a single B-cell screening platform to isolate the cross-protective monoclonal antibody (mAb) 651 from mice immunized with the HAmg of A/Brisbane/59/2007 (H1N1) influenza virus (Bris/07). The mAb 651 recognized the head domain of a broad spectrum of HAs from groups 1 and 2 influenza A viruses and offered prophylactic and therapeutic efficacy against A/California/07/2009 (H1N1) (Cal/09) and Bris/07 infections in mice. The antibody did not possess neutralizing activity; however, antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis mediated by natural killer cells and alveolar macrophages were important in the protective efficacy of mAb 651. Together, this study highlighted the significance of effector functions for non-neutralizing antibodies to exhibit protection against influenza virus infection.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Citotoxicidade Celular Dependente de Anticorpos , Vírus da Influenza A/imunologia , Células Matadoras Naturais/imunologia , Macrófagos Alveolares/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia
6.
Proc Natl Acad Sci U S A ; 117(30): 17757-17763, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669430

RESUMO

Vaccination has been used to control the spread of seasonal flu; however, the virus continues to evolve and escape from host immune response through mutation and increasing glycosylation. Efforts have been directed toward development of a universal vaccine with broadly protective activity against multiple influenza strains and subtypes. Here we report the design and evaluation of various chimeric vaccines based on the most common avian influenza H5 and human influenza H1 sequences. Of these constructs, the chimeric HA (cHA) vaccine with consensus H5 as globular head and consensus H1 as stem was shown to elicit broadly protective CD4+ and CD8+ T cell responses. Interestingly, the monoglycosylated cHA (cHAmg) vaccine with GlcNAc on each glycosite induced more stem-specific antibodies, with higher antibody-dependent cellular cytotoxicity (ADCC), and better neutralizing and stronger cross-protection activities against H1, H3, H5, and H7 strains and subtypes. Moreover, the cHAmg vaccine combined with a glycolipid adjuvant designed for class switch further enhanced the vaccine efficacy with more IFN-γ, IL-4, and CD8+ memory T cells produced.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteção Cruzada/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Influenza Humana/virologia , Camundongos , Modelos Moleculares , Testes de Neutralização , Orthomyxoviridae/classificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-Atividade , Vacinação
7.
J Immunol ; 204(5): 1158-1172, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31969388

RESUMO

Galectin-9 is a risk gene in inflammatory bowel disease. By transcriptomic analyses of ileal biopsies and PBMCs from inflammatory bowel disease patients, we identified a positive correlation between galectin-9 expression and colitis severity. We observed that galectin-9-deficient T cells were less able to induce T cell-mediated colitis. However, several mouse-based studies reported that galectin-9 treatment induces T cell apoptosis and ameliorates autoimmune diseases in an exogenously modulated manner, indicating a complicated regulation of galectin-9 in T cells. We found that galectin-9 is expressed mainly inside T cells, and its secreted form is barely detected under physiological conditions. Endogenous galectin-9 was recruited to immune synapses upon T cell activation. Moreover, proximal TCR signaling was impaired in galectin-9-deficient T cells, and proliferation of these cells was decreased through an intracellularly modulated manner. Th17 cell differentiation was downregulated in galectin-9-deficient T cells, and this impairment can be rescued by strong TCR signaling. Taken together, these findings suggest that intracellular galectin-9 is a positive regulator of T cell activation and modulates the pathogenesis of autoimmune diseases.


Assuntos
Doenças Autoimunes/imunologia , Diferenciação Celular/imunologia , Galectinas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Diferenciação Celular/genética , Galectinas/genética , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Células Th17/patologia
8.
Proc Natl Acad Sci U S A ; 116(10): 4200-4205, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30782805

