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1.
Nat Commun ; 11(1): 4227, 2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32839441

RESUMO

In hematopoietic cell transplants, alloreactive T cells mediate the graft-versus-leukemia (GVL) effect. However, leukemia relapse accounts for nearly half of deaths. Understanding GVL failure requires a system in which GVL-inducing T cells can be tracked. We used such a model wherein GVL is exclusively mediated by T cells that recognize the minor histocompatibility antigen H60. Here we report that GVL fails due to insufficient H60 presentation and T cell exhaustion. Leukemia-derived H60 is inefficiently cross-presented whereas direct T cell recognition of leukemia cells intensifies exhaustion. The anti-H60 response is augmented by H60-vaccination, an agonist αCD40 antibody (FGK45), and leukemia apoptosis. T cell exhaustion is marked by inhibitory molecule upregulation and the development of TOX+ and CD39-TCF-1+ cells. PD-1 blockade diminishes exhaustion and improves GVL, while blockade of Tim-3, TIGIT or LAG3 is ineffective. Of all interventions, FGK45 administration at the time of transplant is the most effective at improving memory and naïve T cell anti-H60 responses and GVL. Our studies define important causes of GVL failure and suggest strategies to overcome them.


Assuntos
Apresentação do Antígeno/imunologia , Efeito Enxerto vs Leucemia/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Leucemia/terapia , Linfócitos T/imunologia , Animais , Células Cultivadas , Humanos , Leucemia/imunologia , Leucemia/patologia , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Recidiva , Transplante Homólogo
2.
PLoS Genet ; 16(8): e1008906, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804949

RESUMO

The killer immunoglobulin-like receptors (KIRs), found predominantly on the surface of natural killer (NK) cells and some T-cells, are a collection of highly polymorphic activating and inhibitory receptors with variable specificity for class I human leukocyte antigen (HLA) ligands. Fifteen KIR genes are inherited in haplotypes of diverse gene content across the human population, and the repertoire of independently inherited KIR and HLA alleles is known to alter risk for immune-mediated and infectious disease by shifting the threshold of lymphocyte activation. We have conducted the largest disease-association study of KIR-HLA epistasis to date, enabled by the imputation of KIR gene and HLA allele dosages from genotype data for 12,214 healthy controls and 8,107 individuals with the HLA-B*27-associated immune-mediated arthritis, ankylosing spondylitis (AS). We identified epistatic interactions between KIR genes and their ligands (at both HLA subtype and allele resolution) that increase risk of disease, replicating analyses in a semi-independent cohort of 3,497 cases and 14,844 controls. We further confirmed that the strong AS-association with a pathogenic variant in the endoplasmic reticulum aminopeptidase gene ERAP1, known to alter the HLA-B*27 presented peptidome, is not modified by carriage of the canonical HLA-B receptor KIR3DL1/S1. Overall, our data suggests that AS risk is modified by the complement of KIRs and HLA ligands inherited, beyond the influence of HLA-B*27 alone, which collectively alter the proinflammatory capacity of KIR-expressing lymphocytes to contribute to disease immunopathogenesis.


Assuntos
Epistasia Genética , Antígenos HLA/genética , Receptores KIR/genética , Espondilite Anquilosante/genética , Alelos , Aminopeptidases/genética , Humanos , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único
3.
Cells ; 9(9)2020 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-32847031

RESUMO

Following influenza infection, rs2248374-G ERAP2 expressing cells may transcribe an alternative spliced isoform: ERAP2/Iso3. This variant, unlike ERAP2-wt, is unable to trim peptides to be loaded on MHC class I molecules, but it can still dimerize with both ERAP2-wt and ERAP1-wt, thus contributing to profiling an alternative cellular immune-peptidome. In order to verify if the expression of ERAP2/Iso3 may be induced by other pathogens, PBMCs and MDMs isolated from 20 healthy subjects were stimulated with flu, LPS, CMV, HIV-AT-2, SARS-CoV-2 antigens to analyze its mRNA and protein expression. In parallel, Calu3 cell lines and PBMCs were in vitro infected with growing doses of SARS-CoV-2 (0.5, 5, 1000 MOI) and HIV-1BAL (0.1, 1, and 10 ng p24 HIV-1Bal/1 × 106 PBMCs) viruses, respectively. Results showed that: (1) ERAP2/Iso3 mRNA expression can be prompted by many pathogens and it is coupled with the modulation of several determinants (cytokines, interferon-stimulated genes, activation/inhibition markers, antigen-presentation elements) orchestrating the anti-microbial immune response (Quantigene); (2) ERAP2/Iso3 mRNA is translated into a protein (western blot); (3) ERAP2/Iso3 mRNA expression is sensitive to SARS-CoV-2 and HIV-1 concentration. Considering the key role played by ERAPs in antigen processing and presentation, it is conceivable that these enzymes may be potential targets and modulators of the pathogenicity of infectious diseases and further analyses are needed to define the role played by the different isoforms.


