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1.
Immunity ; 51(5): 949-965.e6, 2019 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-31653482

RESUMEN

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.


Asunto(s)
Cromatina/genética , Susceptibilidad a Enfermedades , Estudio de Asociación del Genoma Completo , Genómica , Mastocitos/inmunología , Mastocitos/metabolismo , Biomarcadores , Células Cultivadas , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Fibrinógeno/genética , Fibrinógeno/metabolismo , Perfilación de la Expresión Génica , Genómica/métodos , Histonas/metabolismo , Humanos , Hipersensibilidad/etiología , Hipersensibilidad/metabolismo , Inmunoglobulina E/inmunología , Inflamación/etiología , Inflamación/metabolismo , Polimorfismo de Nucleótido Simple
2.
Nature ; 605(7909): 349-356, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35477763

RESUMEN

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.


Asunto(s)
Mutación con Ganancia de Función , Lupus Eritematoso Sistémico , Receptor Toll-Like 7 , Animales , Autoinmunidad/genética , Linfocitos B , GMP Cíclico/análogos & derivados , Guanosina , Humanos , Lupus Eritematoso Sistémico/genética , Ratones , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo
3.
Semin Immunol ; 54: 101513, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-34836771

RESUMEN

Our understanding of the biological role of the ßc family of cytokines has evolved enormously since their initial identification as bone marrow colony stimulating factors in the 1960's. It has become abundantly clear over the intervening decades that this family of cytokines has truly astonishing pleiotropic capacity, capable of regulating not only hematopoiesis but also many other normal and pathological processes such as development, inflammation, allergy and cancer. As noted in the current pandemic, ßc cytokines contribute to the cytokine storm seen in acutely ill COVID-19 patients. Ongoing studies to discover how these cytokines activate their receptor are revealing insights into the fundamental mechanisms that give rise to cytokine pleiotropy and are providing tantalizing glimpses of how discrete signaling pathways may be dissected for activation with novel ligands for therapeutic benefit.


Asunto(s)
COVID-19 , Objetivos , Humanos , SARS-CoV-2
4.
J Allergy Clin Immunol ; 153(3): 672-683.e6, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37931708

RESUMEN

BACKGROUND: Patients with severe asthma can present with eosinophilic type 2 (T2), neutrophilic, or mixed inflammation that drives airway remodeling and exacerbations and represents a major treatment challenge. The common ß (ßc) receptor signals for 3 cytokines, GM-CSF, IL-5, and IL-3, which collectively mediate T2 and neutrophilic inflammation. OBJECTIVE: To determine the pathogenesis of ßc receptor-mediated inflammation and remodeling in severe asthma and to investigate ßc antagonism as a therapeutic strategy for mixed granulocytic airway disease. METHODS: ßc gene expression was analyzed in bronchial biopsy specimens from patients with mild-to-moderate and severe asthma. House dust mite extract and Aspergillus fumigatus extract (ASP) models were used to establish asthma-like pathology and airway remodeling in human ßc transgenic mice. Lung tissue gene expression was analyzed by RNA sequencing. The mAb CSL311 targeting the shared cytokine binding site of ßc was used to block ßc signaling. RESULTS: ßc gene expression was increased in patients with severe asthma. CSL311 potently reduced lung neutrophils, eosinophils, and interstitial macrophages and improved airway pathology and lung function in the acute steroid-resistant house dust mite extract model. Chronic intranasal ASP exposure induced airway inflammation and fibrosis and impaired lung function that was inhibited by CSL311. CSL311 normalized the ASP-induced fibrosis-associated extracellular matrix gene expression network and strongly reduced signatures of cellular inflammation in the lung. CONCLUSIONS: ßc cytokines drive steroid-resistant mixed myeloid cell airway inflammation and fibrosis. The anti-ßc antibody CSL311 effectively inhibits mixed T2/neutrophilic inflammation and severe asthma-like pathology and reverses fibrosis gene signatures induced by exposure to commonly encountered environmental allergens.


