RESUMEN
Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.
Asunto(s)
Autoanticuerpos/genética , Enfermedades Autoinmunes/genética , Linfocitos B/inmunología , Linfoma/genética , Animales , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos B/patología , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas Portadoras/genética , Evolución Clonal/genética , Evolución Clonal/inmunología , Ciclina D3/genética , Guanilato Ciclasa/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Proteínas Inhibidoras de la Diferenciación/genética , Linfoma/inmunología , Linfoma/patología , Ratones , Mutación/genética , Mutación/inmunología , Proteínas de Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteínas Supresoras de Tumor/genética , Recombinación V(D)J/genéticaRESUMEN
Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.
Asunto(s)
Infecciones por Poxviridae/inmunología , Poxviridae/fisiología , Dominios Proteicos/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Alelos , Animales , Extinción Biológica , Humanos , Inmunidad , Inflamación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación Missense/genética , FosforilaciónRESUMEN
Sterile α motif domain-containing protein 9-like (SAMD9L) is encoded by a hallmark interferon-induced gene with a role in controlling virus replication that is not well understood. Here, we analyze SAMD9L function from the perspective of human mutations causing neonatal-onset severe autoinflammatory disease. Whole-genome sequencing of two children with leukocytoclastic panniculitis, basal ganglia calcifications, raised blood inflammatory markers, neutrophilia, anemia, thrombocytopaenia, and almost no B cells revealed heterozygous de novo SAMD9L mutations, p.Asn885Thrfs*6 and p.Lys878Serfs*13. These frameshift mutations truncate the SAMD9L protein within a domain a region of homology to the nucleotide-binding and oligomerization domain (NOD) of APAF1, â¼80 amino acids C-terminal to the Walker B motif. Single-cell analysis of human cells expressing green fluorescent protein (GFP)-SAMD9L fusion proteins revealed that enforced expression of wild-type SAMD9L repressed translation of red fluorescent protein messenger RNA and globally repressed endogenous protein translation, cell autonomously and in proportion to the level of GFP-SAMD9L in each cell. The children's truncating mutations dramatically exaggerated translational repression even at low levels of GFP-SAMD9L per cell, as did a missense Arg986Cys mutation reported recurrently as causing ataxia pancytopenia syndrome. Autoinflammatory disease associated with SAMD9L truncating mutations appears to result from an interferon-induced translational repressor whose activity goes unchecked by the loss of C-terminal domains that may normally sense virus infection.
Asunto(s)
Ataxia/patología , Regulación de la Expresión Génica , Mutación Missense , Síndromes Mielodisplásicos/patología , Pancitopenia/patología , Biosíntesis de Proteínas , Proteínas Supresoras de Tumor/genética , Ataxia/genética , Niño , Femenino , Heterocigoto , Humanos , Recién Nacido , Masculino , Síndromes Mielodisplásicos/genética , Pancitopenia/genéticaRESUMEN
Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.
Asunto(s)
Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Genes myc/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patología , Resistencia a Antineoplásicos/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Pronóstico , ARN Interferente Pequeño/genética , Tasa de SupervivenciaRESUMEN
Overweight and obesity compose a chronic disease process of epidemic proportions that presents on a continuum, likely affecting nearly two out of every three patients treated by physician assistants (PAs). However, meaningful and actionable definitions, including but not limited to anthropometric and clinical descriptors, are needed. The effective treatment of overweight and obesity requires an efficient and timely process of screening, diagnosis, evaluation of complications, staging, and clear algorithmic management. PAs are trained as primary care providers and can diagnose and treat overweight and obese patients regardless of practice setting and across the spectrum of the disease and patient's age.
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Obesidad , Sobrepeso , Asistentes Médicos , Guías de Práctica Clínica como Asunto , Atención Primaria de Salud/métodos , HumanosRESUMEN
MYCN oncogene amplification is frequently observed in aggressive childhood neuroblastoma. Using an unbiased large-scale mutagenesis screen in neuroblastoma-prone transgenic mice, we identify a single germline point mutation in the transcriptional corepressor Runx1t1, which abolishes MYCN-driven tumorigenesis. This loss-of-function mutation disrupts a highly conserved zinc finger domain within Runx1t1. Deletion of one Runx1t1 allele in an independent Runx1t1 knockout mouse model is also sufficient to prevent MYCN-driven neuroblastoma development, and reverse ganglia hyperplasia, a known pre-requisite for tumorigenesis. Silencing RUNX1T1 in human neuroblastoma cells decreases colony formation in vitro, and inhibits tumor growth in vivo. Moreover, RUNX1T1 knockdown inhibits the viability of PAX3-FOXO1 fusion-driven rhabdomyosarcoma and MYC-driven small cell lung cancer cells. Despite the role of Runx1t1 in MYCN-driven tumorigenesis neither gene directly regulates the other. We show RUNX1T1 forms part of a transcriptional LSD1-CoREST3-HDAC repressive complex recruited by HAND2 to enhancer regions to regulate chromatin accessibility and cell-fate pathway genes.