Assuntos
Aminopeptidases/genética , Betacoronavirus/imunologia , Infecções por Coronavirus/genética , Imunização/métodos , Leucócitos Mononucleares/virologia , Macrófagos/virologia , Pneumonia Viral/genética , Isoformas de Proteínas/genética , Apresentação do Antígeno/genética , Doadores de Sangue , Linhagem Celular Tumoral , Infecções por Coronavirus/virologia , Expressão Gênica/imunologia , Genótipo , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Pneumonia Viral/virologia , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/imunologia
4.
Mol Biol Rep ; 47(9): 7283-7289, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32772343

RESUMO

It is well established that Escherichia coli represents a powerful tool for the over-expression of human proteins for structure/function studies. In many cases, such as for membrane transporters, the bacterial toxicity or the aggregation of the target protein hamper the expression limiting the application of this tool. The aim of this study was finding the appropriate conditions for the expression of reluctant proteins that is the human neutral amino acid transporters ASCT2 and B0AT1, that have great relevance to human health in cancer therapy and in COVID-19 research, respectively. The cDNAs coding for the proteins of interest were cloned in the pCOLD I vector and different E. coli strains (BL21 codon plus RIL, and RosettaGami2) were cultured in absence or in presence of glucose (0.5-1%), at low temperature (15 °C), and low inducer concentrations (10-100 µM). Cell growth and protein production were monitored by optical density measurements and western blotting assay, respectively. Even though in different conditions, the expression of both amino acid transporters was obtained.Reducing the growth rate of specific E. coli strains by lowering the temperature and the IPTG concentration, together with the addition of glucose, two reluctant human neutral amino acid transporters have been expressed in E. coli. The results have a potentially great interest in drug discovery since ASCT2 is an acknowledged target of anticancer therapy, and B0AT1 together with ACE2 is part of a receptor for the SARS-Cov-2 RBD proteins.


Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Escherichia coli/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Pneumonia Viral/virologia , Sistema ASC de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos Neutros/genética , Temperatura Baixa , DNA Complementar/genética , Descoberta de Drogas , Escherichia coli/genética , Expressão Gênica , Humanos , Antígenos de Histocompatibilidade Menor/genética , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo
5.
Mol Cell ; 79(5): 728-740.e6, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32721385

RESUMO

Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.


Assuntos
Citidina Desaminase/genética , Citosina , Edição de Genes/métodos , Antígenos de Histocompatibilidade Menor/genética , Oryza/genética , Citosina/química , Genes de Plantas , Humanos , Mutação , RNA Guia/química , RNA de Plantas/química , Reprodutibilidade dos Testes
6.
Mol Cell ; 78(3): 493-505.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32353257

RESUMO

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica , Corpos de Inclusão Intranuclear/genética , Cromossomo Y/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , RNA Helicases DEAD-box/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Células-Tronco Embrionárias/fisiologia , Endonucleases/genética , Sequenciamento de Nucleotídeos em Larga Escala , Corpos de Inclusão Intranuclear/metabolismo , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Enzimas Multifuncionais/genética , Família Multigênica , Estresse Oxidativo , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/metabolismo , Proteínas/genética , Fatores de Transcrição/genética , Cromossomo Y/metabolismo
7.
J Cancer Res Clin Oncol ; 146(6): 1523-1532, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32285256