Asunto(s)
Asma , Receptores de Citocinas , Ratones , Animales , Humanos , Receptores de Citocinas/metabolismo , Remodelación de las Vías Aéreas (Respiratorias) , Pulmón , Citocinas/metabolismo , Ratones Transgénicos , Inflamación , Alérgenos , Esteroides/uso terapéutico , Fibrosis , Pyroglyphidae
5.
Growth Factors ; 42(2): 49-61, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38299881

RESUMEN

Breast cancer represents a collection of pathologies with different molecular subtypes, histopathology, risk factors, clinical behavior, and responses to treatment. "Basal-like" breast cancers predominantly lack the receptors for estrogen and progesterone (ER/PR), lack amplification of human epidermal growth factor receptor 2 (HER2) but account for 10-15% of all breast cancers, are largely insensitive to targeted treatment and represent a disproportionate number of metastatic cases and deaths. Analysis of interleukin (IL)-3 and the IL-3 receptor subunits (IL-3RA + CSF2RB) reveals elevated expression in predominantly the basal-like group. Further analysis suggests that IL-3 itself, but not the IL-3 receptor subunits, associates with poor patient outcome. Histology on patient-derived xenografts supports the notion that breast cancer cells are a significant source of IL-3 that may promote disease progression. Taken together, these observations suggest that IL-3 may be a useful marker in solid tumors, particularly triple negative breast cancer, and warrants further investigation into its contribution to disease pathogenesis.


Asunto(s)
Neoplasias de la Mama , Interleucina-3 , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Interleucina-3/metabolismo , Animales , Pronóstico , Ratones , Línea Celular Tumoral
6.
Trends Immunol ; 42(6): 523-535, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33962887

RESUMEN

Mast cells (MC)s are evolutionarily conserved, tissue-resident immune cells with diverse roles in allergy, cancer, and protection from infection by helminths and microorganisms. The significant diversity in MC development and tissue-specific functional characteristics has recently begun to be understood. Exciting developments in single-cell-based RNA, protein, and chromatin profiling technologies offer new opportunities to characterize MC heterogeneity and to uncover novel MC functions and subtypes; these developments might lead to new and clinically effective therapies for certain pathologies. In this review, we provide an overview of the current understanding of MC development and heterogeneity and discuss new insights gained from single-cell-based studies that may lead to future research directions and therapeutic opportunities.


Asunto(s)
Mastocitos , ARN , Diferenciación Celular
7.
EMBO Rep ; 23(4): e52904, 2022 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-35156745

RESUMEN

Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.


Asunto(s)
Calreticulina , Trastornos Mieloproliferativos , Animales , Anticuerpos Monoclonales , Calreticulina/genética , Calreticulina/metabolismo , Humanos , Janus Quinasa 2/metabolismo , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Ratas
8.
J Allergy Clin Immunol ; 151(2): 324-344, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36424209

RESUMEN

The family of cytokines that comprises IL-3, IL-5, and GM-CSF was discovered over 30 years ago, and their biological activities and resulting impact in clinical medicine has continued to expand ever since. Originally identified as bone marrow growth factors capable of acting on hemopoietic progenitor cells to induce their proliferation and differentiation into mature blood cells, these cytokines are also recognized as key mediators of inflammation and the pathobiology of diverse immunologic diseases. This increased understanding of the functional repertoire of IL-3, IL-5, and GM-CSF has led to an explosion of interest in modulating their functions for clinical management. Key to the successful clinical translation of this knowledge is the recognition that these cytokines act by engaging distinct dimeric receptors and that they share a common signaling subunit called ß-common or ßc. The structural determination of how IL-3, IL-5, and GM-CSF interact with their receptors and linking this to their differential biological functions on effector cells has unveiled new paradigms of cell signaling. This knowledge has paved the way for novel mAbs and other molecules as selective or pan inhibitors for use in different clinical settings.


Asunto(s)
Medicina Clínica , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Humanos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Citocinas/metabolismo , Interleucina-3/metabolismo , Interleucina-5/metabolismo , Eosinófilos , Biología
9.
J Allergy Clin Immunol ; 152(3): 725-735.e10, 2023 09.
Artículo en Inglés | MEDLINE | ID: mdl-37127225

RESUMEN

BACKGROUND: Mast cells (MCs) are tissue-resident immune cells that mediate IgE-dependent allergic responses. Downstream of FcεRI, an intricate network of receptor-specific signaling pathways and adaptor proteins govern MC function. The 14-3-3 family of serine-threonine phosphorylation-dependent adapter proteins are known to organize intracellular signaling. However, the role of 14-3-3 in IgE-dependent activation remains poorly defined. OBJECTIVE: We sought to determine whether 14-3-3 proteins are required for IgE-dependent MC activation and whether 14-3-3 is a viable target for the treatment of MC-mediated inflammatory diseases. METHODS: Genetic manipulation of 14-3-3ζ expression in human and mouse MCs was performed and IgE-dependent mediator release assessed. Pharmacologic inhibitors of 14-3-3 and 14-3-3ζ knockout mice were used to assess 14-3-3ζ function in a MC-dependent in vivo passive cutaneous anaphylaxis (PCA) model of allergic inflammation. Expression and function of 14-3-3ζ were assessed in human nasal polyp tissue MCs. RESULTS: IgE-dependent mediator release from human MCs was decreased by 14-3-3ζ knockdown and increased by 14-3-3ζ overexpression. Deletion of the 14-3-3ζ gene decreased IgE-dependent activation of mouse MCs in vitro and PCA responses in vivo. Furthermore, the 14-3-3 inhibitor, RB-11, which impairs dimerization of 14-3-3, inhibited cultured MC and polyp tissue MC activation and signaling downstream of the FcεRI receptor and dose-dependently attenuated PCA responses. CONCLUSION: IgE/FcεRI-mediated MC activation is positively regulated by 14-3-3ζ. We identify a critical role for this p-Ser/Thr-binding protein in the regulation of MC FcεRI signaling and IgE-dependent immune responses and show that this pathway may be amenable to pharmacologic targeting.