RESUMO

Each year influenza virus infections cause hundreds of thousands of deaths worldwide and a significant level of morbidity with major economic burden. At the present time, vaccination with inactivated virus vaccine produced from embryonated chicken eggs is the most prevalent method to prevent the infections. However, current influenza vaccines are only effective against closely matched circulating strains and must be updated and administered yearly. Therefore, generating a vaccine that can provide broad protection is greatly needed for influenza vaccine development. We have previously shown that vaccination of the major surface glycoprotein hemagglutinin (HA) of influenza virus with a single N-acetylglucosamine at each of the N-glycosylation sites [monoglycosylated HA (HAmg)] can elicit better cross-protection compared with the fully glycosylated HA (HAfg). In the current study, we produced monoglycosylated inactivated split H1N1 virus vaccine from chicken eggs by the N-glycosylation process inhibitor kifunensine and the endoglycosidase Endo H, and intramuscularly immunized mice to examine its efficacy. Compared with vaccination of the traditional influenza vaccine with complex glycosylations from eggs, the monoglycosylated split virus vaccine provided better cross-strain protection against a lethal dose of virus challenge in mice. The enhanced antibody responses induced by the monoglycosylated vaccine immunization include higher neutralization activity, higher hemagglutination inhibition, and more HA stem selectivity, as well as, interestingly, higher antibody-dependent cellular cytotoxicity. This study provides a simple and practical procedure to enhance the cross-strain protection of influenza vaccine by removing the outer part of glycans from the virus surface through modifications of the current egg-based process.


Assuntos
Proteção Cruzada/imunologia , Ovos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinação , Animais , Galinhas/anormalidades , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/prevenção & controle , Injeções Intramusculares , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia
9.
J Biomed Sci ; 27(1): 57, 2020 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-32349769

RESUMO

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) is a type of glycosylation that occurs when a monosaccharide, O-GlcNAc, is added onto serine or threonine residues of nuclear or cytoplasmic proteins by O-GlcNAc transferase (OGT) and which can be reversibly removed by O-GlcNAcase (OGA). O-GlcNAcylation couples the processes of nutrient sensing, metabolism, signal transduction and transcription, and plays important roles in development, normal physiology and physiopathology. Cumulative studies have indicated that O-GlcNAcylation affects the functions of protein substrates in a number of ways, including protein cellular localization, protein stability and protein/protein interaction. Particularly, O-GlcNAcylation has been shown to have intricate crosstalk with phosphorylation as they both modify serine or threonine residues. Aberrant O-GlcNAcylation on various protein substrates has been implicated in many diseases, including neurodegenerative diseases, diabetes and cancers. However, the role of protein O-GlcNAcylation in immune cell lineages has been less explored. This review summarizes the current understanding of the fundamental biochemistry of O-GlcNAcylation, and discusses the molecular mechanisms by which O-GlcNAcylation regulates the development, maturation and functions of immune cells. In brief, O-GlcNAcylation promotes the development, proliferation, and activation of T and B cells. O-GlcNAcylation regulates inflammatory and antiviral responses of macrophages. O-GlcNAcylation promotes the function of activated neutrophils, but inhibits the activity of nature killer cells.


Assuntos
Sistema Imunitário/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Acilação , Animais , Humanos , Sistema Imunitário/crescimento & desenvolvimento
10.
Nucleic Acids Res ; 46(11): 5547-5560, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29718303

RESUMO

T follicular helper (Tfh) cell-derived signals promote activation and proliferation of antigen-primed B cells. It remains unclear whether epigenetic regulation is involved in the B cell responses to Tfh cell-derived signals. Here, we demonstrate that Tfh cell-mimicking signals induce the expression of histone demethylases KDM4A and KDM4C, and the concomitant global down-regulation of their substrates, H3K9me3/me2, in B cells. Depletion of KDM4A and KDM4C potentiates B cell activation and proliferation in response to Tfh cell-derived signals. ChIP-seq and de novo motif analysis reveals NF-κB p65 as a binding partner of KDM4A and KDM4C. Their co-targeting to Wdr5, a MLL complex member promoting H3K4 methylation, up-regulates cell cycle inhibitors Cdkn2c and Cdkn3. Thus, Tfh cell-derived signals trigger KDM4A/KDM4C - WDR5 - Cdkn2c/Cdkn3 cascade in vitro, an epigenetic mechanism regulating proper proliferation of activated B cells. This pathway is dysregulated in B cells from systemic lupus erythematosus patients and may represent a pathological link.