RESUMO

PURPOSE: APOBEC3A and APOBEC3B cytidine deaminases have been implicated in the pathogenesis of multiple cancers, including breast cancer (BC). A germline deletion linking APOBEC3A and APOBEC3B loci (A3A/B) has been associated with higher APOBEC-mediated mutational burden, but its association with BC risk have been controversial. Therefore, this study investigated the association between A3A/B and BC susceptibility and clinical presentation in a Brazilian cohort. METHODS: A3A/B deletion was evaluated through allele-specific PCR in 341 BC patients and 397 women without familial or personal history of neoplasia from Brazil and associations with susceptibility to BC subtypes were tested through age-adjusted logistic models while correlations with clinicopathological parameters were tested using Kendall's tests. RESULTS: No association was found between A3A/B and BC susceptibility; however, in Luminal-A BCs, it was positively correlated with tumor size (Tau-c = 0.125) and Ki67 (Tau-c = 0.116) and negatively correlated with lymph node metastasis (LNM) (Tau-c = - 0.162). The negative association between A3A/B with LNM in Luminal-A BCs remained significant even after adjusting for tumor size and Ki67 in logistic models (OR = 0.22; p = 0.008). CONCLUSION: These results show that although A3A/B may not modify BC susceptibility in Brazilian population, it may affect clinicopathological features in BC subtypes, promoting tumor cell proliferation while being negatively associated with LNM in Luminal-A BCs.


Assuntos
Neoplasias da Mama/genética , Citidina Desaminase/genética , Deleção de Genes , Mutação em Linhagem Germinativa , Antígenos de Histocompatibilidade Menor/genética , Adulto , Idoso , Brasil , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade
8.
Mol Immunol ; 121: 7-19, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32135401

RESUMO

Autoimmunity and cancer affect millions worldwide and both, in principal, result from dysregulated immune responses. There are many well-known molecules involved in immunological process playing as a double-edged sword, by which associating autoimmune diseases and cancer. In this regard, Endoplasmic reticulum aminopeptidases (ERAP) 1, which belongs to the M1 family of aminopeptidases, plays a central role as a "molecular ruler", proteolyzing of N-terminal of the antigenic peptides before their loading onto HLA-I molecules for antigen presentation in the Endoplasmic Reticulum (ER). Several genome-wide association studies (GWAS) highlighted the significance of ERAP1 and ERAP2 in autoimmune diseases, including Ankylosing spondylitis, Psoriasis, Bechet's disease, and Birdshot chorioretinopathy, as well as in cancers. The expression of ERAP1/2 is mostly altered in different cancers compared to normal cells, but how this affects anti-cancer immune responses and cancer growth has been little explored. Recent studies on the immunological outcomes and the catalytic functions of ERAP1 and ERAP2 have provided a better understanding of their potential pathogenetic role in autoimmunity and cancer. In this review, we summarize the role of ERAP1 and ERAP2 in the autoimmune diseases and cancer immunity based on the recent advances in GWAS studies.


Assuntos
Aminopeptidases/genética , Apresentação do Antígeno/genética , Doenças Autoimunes/genética , Antígenos de Histocompatibilidade Menor/genética , Neoplasias/imunologia , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Modelos Animais de Doenças , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Antígenos de Histocompatibilidade Menor/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Polimorfismo de Nucleotídeo Único/imunologia
9.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211893

RESUMO

The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.


Assuntos
Membrana Celular/metabolismo , Membranas Intracelulares/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Células COS , Chlorocebus aethiops , Diglicerídeos/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
10.
PLoS One ; 15(3): e0230261, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32176735

RESUMO

BACKGROUND: We aimed to evaluate the expression of APOBEC3A (A3A), 3B (A3B) mRNA, and germline APOBEC3A/B deletion polymorphism in patients with breast cancers and to investigate the correlation between their expressions and clinicopathological characteristics. METHODS: RNA and DNA samples were extracted from 138 breast cancer tissues and adjacent normal breast tissues. The levels of A3A and A3B mRNA transcripts were determined using quantitative real-time polymerase chain reaction. Insertion and deletion PCR assays were performed to detect the A3B deletion allele. The serum concentrations of soluble programmed death-ligand 1 (sPD-L1) and interferon gamma were determined using enzyme-linked immunosorbent assays. RESULTS: A3B mRNA expression levels were significantly higher in triple-negative breast cancers compared to hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancers. Older age of the patient and high ki-67 expression were associated with increased expression levels of A3A and A3B mRNA. Advanced tumor stage, presence of lymph node involvement, and high histological grade were associated with increased expression levels of A3A mRNA. The APOBEC3A/B deletion allele was found in 77 (55.8%) patients. TP53 and PIK3CA mutations were detected in 62 (44.9%) and 31 (22.5%) patients, respectively. The presence of a PIK3CA mutation was associated with lower A3A mRNA expression levels. There was a weak positive relationship between A3A mRNA expression levels and serum sPD-L1 levels. CONCLUSIONS: There was a difference in A3B mRNA expression levels according to breast cancer subtypes, and high levels of A3A and A3B mRNA expressions were associated with an aggressive phenotype. There was a high incidence of APOBEC3A/B deletion allele. Further studies are needed to identify the clinical significance of APOBEC in Asian patients with breast cancer.