Asunto(s)
Anafilaxia , Receptores de IgE , Humanos , Ratones , Animales , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Mastocitos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inmunoglobulina E , Inflamación/metabolismo , Degranulación de la Célula
10.
Cell ; 134(3): 496-507, 2008 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-18692472

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a betac subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Secuencia de Aminoácidos , Cristalografía , Humanos , Modelos Moleculares , Datos de Secuencia Molecular
11.
Respirology ; 27(8): 617-629, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35599245

RESUMEN

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a devastating disease commonly caused by cigarette smoke (CS) exposure that drives tissue injury by persistently recruiting myeloid cells into the lungs. A significant portion of COPD patients also present with overlapping asthma pathology including eosinophilic inflammation. The ßc cytokine family includes granulocyte monocyte-colony-stimulating factor, IL-5 and IL-3 that signal through their common receptor subunit ßc to promote the expansion and survival of multiple myeloid cells including monocytes/macrophages, neutrophils and eosinophils. METHODS: We have used our unique human ßc receptor transgenic (hßc Tg) mouse strain that expresses human ßc instead of mouse ßc and ßIL3 in an acute CS exposure model. Lung tissue injury was assessed by histology and measurement of albumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid. Transgenic mice were treated with an antibody (CSL311) that inhibits human ßc signalling. RESULTS: hßc Tg mice responded to acute CS exposure by expanding blood myeloid cell numbers and recruiting monocyte-derived macrophages (cluster of differentiation 11b+ [CD11b+ ] interstitial and exudative macrophages [IM and ExM]), neutrophils and eosinophils into the lungs. This inflammatory response was associated with lung tissue injury and oedema. Importantly, CSL311 treatment in CS-exposed mice markedly reduced myeloid cell numbers in the blood and BAL compartment. Furthermore, CSL311 significantly reduced lung CD11b+ IM and ExM, neutrophils and eosinophils, and this decline was associated with a significant reduction in matrix metalloproteinase-12 (MMP-12) and IL-17A expression, tissue injury and oedema. CONCLUSION: This study identifies CSL311 as a therapeutic antibody that potently inhibits immunopathology and lung injury caused by acute CS exposure.


Asunto(s)
Fumar Cigarrillos , Lesión Pulmonar , Enfermedad Pulmonar Obstructiva Crónica , Animales , Líquido del Lavado Bronquioalveolar , Fumar Cigarrillos/efectos adversos , Eosinófilos , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad Pulmonar Obstructiva Crónica/metabolismo
12.
J Biol Chem ; 293(1): 89-99, 2018 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-29109150

RESUMEN

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.


Asunto(s)
Proteínas 14-3-3/química , Chaperonas Moleculares/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación Puntual , Agregado de Proteínas , Conformación Proteica en Hélice alfa , Multimerización de Proteína , Estabilidad Proteica , Sales (Química)/química , Sales (Química)/metabolismo , Alineación de Secuencia
13.
Blood ; 129(6): 771-782, 2017 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-27956387