Asunto(s)
Carcinogénesis , Proteína Proto-Oncogénica N-Myc , Neuroblastoma , Animales , Humanos , Ratones , Carcinogénesis/genética , Línea Celular Tumoral , Proteínas Co-Represoras/metabolismo , Proteínas Co-Represoras/genética , Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/metabolismo , Histona Demetilasas/genética , Ratones Noqueados , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc/genética , Proteína Proto-Oncogénica N-Myc/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
OBJECTIVE: ABCB1 encodes the multi-drug efflux pump P-glycoprotein (P-gp) and has been implicated in multi-drug resistance. We comprehensively evaluated this gene and flanking regions for an association with clinical outcome in epithelial ovarian cancer (EOC). METHODS: The best candidates from fine-mapping analysis of 21 ABCB1 SNPs tagging C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642) were analysed in 4616 European invasive EOC patients from thirteen Ovarian Cancer Association Consortium (OCAC) studies and The Cancer Genome Atlas (TCGA). Additionally we analysed 1,562 imputed SNPs around ABCB1 in patients receiving cytoreductive surgery and either 'standard' first-line paclitaxel-carboplatin chemotherapy (n=1158) or any first-line chemotherapy regimen (n=2867). We also evaluated ABCB1 expression in primary tumours from 143 EOC patients. RESULT: Fine-mapping revealed that rs1128503, rs2032582, and rs1045642 were the best candidates in optimally debulked patients. However, we observed no significant association between any SNP and either progression-free survival or overall survival in analysis of data from 14 studies. There was a marginal association between rs1128503 and overall survival in patients with nil residual disease (HR 0.88, 95% CI 0.77-1.01; p=0.07). In contrast, ABCB1 expression in the primary tumour may confer worse prognosis in patients with sub-optimally debulked tumours. CONCLUSION: Our study represents the largest analysis of ABCB1 SNPs and EOC progression and survival to date, but has not identified additional signals, or validated reported associations with progression-free survival for rs1128503, rs2032582, and rs1045642. However, we cannot rule out the possibility of a subtle effect of rs1128503, or other SNPs linked to it, on overall survival.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Neoplasias Glandulares y Epiteliales/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Carboplatino/administración & dosificación , Carcinoma Epitelial de Ovario , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Neoplasias Glandulares y Epiteliales/cirugía , Neoplasias Ováricas/cirugía , Paclitaxel/administración & dosificación , Farmacogenética , Polimorfismo de Nucleótido Simple , Modelos de Riesgos ProporcionalesRESUMEN
Chronic lymphocytic leukaemia (CLL) cells can express unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain (IGHV) genes with differing clinical behaviours, variable B cell receptor (BCR) signalling capacity and distinct transcriptional profiles. As it remains unclear how these differences reflect the tumour cells' innate pre/post germinal centre origin or their BCR signalling competence, we applied mRNA/miRNA sequencing to 38 CLL cases categorised into three subsets by IGHV mutational status and BCR signalling capacity. We identified 492 mRNAs and 38 miRNAs differentially expressed between U-CLL and M-CLL, but only 9 mRNAs and 0 miRNAs associated with BCR competence within M-CLL. Of the IGHV-associated miRNAs, (14/38 (37%)) derived from chr14q32 clusters where all miRNAs were co-expressed with the MEG3 lncRNA from a cancer associated imprinted locus. Integrative analysis of miRNA/mRNA data revealed pronounced regulatory potential for the 14q32 miRNAs, potentially accounting for up to 25% of the IGHV-related transcriptome signature. GAB1, a positive regulator of BCR signalling, was potentially regulated by five 14q32 miRNAs and we confirmed that two of these (miR-409-3p and miR-411-3p) significantly repressed activity of the GAB1 3'UTR. Our analysis demonstrates a potential key role of the 14q32 miRNA locus in the regulation of CLL-related gene regulation.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , MicroARNs , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , MicroARNs/genética , Mutación , ARN Mensajero/genéticaRESUMEN
