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1.
Pathol Res Pract ; 253: 155026, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38118219

RESUMEN

As patients continue to suffer from lymphoproliferative and myeloproliferative diseases known as haematopoietic malignancies can affect the bone marrow, blood, lymph nodes, and lymphatic and non-lymphatic organs. Despite advances in the current treatment, there is still a significant challenge for physicians to improve the therapy of HMs. WASp is an important regulator of actin polymerization and the involvement of WASp in transcription is thought to be linked to the DNA damage response and repair. In some studies, severe immunodeficiency and lymphoid malignancy are caused by WASp mutations or the absence of WASp and these mutations in WAS can alter the function and/or expression of the intracellular protein. Loss-of-function and Gain-of-function mutations in WASp have an impact on cancer malignancies' incidence and onset. Recent studies suggest that depending on the clinical or experimental situation, WASPs and WAVEs can operate as a suppressor or enhancers for cancer malignancy. These dual functions of WASPs and WAVEs in cancer likely arose from their multifaceted role in cells that could be targeted for anticancer drug development. The significant role and their association of WASp in Chronic myeloid leukaemia, Juvenile myelomonocytic leukaemia and T-cell lymphoma is discussed. In this review, we described the structure and function of WASp and its family mechanism, analysing major regulatory effectors and summarising the clinical relevance and drugs that specifically target WASp in disease treatment in various hematopoietic malignancies by different approaches.


Asunto(s)
Neoplasias Hematológicas , Neoplasias , Síndrome de Wiskott-Aldrich , Humanos , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/terapia , Neoplasias Hematológicas/genética , Biología Molecular , Actinas/metabolismo
2.
Clin Exp Immunol ; 212(2): 137-146, 2023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-36617178

RESUMEN

Primary immune deficiencies (PIDs) are genetic disorders impacting the appropriate development or functioning of any portion of the immune system. The broad adoption of high-throughput sequencing has driven discovery of new genes as well as expanded phenotypes associated with known genes. Beginning with the identification of WAS mutations in patients with severe Wiskott-Aldrich Syndrome, recognition of WAS mutations in additional patients has revealed phenotypes including isolated thrombocytopenia and X-linked neutropenia. Likewise RAC2 patients present with vastly different phenotypes depending on the mutation-ranging from reticular dysgenesis or severe neutrophil dysfunction with neonatal presentation to later onset common variable immune deficiency. This review examines genotype-phenotype correlations in patients with WAS (Wiskott-Aldrich Syndrome) and RAC2 mutations, highlighting functional protein domains, how mutations alter protein interactions, and how specific mutations can affect isolated functions of the protein leading to disparate phenotypes.


Asunto(s)
Trombocitopenia , Síndrome de Wiskott-Aldrich , Humanos , Mutación/genética , Fenotipo , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína RCA2 de Unión a GTP
3.
Nat Commun ; 13(1): 3646, 2022 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-35752626

RESUMEN

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.


Asunto(s)
Proteína del Síndrome de Wiskott-Aldrich , Síndrome de Wiskott-Aldrich , Empalme Alternativo , Núcleo Celular/metabolismo , Humanos , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo
4.
Front Immunol ; 12: 607836, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33717090

RESUMEN

Wiskott-Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the WAS gene, which together with its homolog, N-WASP, regulates actin cytoskeleton remodeling and cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of WAS and its binding partner WIP. Depletion of either WAS or WIP in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of WAS/WIP also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott-Aldrich syndrome.


Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Trombopoyesis/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/etiología , Síndrome de Wiskott-Aldrich/metabolismo , Animales , Secuencia de Bases , Biomarcadores , Línea Celular , Secuenciación de Inmunoprecipitación de Cromatina , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación de la Expresión Génica , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/genética , Transducción de Señal
5.
Eur J Immunol ; 51(5): 1285-1288, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33448368

RESUMEN

Analysis of serum cytokine levels in Wiskott-Aldrich syndrome patients pre- and post- treatment reveals IL-18 as a stable and reliable marker of inflammation. Definitive stem cell treatment with good myeloid correction correlates with resolution of inflammation and reduction of circulating IL-18, highlighting the importance of actin cytoskeletal regulation of myeloid cells in control of inflammation.