RESUMEN

Acute myeloid leukemia (AML) is an aggressive malignancy where despite improvements in conventional chemotherapy and bone marrow transplantation, overall survival remains poor. Sphingosine kinase 1 (SPHK1) generates the bioactive lipid sphingosine 1-phosphate (S1P) and has established roles in tumor initiation, progression, and chemotherapy resistance in a wide range of cancers. The role and targeting of SPHK1 in primary AML, however, has not been previously investigated. Here we show that SPHK1 is overexpressed and constitutively activated in primary AML patient blasts but not in normal mononuclear cells. Subsequent targeting of SPHK1 induced caspase-dependent cell death in AML cell lines, primary AML patient blasts, and isolated AML patient leukemic progenitor/stem cells, with negligible effects on normal bone marrow CD34+ progenitors from healthy donors. Furthermore, administration of SPHK1 inhibitors to orthotopic AML patient-derived xenografts reduced tumor burden and prolonged overall survival without affecting murine hematopoiesis. SPHK1 inhibition was associated with reduced survival signaling from S1P receptor 2, resulting in selective downregulation of the prosurvival protein MCL1. Subsequent analysis showed that the combination of BH3 mimetics with either SPHK1 inhibition or S1P receptor 2 antagonism triggered synergistic AML cell death. These results support the notion that SPHK1 is a bona fide therapeutic target for the treatment of AML.


Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Clorometilcetonas de Aminoácidos/farmacología , Amino Alcoholes/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Inhibidores de Caspasas/farmacología , Caspasas/genética , Caspasas/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Lisofosfolípidos/metabolismo , Ratones , Ratones Endogámicos NOD , Terapia Molecular Dirigida , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Receptores de Lisoesfingolípidos/genética , Receptores de Lisoesfingolípidos/metabolismo , Transducción de Señal , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Bioconjug Chem ; 29(4): 1352-1363, 2018 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-29433309

RESUMEN

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64Cu-A14-ChAcNLS increased 64Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64Cu-A14. However, 64Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64Cu-A14. This resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64Cu-A14. In addition, 64Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.


Asunto(s)
Ácido Cólico/química , Ácido Cólico/farmacocinética , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Subunidad alfa del Receptor de Interleucina-5/análisis , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Animales , Línea Celular Tumoral , Ácido Cólico/administración & dosificación , Radioisótopos de Cobre/administración & dosificación , Radioisótopos de Cobre/química , Radioisótopos de Cobre/farmacocinética , Sistemas de Liberación de Medicamentos , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/inmunología , Subunidad alfa del Receptor de Interleucina-5/inmunología , Ratones Endogámicos NOD , Ratones SCID , Tomografía de Emisión de Positrones/métodos , Distribución Tisular , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/terapia
15.
J Proteome Res ; 16(5): 1976-1987, 2017 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-28365999

RESUMEN

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.


Asunto(s)
Proteínas 14-3-3/metabolismo , Drosophila/genética , Redes y Vías Metabólicas/fisiología , Proteoma/análisis , Animales , Cuerpo Adiposo/química , Regulación del Desarrollo de la Expresión Génica , Larva/anatomía & histología , Estadios del Ciclo de Vida , Proteómica/métodos , Receptores de Esteroides/metabolismo
16.
Angiogenesis ; 19(4): 463-86, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27338829

RESUMEN

Desmogleins (DSG) are a family of cadherin adhesion proteins that were first identified in desmosomes and provide cardiomyocytes and epithelial cells with the junctional stability to tolerate mechanical stress. However, one member of this family, DSG2, is emerging as a protein with additional biological functions on a broader range of cells. Here we reveal that DSG2 is expressed by non-desmosome-forming human endothelial progenitor cells as well as their mature counterparts [endothelial cells (ECs)] in human tissue from healthy individuals and cancer patients. Analysis of normal blood and bone marrow showed that DSG2 is also expressed by CD34(+)CD45(dim) hematopoietic progenitor cells. An inability to detect other desmosomal components, i.e., DSG1, DSG3 and desmocollin (DSC)2/3, on these cells supports a solitary role for DSG2 outside of desmosomes. Functionally, we show that CD34(+)CD45(dim)DSG2(+) progenitor cells are multi-potent and pro-angiogenic in vitro. Using a 'knockout-first' approach, we generated a Dsg2 loss-of-function strain of mice (Dsg2 (lo/lo)) and observed that, in response to reduced levels of Dsg2: (i) CD31(+) ECs in the pancreas are hypertrophic and exhibit altered morphology, (ii) bone marrow-derived endothelial colony formation is impaired, (iii) ex vivo vascular sprouting from aortic rings is reduced, and (iv) vessel formation in vitro and in vivo is attenuated. Finally, knockdown of DSG2 in a human bone marrow EC line reveals a reduction in an in vitro angiogenesis assay as well as relocalisation of actin and VE-cadherin away from the cell junctions, reduced cell-cell adhesion and increased invasive properties by these cells. In summary, we have identified DSG2 expression in distinct progenitor cell subpopulations and show that, independent from its classical function as a component of desmosomes, this cadherin also plays a critical role in the vasculature.