Assuntos
Neoplasias da Mama/genética , Citidina Desaminase/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Proteínas/genética , Antígeno B7-H1/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/classificação , Classe I de Fosfatidilinositol 3-Quinases/genética , Feminino , Predisposição Genética para Doença , Humanos , Interferon gama/sangue , Pessoa de Meia-Idade , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência , Proteína Supressora de Tumor p53/genética
11.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045479

RESUMO

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de Tempo
12.
PLoS Negl Trop Dis ; 14(2): e0008080, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32078636

RESUMO

Schistosoma mansoni adaptive success is related to regulation of replication, transcription and translation inside and outside the intermediate and definitive host. We hypothesize that S. mansoni alters its epigenetic state in response to the mammalian host immune system, reprogramming gene expression and altering the number of eggs. In response, a change in the DNA methylation profile of hepatocytes could occurs, modulating the extent of hepatic granuloma. To investigate this hypothesis, we used the EBi3-/- murine (Mus musculus) model of S. mansoni infection and evaluated changes in new and maintenance DNA methylation profiles in the liver after 55 days of infection. We evaluated expression of epigenetic genes and genes linked to histone deubiquitination in male and female S. mansoni worms. Comparing TET expression with DNMT expression indicated that DNA demethylation exceeds methylation in knockout infected and uninfected mice and in wild-type infected and uninfected mice. S. mansoni infection provokes activation of demethylation in EBi3-/-I mice (knockout infected). EBi3-/-C (knockout uninfected) mice present intrinsically higher DNA methylation than WTC (control uninfected) mice. EBi3-/-I mice show decreased hepatic damage considering volume and reduced number of granulomas compared to WTI mice; the absence of IL27 and IL35 pathways decreases the Th1 response resulting in minor liver damage. S. mansoni males and females recovered from EBi3-/-I mice have reduced expression of a deubiquitinating enzyme gene, orthologs of which target histones and affect chromatin state. SmMBD and SmHDAC1 expression levels are downregulated in male and female parasites recovered from EBi3-/-, leading to epigenetic gene downregulation in S. mansoni. Changes to the immunological background thus induce epigenetic changes in hepatic tissues and alterations in S. mansoni gene expression, which attenuate liver symptoms in the acute phase of schistosomiasis.


Assuntos
Epigênese Genética , Antígenos de Histocompatibilidade Menor/genética , Receptores de Citocinas/genética , Esquistossomose mansoni/imunologia , Animais , Metilação de DNA , Feminino , Regulação da Expressão Gênica/imunologia , Fígado/metabolismo , Fígado/parasitologia , Masculino , Camundongos , Camundongos Knockout , MicroRNAs , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Schistosoma mansoni/imunologia , Esquistossomose mansoni/parasitologia
13.
PLoS One ; 15(2): e0227997, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32023277

RESUMO

BACKGROUND: Behçet's disease (BD) is a chronic multi-systemic vasculitis with a considerable prevalence in Asian countries. There are many genes associated with a higher risk of developing BD, one of which is endoplasmic reticulum aminopeptidase-1 (ERAP1). In this study, we aimed to investigate the interactions of ERAP1 single nucleotide polymorphisms (SNPs) using a novel data mining method called Model-based multifactor dimensionality reduction (MB-MDR). METHODS: We have included 748 BD patients and 776 healthy controls. A peripheral blood sample was collected, and eleven SNPs were assessed. Furthermore, we have applied the MB-MDR method to evaluate the interactions of ERAP1 gene polymorphisms. RESULTS: The TT genotype of rs1065407 had a synergistic effect on BD susceptibility, considering the significant main effect. In the second order of interactions, CC genotype of rs2287987 and GG genotype of rs1065407 had the most prominent synergistic effect (ß = 12.74). The mentioned genotypes also had significant interactions with CC genotype of rs26653 and TT genotype of rs30187 in the third-order (ß = 12.74 and ß = 12.73, respectively). CONCLUSION: To the best of our knowledge, this is the first study investigating the interaction of a particular gene's SNPs in BD patients by applying a novel data mining method. However, future studies investigating the interactions of various genes could clarify this issue.