Asunto(s)
Biomarcadores , Mediadores de Inflamación/metabolismo , Interleucina-18/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Humanos , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/etiología
6.
Front Immunol ; 12: 794795, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35116029

RESUMEN

Background: The T cell receptor (TCR) diversity is essential for effective T cell immunity. Previous studies showed that TCR diversity in Wiskott-Aldrich Syndrome (WAS) patients was severely impaired, especially in the memory T cell populations. Whether this defect was caused by intrinsic WASp deficiency or extrinsic reasons is still unclear. Methods: We sorted different T cell subsets from the bone marrow chimeric mice model using both magnetic beads and flow cytometry. TCR repertoires of memory T cells, especially CD4+ effector memory T (TEM) cells and CD8+ central memory T (TCM) cells, were analyzed using the UMI quantitative high-throughput sequencing (HTS). Results: An average of 5.51 million sequencing reads of 32 samples was obtained from the Illumina sequencing platform. Bioinformatic analyses showed that compared with wild type (WT), WAS knock out (KO)-CD4+ TEM cells exhibited increased Simpson index and decreased D50 index (P <0.05); The rank abundance curve of KO-CD4+ TEM cells was shorter and steeper than that of WT, and the angle of qD and q in KO-CD4+ TEM cells was lower than that of WT, while these indexes showed few changes between WT and KO chimeric mice in the CD8+TCM population. Therefore, it indicated that the restriction on the TCRVß repertoires is majorly in KO-CD4+ TEM cells but not KO- CD8+ TCM cells. Principal Component Analysis (PCA), a comprehensive parameter for TCRVß diversity, successfully segregated CD4+ TEM cells from WT and KO, but failed in CD8+ TCM cells. Among the total sequences of TRB, the usage of TRBV12.2, TRBV30, TRBV31, TRBV4, TRBD1, TRBD2, TRBJ1.1, and TRBJ1.4 showed a significant difference between WT-CD4+ TEM cells and KO-CD4+ TEM cells (P <0.05), while in CD8+ TCM cells, only the usage of TRBV12.2 and TRBV20 showed a substantial difference between WT and KO (P <0.05). No significant differences in the hydrophobicity and sequence length of TCRVß were found between the WT and KO groups. Conclusion: WASp deficiency selectively affected the TCR diversity of different memory T cell subsets, and it had more impact on the TCRVß diversity of CD4+ TEM cells than CD8+ TCM cells. Moreover, the limitation of TCRVß diversity of CD4+ TEM cells and CD8+ TCM cells in WAS was not severe but intrinsic.


Asunto(s)
Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Síndrome de Wiskott-Aldrich/etiología , Síndrome de Wiskott-Aldrich/metabolismo , Secuencia de Aminoácidos , Animales , Trasplante de Médula Ósea , Biología Computacional/métodos , Modelos Animales de Enfermedad , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/metabolismo , Quimera por Trasplante , Recombinación V(D)J
7.
Oncogene ; 40(2): 345-354, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33139832

RESUMEN

Immunodeficiency is associated with cancer risk. Accordingly, hematolymphoid cancers develop in Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder (PID) resulting from the deficiency of WAS-protein (WASp) expressed predominantly in the hematolymphoid cell lineages. Despite the correlation between WASp deficiency and hematolymphoid cancers, the molecular mechanism underlying the oncogenic role of WASp is incompletely understood. Employing the WASp-sufficient and WASp-deficient cell-pair model of human T and B lymphocytes, we show that WASp deficiency differentially influences hyperactivation versus inhibition of both CDC42:ERK1/2 and NF-κB:AP-1 pro-oncogenic signaling pathways in nonmalignant versus malignant T and B lymphocytes. Furthermore, WASp deficiency induces a cell-type specific up/down-modulation of the DNA-binding activities of NF-κB, AP-1, and multiple other transcription factors with known roles in oncogenesis. We propose that WASp functions as a putative "tumor-suppressor" protein in normal T and B cells, and "oncoprotein" in a subset of established T and B cell malignancies that are not associated with the NPM-ALK fusion.