Asunto(s)
Desmogleína 2/metabolismo , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Animales , Diferenciación Celular , Células Cultivadas , Desmogleína 2/deficiencia , Desmogleína 2/genética , Células Endoteliales/citología , Femenino , Técnicas de Silenciamiento del Gen , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Neovascularización Fisiológica/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética
17.
Blood ; 123(8): 1218-28, 2014 Feb 20.
Artículo en Inglés | MEDLINE | ID: mdl-24363400

RESUMEN

Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Receptores de Interleucina-3/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Adulto , Anciano , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Antígenos CD34/metabolismo , Línea Celular , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Leucemia Eritroblástica Aguda/inmunología , Leucemia Eritroblástica Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patología
18.
PLoS Biol ; 11(3): e1001515, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23526884

RESUMEN

The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in cancer.


Asunto(s)
Supervivencia Celular/fisiología , Citocinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/genética , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Humanos , Leucemia Mieloide Aguda , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Transducción de Señal/genética , Transducción de Señal/fisiología
19.
Immunol Rev ; 250(1): 277-302, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23046136

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are members of a discrete family of cytokines that regulates the growth, differentiation, migration and effector function activities of many hematopoietic cells and immunocytes. These cytokines are involved in normal responses to infectious agents, bridging innate and adaptive immunity. However, in certain cases, the overexpression of these cytokines or their receptors can lead to excessive or aberrant initiation of signaling resulting in pathological conditions, with chronic inflammatory diseases and myeloid leukemias the most notable examples. Recent crystal structures of the GM-CSF receptor ternary complex and the IL-5 binary complex have revealed new paradigms of cytokine receptor activation. Together with a wealth of associated structure-function studies, they have significantly enhanced our understanding of how these receptors recognize cytokines and initiate signals across cell membranes. Importantly, these structures provide opportunities for structure-based approaches for the discovery of novel and disease-specific therapeutics. In addition, recent biochemical evidence has suggested that the GM-CSF/IL-3/IL-5 receptor family is capable of interacting productively with other membrane proteins at the cell surface. Such interactions may afford additional or unique biological activities and might be harnessed for selective modulation of the function of these receptors in disease.


Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Interleucina-3/química , Interleucina-5/química , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/química , Receptores de Interleucina-3/química , Receptores de Interleucina-5/química , Cristalografía por Rayos X , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-3/inmunología , Interleucina-3/metabolismo , Interleucina-5/inmunología , Interleucina-5/metabolismo , Leucemia Mieloide/inmunología , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Linfocitos/inmunología , Linfocitos/metabolismo , Linfocitos/patología , Modelos Moleculares , Unión Proteica , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Receptores de Interleucina-3/inmunología , Receptores de Interleucina-3/metabolismo , Receptores de Interleucina-5/inmunología , Receptores de Interleucina-5/metabolismo , Transducción de Señal , Relación Estructura-Actividad
20.
J Neurosci ; 34(36): 12168-81, 2014 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-25186760

RESUMEN

During brain development, neural progenitor cells proliferate and differentiate into neural precursors. These neural precursors migrate along the radial glial processes and localize at their final destination in the cortex. Numerous reports have revealed that 14-3-3 proteins are involved in many neuronal activities, although their functions in neurogenesis remain unclear. Here, using 14-3-3ε/ζ double knock-out mice, we found that 14-3-3 proteins are important for proliferation and differentiation of neural progenitor cells in the cortex, resulting in neuronal migration defects and seizures. 14-3-3 deficiency resulted in the increase of δ-catenin and the decrease of ß-catenin and αN-catenin. 14-3-3 proteins regulated neuronal differentiation into neurons via direct interactions with phosphorylated δ-catenin to promote F-actin formation through a catenin/Rho GTPase/Limk1/cofilin signaling pathway. Conversely, neuronal migration defects seen in the double knock-out mice were restored by phosphomimic Ndel1 mutants, but not δ-catenin. Our findings provide new evidence that 14-3-3 proteins play important roles in neurogenesis and neuronal migration via the regulation of distinct signaling cascades.


Asunto(s)
Proteínas 14-3-3/metabolismo , Corteza Cerebral/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis , Proteínas 14-3-3/genética , Actinas/metabolismo , Animales , Cateninas/metabolismo , Movimiento Celular , Proliferación Celular , Corteza Cerebral/crecimiento & desarrollo , Corteza Cerebral/fisiología , Ratones , Ratones Endogámicos C57BL , Células-Madre Neurales/citología , Células-Madre Neurales/fisiología , Neuronas/citología , Neuronas/metabolismo , Neuronas/fisiología , Unión Proteica
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