The intent of this investigation was to determine the effect of varying the side chain length of the basic amino acids residues on the binding of a series of antimicrobial peptides (AMPs) to zwitterionic and anionic LUVs, SUVs and micelles. These AMPs are based on the incorporation of three dipeptide units consisting of the unnatural amino acids Tic-Oic in the sequence, Ac-GF-Tic-Oic-GX-Tic-Oic-GF-Tic-Oic-GX-Tic-XXXX-CONH(2), where X (Spacer #2) may be one of the following amino acids, Lys, Orn, Dab, Dpr or Arg. A secondary focus of this study was to attempt to correlate the possible mechanisms of membrane binding of these AMPs to their bacterial strain potency and selectivity. These AMPs produced different CD spectra in the presence of zwitterionic DPC and anionic SDS micelles. This observation indicates that these AMPs adopt different conformations on binding to the surface of zwitterionic and anionic membrane model systems. The CD spectra of these AMPs in the presence of zwitterionic POPC and anionic 4:1 POPC/POPG LUVs and SUVs also were different, indicating that they adopt different conformations on interaction with the zwitterionic and anionic liposomes. This observation was supported by ITC and calcein leakage data that indicated that these AMPs interact via very different mechanisms with anionic and zwitterionic LUVs. The enthalpy for the binding of these AMPs to POPC directly correlates to the length of Spacer #2. The enthalpy of binding of these AMPs to 4:1 POPC/POPG, however do not correlate with the length of Spacer #2. Clear evidence exists that the AMP containing the Dpr residues (the shortest length spacer) interacts very differently with both POPC and 4:1 POPC/POPG LUVs compared to the other four compounds. Data indicates that both the hydrophobicity and the charge distribution of Spacer #2, contribute to defining antibacterial activity. These observations have major implications on the development of these analogs as potential therapeutic agents.
Asunto(s)
Aminoácidos Básicos/química , Aminoácidos Básicos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Secuencia de Aminoácidos , Dicroismo Circular , Liposomas/química , Micelas , Modelos Biológicos , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Relación Estructura-Actividad Cuantitativa , Espectrometría de FluorescenciaRESUMEN
BACKGROUND: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients. METHODS: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification. RESULTS: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells. CONCLUSIONS: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.
RESUMEN
High-level expression of the transcription factor T-bet characterizes a phenotypically distinct murine B cell population known as "age-associated B cells" (ABCs). T-bet-deficient mice have reduced ABCs and impaired humoral immunity. We describe a patient with inherited T-bet deficiency and largely normal humoral immunity including intact somatic hypermutation, affinity maturation and memory B cell formation in vivo, and B cell differentiation into Ig-producing plasmablasts in vitro. Nevertheless, the patient exhibited skewed class switching to IgG1, IgG4, and IgE, along with reduced IgG2, both in vivo and in vitro. Moreover, T-bet was required for the in vivo and in vitro development of a distinct subset of human B cells characterized by reduced expression of CD21 and the concomitantly high expression of CD19, CD20, CD11c, FCRL5, and T-bet, a phenotype that shares many features with murine ABCs. Mechanistically, human T-bet governed CD21loCD11chi B cell differentiation by controlling the chromatin accessibility of lineage-defining genes in these cells: FAS, IL21R, SEC61B, DUSP4, DAPP1, SOX5, CD79B, and CXCR4. Thus, human T-bet is largely redundant for long-lived protective humoral immunity but is essential for the development of a distinct subset of human CD11chiCD21lo B cells.