Assuntos
Algoritmos , Aminopeptidases/genética , Síndrome de Behçet/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Menor/genética , Modelos Genéticos , Redução Dimensional com Múltiplos Fatores , Polimorfismo de Nucleotídeo Único/genética , Adulto , Entropia , Feminino , Frequência do Gene/genética , Redes Reguladoras de Genes , Humanos , Irã (Geográfico) , Masculino
14.
Nat Cell Biol ; 22(2): 167-174, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32029896

RESUMO

Branched-chain amino acid (BCAA) metabolism is potentially linked with development of pancreatic ductal adenocarcinoma (PDAC)1-4. BCAA transaminase 2 (BCAT2) was essential for the collateral lethality conferred by deletion of malic enzymes in PDAC and the BCAA-BCAT metabolic pathway contributed to non-small-cell lung carcinomas (NSCLCs) other than PDAC3,4. However, the underlying mechanism remains undefined. Here we reveal that BCAT2 is elevated in mouse models and in human PDAC. Furthermore, pancreatic tissue-specific knockout of Bcat2 impedes progression of pancreatic intraepithelial neoplasia (PanIN) in LSL-KrasG12D/+; Pdx1-Cre (KC) mice. Functionally, BCAT2 enhances BCAA uptake to sustain BCAA catabolism and mitochondrial respiration. Notably, BCAA enhances growth of pancreatic ductal organoids from KC mice in a dose-dependent manner, whereas addition of branched-chain α-keto acid (BCKA) and nucleobases rescues growth of KC organoids that is suppressed by BCAT2 inhibitor. Moreover, KRAS stabilizes BCAT2, which is mediated by spleen tyrosine kinase (SYK) and E3 ligase tripartite-motif-containing protein 21 (TRIM21). In addition, BCAT2 inhibitor ameliorates PanIN formation in KC mice. Of note, a lower-BCAA diet also impedes PDAC development in mouse models of PDAC. Thus, BCAT2-mediated BCAA catabolism is critical for development of PDAC harbouring KRAS mutations. Targeting BCAT2 or lowering dietary BCAA may have translational significance.


Assuntos
Adenocarcinoma/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Carcinoma Ductal Pancreático/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias Pancreáticas/genética , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transaminases/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Aminoácidos de Cadeia Ramificada/farmacologia , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Cetoácidos/metabolismo , Cetoácidos/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Antígenos de Histocompatibilidade Menor/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Ductos Pancreáticos/efeitos dos fármacos , Ductos Pancreáticos/metabolismo , Ductos Pancreáticos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Transaminases/metabolismo
15.
Nat Commun ; 11(1): 790, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034147

RESUMO

APOBEC3B, an anti-viral cytidine deaminase which induces DNA mutations, has been implicated as a mediator of cancer evolution and therapeutic resistance. Mutational plasticity also drives generation of neoepitopes, which prime anti-tumor T cells. Here, we show that overexpression of APOBEC3B in tumors increases resistance to chemotherapy, but simultaneously heightens sensitivity to immune checkpoint blockade in a murine model of melanoma. However, in the vaccine setting, APOBEC3B-mediated mutations reproducibly generate heteroclitic neoepitopes in vaccine cells which activate de novo T cell responses. These cross react against parental, unmodified tumors and lead to a high rate of cures in both subcutaneous and intra-cranial tumor models. Heteroclitic Epitope Activated Therapy (HEAT) dispenses with the need to identify patient specific neoepitopes and tumor reactive T cells ex vivo. Thus, actively driving a high mutational load in tumor cell vaccines increases their immunogenicity to drive anti-tumor therapy in combination with immune checkpoint blockade.


Assuntos
Vacinas Anticâncer/farmacologia , Citidina Desaminase/imunologia , Imunoterapia/métodos , Antígenos de Histocompatibilidade Menor/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular Tumoral , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Resistencia a Medicamentos Antineoplásicos , Epitopos/imunologia , Feminino , Humanos , Células Matadoras Naturais/imunologia , Melanoma/imunologia , Melanoma/terapia , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Mutação , Evasão Tumoral/efeitos dos fármacos
16.
Diabetes ; 69(4): 670-680, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31896552

RESUMO

The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)-mediated ß-cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human ß-cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1α/miR-17-5p axis and demonstrate that inhibition of the IRE1α activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of ß-cells to the immune system and position the IRE1α/miR-17 pathway as a central component in ß-cell destruction processes and as a potential target for the treatment of autoimmune T1D.


Assuntos
Aminopeptidases/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aminopeptidases/genética , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Células HEK293 , Humanos , Inflamação/genética , Inflamação/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/imunologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , MicroRNAs/genética , MicroRNAs/metabolismo , Antígenos de Histocompatibilidade Menor/genética , Proteínas Serina-Treonina Quinases/genética , Regulação para Cima
17.
PLoS One ; 15(1): e0223463, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914134

RESUMO

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.