Asunto(s)
Linfocitos B/patología , Proteínas Oncogénicas/metabolismo , Linfocitos T/patología , Proteínas Supresoras de Tumor/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/deficiencia , Síndrome de Wiskott-Aldrich/patología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/metabolismo , Proteína de Unión al GTP cdc42/metabolismo
8.
EMBO J ; 39(5): e102783, 2020 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-31894880

RESUMEN

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.


Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Sinapsis Inmunológicas/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Movimiento Celular , Citoesqueleto/metabolismo , Humanos , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Linfocitos T/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genética
9.
Haematologica ; 105(4): 1095-1106, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31278208

RESUMEN

Wiskott-Aldrich syndrome (WAS) is associated with thrombocytopenia of unclear origin. We investigated real-time cytosolic calcium dynamics, mitochondrial membrane potential and phoszphatidylserine (PS) exposure in single fibrinogen-bound platelets using confocal microscopy. The WAS platelets had higher resting calcium levels, more frequent spikes, and their mitochondria more frequently lost membrane potential followed by PS exposure (in 22.9% of platelets vs 3.9% in controls; P<0.001) after the collapse of the last mitochondria. This phenomenon was inhibited by the mitochondrial permeability transition pore inhibitor cyclosporine A, as well by xestospongin C and lack of extracellular calcium. Thapsigargin by itself caused accelerated cell death in the WAS platelets. The number of mitochondria was predictive of PS exposure: 33% of platelets from WAS patients with fewer than five mitochondria exposed PS, while only 12% did among those that had five or more mitochondria. Interestingly, healthy donor platelets with fewer mitochondria also more readily became procoagulant upon PAR1/PAR4 stimulation. Collapse of single mitochondria led to greater cytosolic calcium increase in WAS platelets if they had one to three mitochondria compared with platelets containing higher numbers. A computer systems biology model of platelet calcium homeostasis showed that smaller platelets with fewer mitochondria could have impaired calcium homeostasis because of higher surface-to-volume ratio and greater metabolic load, respectively. There was a correlation (C=0.81, P<0.02) between the mean platelet size and platelet count in the WAS patients. We conclude that WAS platelets readily expose PS via a mitochondria-dependent necrotic mechanism caused by their smaller size, which could contribute to the development of thrombocytopenia.


Asunto(s)
Plaquetas , Síndrome de Wiskott-Aldrich , Plaquetas/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Necrosis , Síndrome de Wiskott-Aldrich/metabolismo
10.
Front Immunol ; 10: 1632, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31379835

RESUMEN

Actin nucleators initiate formation of actin filaments. Among them, the Arp2/3 complex has the ability to form branched actin networks. This complex is regulated by members of the Wiscott-Aldrich syndrome protein (WASp) family. Polymerization of actin filaments can be evaluated through flow cytometry by fluorescent phalloidin staining before and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP). We identified a missense mutation in the gene ARPC1B (Arp2/3 activator subunit) resulting in defective actin polymerization in four patients (three of them were related). All patients (1 male, 3 female) developed microthrombocytopenia, cellular immune deficiency, eczema, various autoimmune manifestations, recurrent skin abscesses and elevated IgE antibodies. Besides four patients with homozygous mutation in ARPC1B, we also identified six heterozygous carriers without clinical disease (3 males, 3 females) within the same family. We developed a functional test to evaluate Arp2/3 complex function, which consists of flow cytometric detection of intracellular polymerized actin after in vitro fMLP stimulation of leukocytes. Median fluorescence intensities of FITC-phalloidin stained actin were measured in monocytes, neutrophils and lymphocytes of patients, carriers, and healthy control subjects. We detected non-efficient actin polymerization in monocytes and neutrophils of homozygous patients compared to carriers or the healthy subjects. In monocytes, the increase in median fluorescence intensities was significantly lower in patients compared to carriers (104 vs. 213%; p < 0.01) and healthy controls (104 vs. 289%; p < 0.01). Similarly, the increase in median fluorescence intensities in neutrophils was significantly increased in the group with carriers (208%; p < 0.01) and healthy controls (238%; p < 0.01) and significantly decreased in the patient's group (94%). Our functional fMLP/phalloidin test can therefore be used as a practical tool to separate symptomatic patients from asymptomatic mutation associated to actin polymerization.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Leucocitos/metabolismo , Mutación/genética , Adulto , Femenino , Citometría de Flujo/métodos , Heterocigoto , Homocigoto , Humanos , Lactante , Masculino , Monocitos/metabolismo , Neutrófilos/metabolismo , Polimerizacion , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Adulto Joven
11.
J Allergy Clin Immunol ; 144(5): 1377-1390.e4, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31405607