Assuntos
Linfócitos B/imunologia , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p18/metabolismo , Histona Desmetilases/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteínas/metabolismo , Fator de Transcrição RelA/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Epigênese Genética/genética , Peptídeos e Proteínas de Sinalização Intracelular , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica/genética , Linfócitos T Auxiliares-Indutores/imunologia , Regulação para Cima/genética
11.
J Biomed Sci ; 26(1): 64, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31472685

RESUMO

Terminally differentiated B cell, the plasma cell, is the sole cell type capable of producing antibodies in our body. Over the past 30 years, the identification of many key molecules controlling B cell activation and differentiation has elucidated the molecular pathways for generating antibody-producing plasma cells. Several types of regulation modulating the functions of the important key molecules in B cell activation and differentiation add other layers of complexity in shaping B cell responses following antigen exposure in the absence or presence of T cell help. Further understanding of the mechanisms contributing to the proper activation and differentiation of B cells into antibody-secreting plasma cells may enable us to develop new strategies for managing antibody humoral responses during health and disease. Herein, we reviewed the effect of different types of regulation, including transcriptional regulation, post-transcriptional regulation and epigenetic regulation, on B cell activation, and on mounting memory B cell and antibody responses. We also discussed the link between the dysregulation of the abovementioned regulatory mechanisms and B cell-related disorders.


Assuntos
Linfócitos B/metabolismo , Epigênese Genética , Regulação da Expressão Gênica , Transtornos Leucocíticos/fisiopatologia , Animais , Humanos , Transtornos Leucocíticos/genética
12.
Proc Natl Acad Sci U S A ; 111(7): 2476-81, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24469815

RESUMO

The 2009 H1N1 pandemic and recent human cases of H5N1, H7N9, and H6N1 in Asia highlight the need for a universal influenza vaccine that can provide cross-strain or even cross-subtype protection. Here, we show that recombinant monoglycosylated hemagglutinin (HAmg) with an intact protein structure from either seasonal or pandemic H1N1 can be used as a vaccine for cross-strain protection against various H1N1 viruses in circulation from 1933 to 2009 in mice and ferrets. In the HAmg vaccine, highly conserved sequences that were originally covered by glycans in the fully glycosylated HA (HAfg) are exposed and thus, are better engulfed by dendritic cells (DCs), stimulated better DC maturation, and induced more CD8+ memory T cells and IgG-secreting plasma cells. Single B-cell RT-PCR followed by sequence analysis revealed that the HAmg vaccine activated more diverse B-cell repertoires than the HAfg vaccine and produced antibodies with cross-strain binding ability. In summary, the HAmg vaccine elicits cross-strain immune responses that may mitigate the current need for yearly reformulation of strain-specific inactivated vaccines. This strategy may also map a new direction for universal vaccine design.


Assuntos
Desenho de Fármacos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/farmacologia , Imunidade Celular/imunologia , Vacinas contra Influenza/farmacologia , Influenza Humana/prevenção & controle , Orthomyxoviridae/imunologia , Animais , Sequência de Bases , Cromatografia Líquida , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , ELISPOT , Feminino , Furões , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Testes de Neutralização , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência , Especificidade da Espécie , Espectrometria de Massas em Tandem
13.
J Immunol ; 193(5): 2207-17, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25070843

RESUMO

Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.


Assuntos
Diferenciação Celular/imunologia , Proliferação de Células , Regulação da Expressão Gênica/imunologia , Memória Imunológica , Plasmócitos/imunologia , Fatores de Transcrição/imunologia , Células 3T3 , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Transgênicos , Plasmócitos/citologia , Fatores de Transcrição/genética
14.
Proc Natl Acad Sci U S A ; 110(27): 10928-33, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23776238

RESUMO

Glycosylation, an important posttranslational modification process, can modulate the structure and function of proteins, but its effect on the properties of plasma cells is largely unknown. In this study, we identified a panel of glycoproteins by click reaction with alkynyl sugar analogs in plasma cells coupled with mass spectrometry analysis. The B-cell maturation antigen (BCMA), an essential membrane protein for maintaining the survival of plasma cells, was identified as a glycoprotein exhibiting complex-type N-glycans at a single N-glycosylation site, asparagine 42. We then investigated the effect of N-glycosylation on the function of BCMA and found that the dexamethasone-induced apoptosis in malignant plasma cells can be rescued by treatment with BCMA ligands, such as a proliferation-inducing ligand (APRIL) and B-cell-activating factor (BAFF), whereas removal of terminal sialic acid on plasma cells further potentiated the ligand-mediated protection. This effect is associated with the increased surface retention of BCMA, leading to its elevated level on cell surface. In addition, the α1-3,-4 fucosylation, but not the terminal sialylation, assists the binding of BCMA with ligands in an in vitro binding assay. Together, our results highlight the importance of N-glycosylation on BCMA in the regulation of ligand binding and functions of plasma cells.