Assuntos
Citidina Desaminase/genética , Dano ao DNA/genética , Antígenos de Histocompatibilidade Menor/genética , Mieloma Múltiplo/genética , Anticorpos Monoclonais Humanizados/farmacologia , Bortezomib/farmacologia , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Lenalidomida/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Mieloma Múltiplo/radioterapia , Mutação/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacologia , Ácidos Polimetacrílicos/farmacologia , RNA Interferente Pequeno/genética , Radiação , Transdução de Sinais/efeitos dos fármacos
18.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 1): 14-19, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31929181

RESUMO

This study presents the crystal structure of a thiol variant of the human mitochondrial branched-chain aminotransferase protein. Human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and α-ketoglutarate to their respective α-keto acids and glutamate. hBCAT activity is regulated by a CXXC center located approximately 10 Šfrom the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. Site-directed mutagenesis of the redox sensor (Cys315) results in a significant loss of activity, with no loss of activity reported on the mutation of the resolving cysteine (Cys318), which allows the reversible formation of a disulfide bond between Cys315 and Cys318. The crystal structure of the oxidized form of the C318A variant was used to better understand the contributions of the individual cysteines and their oxidation states. The structure reveals the modified CXXC center in a conformation similar to that in the oxidized wild type, supporting the notion that its regulatory mechanism depends on switching the Cys315 side chain between active and inactive conformations. Moreover, the structure reveals conformational differences in the N-terminal and inter-domain region that may correlate with the inactivated state of the CXXC center.


Assuntos
Cisteína/química , Cisteína/genética , Antígenos de Histocompatibilidade Menor/química , Mitocôndrias/enzimologia , Proteínas da Gravidez/química , Transaminases/química , Sequência de Aminoácidos , Catálise , Domínio Catalítico/genética , Cristalografia por Raios X , Cisteína/metabolismo , Escherichia coli , Expressão Gênica/genética , Humanos , Antígenos de Histocompatibilidade Menor/genética , Modelos Moleculares , Mutação , Oxirredução , Proteínas da Gravidez/genética , Domínios Proteicos/genética , Transaminases/genética
19.
Mol Immunol ; 117: 155-159, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31790864

RESUMO

INTRODUCTION: ERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. MATERIALS AND METHODS: We recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. RESULTS: A novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. CONCLUSIONS: Our study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies.


Assuntos
Aminopeptidases/química , Aminopeptidases/genética , Síndrome de Behçet/genética , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Adulto , Domínio Catalítico/fisiologia , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Relação Estrutura-Atividade
20.
J Hum Genet ; 65(3): 325-335, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31873220

RESUMO

Behçet disease (BD) is an immune-mediated disease. The cause of BD remains unknown, but the existence of multiple pathological pathways is suspected, including different genetic factors. Polymorphisms in ERAP1 gene have been associated with an increased risk of BD. However, while current BD-associated ERAP1 variants are suggested to contribute to disease by altering the activity of the encoded protein, there is no knowledge of variants that alter the expression level of ERAP1, despite previous associations between ERAP1 expression and BD. Here, we used whole-exome sequencing of a patient with a Behçet-like MHC-I-opathy to identify that the patient, unlike its healthy parents, was homozygous for a rare 1-bp deletion, rs140416843, in the promoter region of ERAP1. rs140416843 has not previously been associated with disease, but is linked to ERAP1 haplotype Hap10 which is associated with BD. The expression of ERAP1 by both RT-qPCR and RNA sequencing showed that ERAP1 mRNA expression correlated with the zygosity for the identified deletion and was decreased in comparison to a healthy cohort. In conclusion, we diagnosed the patient as having BD, and hypothesize that rs140416843-mediated changes in ERAP1 expression play a causative role in BD and that this risk factor is contributing to the association between Hap10 and BD. This is the first report to identify a variant that may cause BD by altering the expression of ERAP1, and our findings suggest that downregulation of ERAP1 expression can serve as a diagnostic marker for BD.


Assuntos
Aminopeptidases/genética , Síndrome de Behçet/genética , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Menor/genética , Adulto , Síndrome de Behçet/patologia , Estudos de Coortes , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Genótipo , Haplótipos/genética , Humanos , Masculino , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Sequenciamento Completo do Exoma
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