RESUMEN

BACKGROUND: B-cell receptor (BCR) signaling, combined with CD19 and CD21 signals, imparts specific control of B-cell responses. Dedicator of cytokinesis protein 2 (DOCK2) is critical for the migration and motility of lymphocytes. Although absence of DOCK2 leads to lymphopenia, little is known about the signaling mechanisms and physiologic functions of DOCK2 in B cells. OBJECTIVE: We sought to determine the underlying molecular mechanism of how DOCK2 regulates BCR signaling and peripheral B-cell differentiation. METHODS: In this study we used genetic models for DOCK2, Wiskott-Aldrich syndrome protein (WASP), and lymphoid enhancer-binding factor 1 deficiency to study their interplay in BCR signaling and B-cell differentiation. RESULTS: We found that the absence of DOCK2 led to downregulation of proximal and distal BCR signaling molecules, including CD19, but upregulation of SH2-containing inositol 5 phosphatase 1, a negative signaling molecule. Interestingly, DOCK2 deficiency reduced CD19 and CD21 expression at the mRNA and/or protein levels and was associated with reduced numbers of marginal zone B cells. Additionally, loss of DOCK2 reduced activation of WASP and accelerated degradation of WASP, resulting into reduced actin accumulation and early activation of B cells. Mechanistically, the absence of DOCK2 upregulates the expression of lymphoid enhancer-binding factor 1. These differences were associated with altered humoral responses in the absence of DOCK2. CONCLUSIONS: Overall, our study has provided a novel underlying molecular mechanism of how DOCK2 deficiency regulates surface expression of CD21, which leads to downregulation of CD19-mediated BCR signaling and marginal zone B-cell differentiation.


Asunto(s)
Linfocitos B/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Diferenciación Celular , Células Cultivadas , Proteínas Activadoras de GTPasa/genética , Regulación de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Factor de Unión 1 al Potenciador Linfoide/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/metabolismo , Transducción de Señal , Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/genética
12.
Hum Gene Ther ; 29(3): 366-380, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28922955

RESUMEN

Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Sitios Genéticos , Lentivirus , Transducción Genética , Síndrome de Wiskott-Aldrich/genética , Nucleasas con Dedos de Zinc , Humanos , Células K562 , Síndrome de Wiskott-Aldrich/metabolismo , Nucleasas con Dedos de Zinc/biosíntesis , Nucleasas con Dedos de Zinc/genética
13.
Nat Commun ; 8(1): 1576, 2017 11 17.
Artículo en Inglés | MEDLINE | ID: mdl-29146903

RESUMEN

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Autofagia/inmunología , Inflamasomas/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Animales , Autofagia/genética , Carga Bacteriana/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Escherichia coli Enteropatógena/inmunología , Humanos , Inmunidad Innata/inmunología , Interferón Tipo I/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Nigericina/farmacología , Septinas/metabolismo , Shigella flexneri/inmunología , Células THP-1 , Receptor Toll-Like 4/inmunología , Síndrome de Wiskott-Aldrich/metabolismo
14.
Mol Med Rep ; 16(5): 6526-6531, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28901403

RESUMEN

Wiskott­Aldrich syndrome (WAS) is a rare X­linked recessive immunodeficiency disorder, characterized by thrombocytopenia, small platelets, eczema and recurrent infections associated with increased risk of autoimmunity and malignancy disorders. Mutations in the WAS protein (WASP) gene are responsible for WAS. To date, WASP mutations, including missense/nonsense, splicing, small deletions, small insertions, gross deletions, and gross insertions have been identified in patients with WAS. In addition, WASP­interacting proteins are suspected in patients with clinical features of WAS, in whom the WASP gene sequence and mRNA levels are normal. The present study aimed to investigate the application of next generation sequencing in definitive diagnosis and clinical therapy for WAS. A 5 month­old child with WAS who displayed symptoms of thrombocytopenia was examined. Whole exome sequence analysis of genomic DNA showed that the coverage and depth of WASP were extremely low. Quantitative polymerase chain reaction indicated total WASP gene deletion in the proband. In conclusion, high throughput sequencing is useful for the verification of WAS on the genetic profile, and has implications for family planning guidance and establishment of clinical programs.