Asunto(s)
Linfocitos B , Células Plasmáticas , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígeno CD11c/metabolismo , Regulación de la Expresión Génica , Humanos , Inmunoglobulina G , Lipoproteínas/metabolismo , Activación de Linfocitos , RatonesRESUMEN
Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Línea Celular , Chlorocebus aethiops , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Regiones Promotoras Genéticas , Estabilidad Proteica , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transactivadores/genética , Activación Transcripcional , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Extensive circular dichroism, isothermal titration calorimetry and induced calcein leakage studies were conducted on a series of antimicrobial peptides (AMPs), with a varying number of Lys residues located at either the C-terminus or the N-terminus to gain insight into their effect on the mechanisms of binding with zwitterionic and anionic membrane model systems. Different CD spectra were observed for these AMPs in the presence of zwitterionic DPC and anionic SDS micelles indicating that they adopt different conformations on binding to the surfaces of zwitterionic and anionic membrane models. Different CD spectra were observed for these AMPs in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG LUVs and SUVs, indicating that they adopt very different conformations on interaction with these two types of LUVs and SUVs. In addition, ITC and calcein leakage data indicated that all the AMPs studied interact via very different mechanisms with anionic and zwitterionic LUVs. ITC data suggest these peptides interact primarily with the surface of zwitterionic LUVs while they insert into and form pores in anionic LUVs. CD studies indicated that these compounds adopt different conformations depending on the ratio of POPC to POPG lipids present in the liposome. There are detectable spectroscopic and thermodynamic differences between how each of these AMPs interacts with membranes, that is position and total charge density defines how these AMPs interact with specific membrane models and thus partially explain the resulting diversity of antibacterial activity of these compounds.
Asunto(s)
Aminoácidos Básicos/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Calorimetría , Dicroismo Circular , Datos de Secuencia Molecular , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Unión Proteica , Relación Estructura-ActividadRESUMEN
Regulation of the anti-apoptotic BCL2 protein determines cell survival and is frequently abnormal in B cell lymphomas. An evolutionarily conserved post-translational mechanism for over-expression of BCL2 in human B cell lymphomas and the BCL2 paralogue CED-9 in Caenorhabditis elegans results from loss-of-function mutations in human FBXO10 and its C.elegans paralogue DRE-1, a BCL2/CED-9-binding subunit of the SKP-CULLIN-FBOX (SCF) ubiquitin ligase. Here, we tested the role of FBXO10 in BCL2 regulation by producing mice with two different CRISPR/Cas9-engineered Fbxo10 mutations: an Asp54Lys (E54K) missense mutation in the FBOX domain and a Cys55SerfsTer55 frameshift (fs) truncating mutation. Mice homozygous for either mutant allele were born at the expected Mendelian frequency and appeared normal in body weight and appearance as adults. Spleen B cells from homozygous mutant mice did not have increased BCL2 protein, nor were the numbers of mature B cells or germinal centre B cells increased as would be expected if BCL2 was increased. Other lymphocyte subsets that are also regulated by BCL2 levels also displayed no difference in frequency in homozygous Fbxo10 mutant mice. These results support one of two conclusions: either FBXO10 does not regulate BCL2 in mice, or it does so redundantly with other ubiquitin ligase complexes. Possible candidates for the latter include FBXO11 or ARTS-XIAP. The difference between the role of FBXO10 in regulating BCL2 protein levels in C. elegans and in human DLBCL, relative to single-gene deficient mouse leukocytes, should be further investigated.
Asunto(s)
Linfocitos B/metabolismo , Linfoma/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Mutación de Línea Germinal , Humanos , Mutación con Pérdida de Función , Linfoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Procesamiento Proteico-PostraduccionalRESUMEN
Ornithine decarboxylase (ODC1), a critical regulatory enzyme in polyamine biosynthesis, is a direct transcriptional target of MYCN, amplification of which is a powerful marker of aggressive neuroblastoma. A single nucleotide polymorphism (SNP), G316A, within the first intron of ODC1, results in genotypes wildtype GG, and variants AG/AA. CRISPR-cas9 technology was used to investigate the effects of AG clones from wildtype MYCN-amplified SK-N-BE(2)-C cells and the effect of the SNP on MYCN binding, and promoter activity was investigated using EMSA and luciferase assays. AG clones exhibited decreased ODC1 expression, growth rates, and histone acetylation and increased sensitivity to ODC1 inhibition. MYCN was a stronger transcriptional regulator of the ODC1 promoter containing the G allele, and preferentially bound the G allele over the A. Two neuroblastoma cohorts were used to investigate the clinical impact of the SNP. In the study cohort, the minor AA genotype was associated with improved survival, while poor prognosis was associated with the GG genotype and AG/GG genotypes in MYCN-amplified and non-amplified patients, respectively. These effects were lost in the GWAS cohort. We have demonstrated that the ODC1 G316A polymorphism has functional significance in neuroblastoma and is subject to allele-specific regulation by the MYCN oncoprotein.
RESUMEN
Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs.