Assuntos
Antígeno de Maturação de Linfócitos B/química , Antígeno de Maturação de Linfócitos B/metabolismo , Polissacarídeos/química , Polissacarídeos/fisiologia , Asparagina/química , Sítios de Ligação , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Química Click , Glicosilação , Humanos , Ligantes , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Plasmócitos/patologia
15.
Proc Natl Acad Sci U S A ; 110(16): 6476-81, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23576729

RESUMO

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor important for the differentiation and function of several types of immune cells. Because skin serves as a physical barrier and acts as an immune sentinel, we investigated whether Blimp-1 is involved in epidermal immune function. We show that Blimp-1 expression is reduced in skin lesions of some human eczema samples and in stimulated primary keratinocytes. Epidermal-specific deletion of PR domain containing 1, with ZNF domain (Prdm1), the gene encoding Blimp-1, in adult mice caused spontaneously inflamed skin characterized by massive dermal infiltration of neutrophils/macrophages and development of chronic inflammation associated with higher levels of cytokines/chemokines, including granulocyte colony-stimulating factor (G-CSF), and enhanced myelopoiesis in bone marrow. Deletion of Prdm1 in the epidermis of adult mice also led to stronger inflammatory reactions in a tape-stripping test and in a disease model of contact dermatitis. The elevated G-CSF produced by keratinocytes after deletion of Prdm1 in vitro was mediated by the transcriptional activation of FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1). Systemic increases in G-CSF contributed to the inflammatory responses, because deletion of the G-CSF gene [colony stimulating factor 3, (Csf3)] prevented neutrophilia and partially ameliorated the inflamed skin in Prdm1-deficient mice. Our findings indicate a previously unreported function for Blimp-1 in restraining steady-state epidermal barrier immunity.


Assuntos
Dermatite/genética , Epiderme/metabolismo , Deleção de Genes , Fatores de Transcrição/genética , Animais , Citocinas/metabolismo , Dermatite/fisiopatologia , Dinitrofluorbenzeno , Citometria de Fluxo , Imunofluorescência , Fator Estimulador de Colônias de Granulócitos/metabolismo , Immunoblotting , Queratinócitos/metabolismo , Macrófagos/imunologia , Camundongos , Infiltração de Neutrófilos/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição/metabolismo
16.
Proc Natl Acad Sci U S A ; 110(34): 13809-14, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23908400

RESUMO

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.


Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos Glicosídicos Associados a Tumores/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Fucose/imunologia , Polissacarídeos Fúngicos/imunologia , Reishi/química , Animais , Linfócitos B/imunologia , Quimiocina CCL2/metabolismo , Polissacarídeos Fúngicos/metabolismo , Imunização , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries
17.
Clin Immunol ; 151(2): 101-13, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24568746

RESUMO

Recently, we demonstrated that B lymphocyte-induced maturation protein 1 (BLIMP-1) has a role in regulating the differentiation and effector function of Th1 and Th17 cells. As these cells play critical roles in the induction and pathogenesis of experimental autoimmune encephalomyelitis (EAE), we investigated the potential role of T cell BLIMP-1 in modulating MOG35-55-induced EAE. We established T cell-specific BLIMP-1 conditional knockout (CKO) NOD mice to dissect the role of BLIMP-1 in EAE using loss-of-function model. Our results indicate that EAE severity is dramatically exacerbated in CKO mice. The numbers of CNS-infiltrating Th1, Th17, IFN-γ(+)IL-17A(+), and IL-21(+)IL-17A(+) CD4(+) T cells are remarkably increased in brain and spinal cord of CKO mice. Moreover, the ratio of Tregs/effectors and IL-10 production of Tregs are significantly downregulated in CNS of CKO mice. We conclude that BLIMP-1 suppresses autoimmune encephalomyelitis via downregulating Th1 and Th17 cells and impairing Treg cells.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica/imunologia , Células Th1/fisiologia , Células Th17/fisiologia , Fatores de Transcrição/metabolismo , Transferência Adotiva , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linfócitos T CD4-Positivos , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT4/genética , Fator de Transcrição STAT4/metabolismo , Organismos Livres de Patógenos Específicos , Medula Espinal/metabolismo , Medula Espinal/patologia , Fatores de Transcrição/genética
18.
Blood ; 120(5): 1039-47, 2012 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-22723553