Asunto(s)
Eliminación de Secuencia/genética , Proteína del Síndrome de Wiskott-Aldrich/genética , Adulto , Codón sin Sentido/genética , Análisis Mutacional de ADN/métodos , Exones/genética , Femenino , Eliminación de Gen , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Masculino , Mutación/genética , Fenotipo , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/metabolismo
15.
Haematologica ; 101(8): 896-908, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27478199

RESUMEN

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.


Asunto(s)
Trombocitopenia/etiología , Trombocitopenia/metabolismo , Animales , Autoantígenos/metabolismo , Síndrome de Bernard-Soulier/etiología , Síndrome de Bernard-Soulier/metabolismo , Plaquetas/metabolismo , Diferenciación Celular/genética , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Yoduro Peroxidasa/metabolismo , Proteínas de Unión a Hierro/metabolismo , Megacariocitos/citología , Megacariocitos/metabolismo , Ratones , Receptores de Trombopoyetina/metabolismo , Transducción de Señal , Trombocitopenia/diagnóstico , Trombopoyesis , Factores de Transcripción/metabolismo , Síndrome de Wiskott-Aldrich/etiología , Síndrome de Wiskott-Aldrich/metabolismo
16.
Blood ; 128(13): 1723-34, 2016 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-27330000

RESUMEN

Wiskott-Aldrich syndrome (WAS) pediatric patients exhibit a deficiency in humoral immune memory. However, the mechanism by which Wiskott-Aldrich syndrome protein (WASP) regulates the differentiation and activation of memory B cells remains elusive. Here we examine the early activation events of memory B cells from the peripheral blood mononuclear cells of WAS patients and age-matched healthy controls (HCs) using total internal reflection fluorescence microscopy. In response to stimulation through the B-cell receptor (BCR), memory B cells from HCs showed significantly higher magnitudes of BCR clustering and cell spreading than naive B cells from the same individuals. This was associated with increases in CD19 recruitment to the BCR and the activation of its downstream signaling molecule Btk and decreases in FcγRIIB recruitment and the activation of its downstream molecule Src homology 2-containing inositol 5' phosphatase (SHIP). However, these enhanced signaling activities mediated by CD19 and Btk are blocked in memory B cells from WAS patients, whereas the activation of FcγRIIB and SHIP was increased. Although the expression levels of CD19, Btk, and FcγRIIB did not change between CD27(-) and CD27(+) B cells of HCs, the protein and mRNA levels of CD19 but not Btk and FcγRIIB were significantly reduced in both CD27(-) and CD27(+) B cells of WAS patients, compared with those of HCs. Overall, our study suggests that WASP is required for memory B-cell activation, promoting the activation by positive regulating CD19 transcription and CD19 recruitment to the BCR.


Asunto(s)
Antígenos CD19/metabolismo , Linfocitos B/inmunología , Memoria Inmunológica , Síndrome de Wiskott-Aldrich/inmunología , Citoesqueleto de Actina/metabolismo , Antígenos CD19/genética , Linfocitos B/metabolismo , Estudios de Casos y Controles , Preescolar , Regulación hacia Abajo , Humanos , Lactante , Activación de Linfocitos , Mutación , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genética
17.
Biol Cell ; 108(8): 205-18, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27061938

RESUMEN

The trans-Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low-level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching-off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot-Marie-Tooth disease, I-cell disease and Wiskott-Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth/metabolismo , Endosomas/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Red trans-Golgi/metabolismo , Animales , Transporte Biológico Activo , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/patología , Endosomas/genética , Endosomas/patología , Humanos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patología , Red trans-Golgi/genética , Red trans-Golgi/patología
18.
Pediatr Int ; 58(1): 4-7, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26331277