Asunto(s)
Chaperonina 60/genética , Enterococcus/clasificación , Animales , Carbono/metabolismo , Recuento de Colonia Microbiana , Enterococcus/genética , Enterococcus/metabolismo , Heces/microbiología , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Porcinos/microbiologíaRESUMEN
One of the greatest challenges facing modern medicine is the evolution of drug resistant strains of bacteria. In addition to traditional methods of exposure to traditional bacterial organisms there is a growing concerned of the use of bacteria as bio-terrorism agents. To counter the evolution of drug resistant and potential bio-terrorism bacterial agents new antibiotic drugs must be developed. One potential source of new therapeutic agents that act via a novel mechanism of action are natural and synthetic antimicrobial peptides (AMPs). In our laboratories we have developed a series of AMPs incorporating the un-natural amino acids Tic-Oic to impart organism selectivity and potency while increasing metabolic stability. Herein the in vitro activity of these peptides, including ten new compounds, against eight potential bio-terrorism bacterial agents and three other bacterial strains is presented and discussed. These peptides exhibit a wide range of organism potency and selectivity. Calcein fluorescence leakage and circular dichroism studies were conducted to confirm that these peptides interact with zwitterionic and anionic liposomes.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana , Péptidos/química , Péptidos/farmacología , Antibacterianos/metabolismo , Fluoresceínas/metabolismo , Liposomas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Péptidos/metabolismoRESUMEN
Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.
Asunto(s)
Formación de Anticuerpos/genética , Autoanticuerpos/inmunología , Fosfatidilinositol 3-Quinasa Clase I/genética , Mutación con Ganancia de Función , Tolerancia Inmunológica/inmunología , Células Plasmáticas/inmunología , Animales , Autoanticuerpos/sangre , Autoantígenos/inmunología , Autoinmunidad/genética , Fosfatidilinositol 3-Quinasa Clase I/sangre , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Femenino , Centro Germinal/inmunología , Humanos , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/genéticaRESUMEN
We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three-dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14-day chondrogenic cultures showed that the Notch ligand Jagged-1 (Jag-1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY-1, was also expressed, with a temporal profile that closely followed the expression of Jag-1, and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut-down in Notch signaling was critical for full chondrogenesis, as adenoviral human Jag-1 transduction of hMSC, which caused continuous elevated expression of Jag-1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures, there was inhibited production of extracellular matrix, and the gene expression of aggrecan and type II collagen were strongly suppressed; this may reflect the retention of a prechondrogenic state. The JAG-1-mediated Notch signaling was also shown to be necessary for chondrogenesis, as N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT) added to cultures on days 0-14 or just days 0-5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag-1-mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed.
Asunto(s)
Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteínas de Unión al Calcio/metabolismo , Condrogénesis , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Células del Estroma/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Agregación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrogénesis/efectos de los fármacos , Dipéptidos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Ligandos , Proteínas de la Membrana/genética , Transporte de Proteínas/efectos de los fármacos , Receptores Notch/genética , Proteínas Serrate-Jagged , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Transducción GenéticaRESUMEN
Estrogen treatment of MCF-7 human breast cancer cells allows the reinitiation of synchronous cell cycle progression in antiestrogen-arrested cells. Here, we report that progestins also reinitiate cell cycle progression in this model. Using clonal cell lines derived from progesterone receptor (PR)-negative MCF-7M13 cells expressing wild-type or mutant forms of PRA and PRB, we show that this effect is mediated via PRB, not PRA. Cell cycle progression did not occur with a DNA-binding domain mutant of PRB but was unaffected by mutation in the NH(2)-terminal, SH3 domain interaction motif, which mediates rapid progestin activation of c-Src. Thus, the progestin-induced proliferative response in antiestrogen-inhibited cells is mediated primarily by the transcriptional activity of PRB. Analysis of selected cell cycle targets showed that progestin treatment induced levels of cyclin D1 expression and retinoblastoma protein (Rb) phosphorylation similar to those induced by estradiol. In contrast, progestin treatment resulted in only a 1.2-fold induction of c-Myc compared with a 10-fold induction by estradiol. These results support the conclusion that progestin, in a PRB-dependent manner, can overcome the growth-inhibitory effects of antiestrogens in estrogen receptor/PR-positive breast cancer cells by the induction of cyclin D1 expression. The mediation of this effect by PRB, but not PRA, further suggests a mechanism whereby abnormal regulation of the normal expression ratios of PR isoforms in breast cancer could lead to the attenuation of antiestrogen-mediated growth arrest.