RESUMO

Although the overproduction of immunoglobulins by short-lived plasma cells accompanying an immune response links with their apoptosis, how long-lived plasma cells adapt to ensure their longevity in this context is obscure. Here, we show that apoptosis signal-regulating kinase 1 (ASK1) contributes to apoptosis of plasma cells because ASK1 activity was induced during differentiation of short-lived plasma cells, and, when produced by ASK1-deficient mice, these cells survived better than those of control mice. Moreover, antigen-specific long-lived plasma cells generated by immunization accumulated in ASK1-deficient mice, suggesting ASK1 also plays a negative role in survival of long-lived plasma cells. In malignant plasma cells, ASK1 transcription was directly suppressed by B lymphocyte-induced maturation protein-1 (Blimp-1). The expression of ASK1 and Blimp-1 showed an inverse correlation between normal human mature B cells and bone marrow plasma cells from patients with multiple myeloma (MM). Suppression of ASK1 is crucial for cell survival because its enforced expression in MM cells caused apoptosis in vitro and lowered MM load in a xenograft animal model; furthermore, alteration of ASK1 activity affected MM cell survival. Our findings indicate a novel mechanism underlying the regulation of survival in normal and malignant plasma cells by ASK1.


Assuntos
Apoptose/genética , MAP Quinase Quinase Quinase 5/fisiologia , Neoplasias de Plasmócitos/patologia , Plasmócitos/fisiologia , Animais , Sobrevivência Celular/genética , Células Cultivadas , Regulação para Baixo/genética , Feminino , Humanos , Contagem de Leucócitos , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Neoplasias de Plasmócitos/genética , Neoplasias de Plasmócitos/metabolismo , Plasmócitos/citologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
19.
EMBO Rep ; 13(7): 631-7, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22555612

RESUMO

Transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) is a master regulator of plasma cell differentiation. Here we show that Blimp-1 is covalently modified by SUMO1 at lysine 816, a modification mediated by SUMO E3 ligase PIAS1. Mutation of Blimp-1 lysine 816 reduces transcriptional repression--correlating with a reduced interaction with a histone deacetylase, HDAC2--and impairs differentiation of antibody-secreting cells. Thus, the SUMO pathway critically regulates Blimp-1 function during plasma cell differentiation.


Assuntos
Diferenciação Celular , Plasmócitos/citologia , Plasmócitos/metabolismo , Proteína SUMO-1/metabolismo , Sumoilação , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Histona Desacetilase 2/metabolismo , Lisina/metabolismo , Camundongos , Camundongos Knockout , Mutação , Fator 1 de Ligação ao Domínio I Regulador Positivo , Proteínas Inibidoras de STAT Ativados/metabolismo , Fatores de Transcrição/genética , Transcrição Gênica
20.
J Immunol ; 188(6): 2914-21, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22308310

RESUMO

Syndecan is the major transmembrane proteoglycan in cells. Of the four syndecans, syndecan-1 is the dominant form expressed in multiple myeloma and is an indicator of poor prognosis. In the current study, we observed that early TRAIL-induced apoptotic processes were accompanied by cleavage of syndecan-1 intracellular region, and explored the possibility whether removal of syndecan-1 promotes apoptotic processes. We found that syndecan-1 knockdown by specific small interfering RNA in multiple myeloma enhanced TRAIL-induced apoptosis, even though the expression of TRAIL receptors and several apoptosis-associated molecules was unaffected. The enhanced TRAIL-mediated apoptosis in syndecan-1-deficient cells was not due to a decrease in surface heparan sulfate or a reduction in TRAIL receptor endocytosis. The increase in TRAIL-induced cell death was accompanied by an elevated caspase-8 activation and an enhanced formation of death-inducing signaling complexes, which could be attributed to an increased expression of TRAIL receptor O-glycosylation enzyme in syndecan-1-deficient cells. We also found that in H9 lymphoma and Jurkat cells, knockdown of the predominant syndecan member also led to an increase in Fas ligand-induced apoptosis. Our results demonstrate that syndecan plays a negative role in death receptor-mediated cell death, suggesting potential application of syndecan downregulation in the treatment of myeloma in combination with TRAIL.


Assuntos
Apoptose/fisiologia , Mieloma Múltiplo/metabolismo , Sindecana-1/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/imunologia , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Mieloma Múltiplo/imunologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Sindecana-1/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/imunologia
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