RESUMEN

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease characterized by recurrent infection, thrombocytopenia, and eczema. The gene responsible for X-linked WAS encodes the Wiskott-Aldrich syndrome protein (WASP), which is expressed in hematopoietic cells and which regulates T-cell activation and cytoskeletal reorganization in T-cell receptor (TCR) signaling. Here, I review my recent research on WASP and the WASP-interacting protein (WIP) complex in T cells. I and my colleagues first established a diagnostic screening method using flow cytometry and genetic analysis, and elucidated the molecular pathogenesis in WAS patients with unique clinical manifestations. We investigated the mechanisms by which WASP is recruited to lipid rafts following TCR stimulation and to immunological synapses between antigen-presenting cells and T cells. Subsequently, we elucidated the molecular mechanisms by which WASP is degraded by calpain and ubiquitinated by Cbl-family proteins, which terminate WASP activation. More importantly, we found that WIP plays a critical role in WASP stability in T cells. These results provide new insights into the molecular pathogenesis of X-linked WAS and have facilitated the identification of WIP deficiency as an autosomal recessive form of WAS.


Asunto(s)
Proteína del Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Humanos , Transducción de Señal , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/biosíntesis
19.
Brain Behav ; 5(11): e00359, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26664784

RESUMEN

INTRODUCTION: Neuronal morphogenesis is governed mainly by two interconnected processes, cytoskeletal reorganization, and signal transduction. The actin-binding molecule WIP (Wiskott-Aldrich syndrome protein [WASP]-interacting protein) was identified as a negative regulator of neuritogenesis. Although WIP controls activity of the actin-nucleation-promoting factor neural WASP (N-WASP) during neuritic differentiation, its implication in signal transduction remains unknown. METHODS: Using primary neurons from WIP-deficient and wild-type mice we did an immunofluorescence, morphometric, and biochemical analysis of the signaling modified by WIP deficiency. RESULTS: Here, we describe the WIP contribution to the regulation of neuritic elaboration and ramification through modification in phosphorylation levels of several kinases that participate in the mammalian target of rapamycin complex 1 (mTORC1)-p70S6K (phosphoprotein 70 ribosomal protein S6 kinase, S6K) intracellular signaling pathway. WIP deficiency induces an increase in the number of neuritic bifurcations and filopodial protrusions in primary embryonic neurons. This phenotype is not due to modifications in the activity of the phosphoinositide 3 kinase (PI3K)-Akt pathway, but to reduced phosphorylation of the S6K residues Ser(411) and Thr(389). The resulting decrease in kinase activity leads to reduced S6 phosphorylation in the absence of WIP. Incubation of control neurons with pharmacological inhibitors of mTORC1 or Abl, two S6K regulators, conferred a morphology resembling that of WIP-deficient neurons. Moreover, the preferential co-distribution of phospho-S6K with polymerized actin is altered in WIP-deficient neurons. CONCLUSION: These experiments identify WIP as a member of a signaling cascade comprised of Abl family kinases, mTORC1 and S6K, which regulates neuron development and specifically, neuritic branching and complexity. Thus, we postulated a new role for WIP protein.


Asunto(s)
Proteínas Portadoras/metabolismo , Hipocampo/metabolismo , Complejos Multiproteicos/metabolismo , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Actinas/metabolismo , Animales , Proteínas del Citoesqueleto , Femenino , Hipocampo/citología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos , Neuronas/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Transducción de Señal , Síndrome de Wiskott-Aldrich/metabolismo
20.
PLoS One ; 10(10): e0139729, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26448644

RESUMEN

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.


Asunto(s)
Envejecimiento , Linfocitos B Reguladores/inmunología , Síndrome de Wiskott-Aldrich/patología , Animales , Antígenos CD19/metabolismo , Antígenos CD1d/metabolismo , Linfocitos B Reguladores/citología , Linfocitos B Reguladores/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD5/metabolismo , Técnicas de Cocultivo , Citocinas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Interleucina-10/análisis , Masculino , Ratones , Ratones Noqueados , Síndrome de Wiskott-Aldrich/metabolismo
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