RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies. A specific mechanism of its metastasis has not been established. In this study, we investigated whether Neural Wiskott-Aldrich syndrome protein (N-WASP) plays a role in distant metastasis of PDAC. We found that N-WASP is markedly expressed in clinical patients with PDAC. Clinical analysis showed a notably more distant metastatic pattern in the N-WASP-high group compared to the N-WASP-low group. N-WASP was noted to be a novel mediator of epithelial-mesenchymal transition (EMT) via gene expression profile studies. Knockdown of N-WASP in pancreatic cancer cells significantly inhibited cell invasion, migration, and EMT. We also observed positive association of lysyl oxidase-like 2 (LOXL2) and focal adhesion kinase (FAK) with the N-WASP-mediated response, wherein EMT and invadopodia function were modulated. Both N-WASP and LOXL2 depletion significantly reduced the incidence of liver and lung metastatic lesions in orthotopic mouse models of pancreatic cancer. These results elucidate a novel role for N-WASP signaling associated with LOXL2 in EMT and invadopodia function, with respect to regulation of intercellular communication in tumor cells for promoting pancreatic cancer metastasis. These findings may aid in the development of therapeutic strategies against pancreatic cancer.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Camundongos , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
BACKGROUND: WAS gene mutational analysis is crucial to establish a definite diagnosis of Wiskott-Aldrich syndrome (WAS). Data on the genetic background of WAS in Vietnamese patients have not been reported. METHODS: We recruited 97 male, unrelated patients with WAS and analyzed WAS gene mutation using Sanger sequencing technology. RESULTS: We identified 36 distinct hemizygous pathogenic mutations, with 17 novel variants, from 38 patients in the entire cohort (39.2%). The mutational spectrum included 14 missense, 12 indel, five nonsense, four splicing, and one non-stop mutations. Most mutations appear only once, with the exception of c.37C>T (p.R13X) and c.374G>A (p.G125E) each of which occurs twice in unrelated patients. CONCLUSION: Our data enrich the mutational spectrum of the WAS gene and are crucial for understanding the genetic background of WAS and for supporting genetic counseling.
Assuntos
Síndrome de Wiskott-Aldrich , Humanos , Masculino , Análise Mutacional de DNA , Mutação , Vietnã , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.
Assuntos
Antígeno B7-H1 , Neoplasias , Proteômica , Microambiente Tumoral , Proteína da Síndrome de Wiskott-Aldrich , Humanos , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Proteômica/métodos , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Neoplasias/metabolismo , Neoplasias/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Linhagem Celular TumoralRESUMO
BACKGROUND: T-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. METHODS: Pediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. RESULTS: We discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. CONCLUSION: Our findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.
Assuntos
Reparo por Excisão , Instabilidade Genômica , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Células Th1 , Proteína da Síndrome de Wiskott-Aldrich , Criança , Humanos , Dano ao DNA , Instabilidade Genômica/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Células Th1/imunologia , Transcrição Gênica , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.
Assuntos
Genótipo , Síndrome de Wiskott-Aldrich , Humanos , Adolescente , Criança , Masculino , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/terapia , Feminino , Pré-Escolar , Adulto , Estudos Retrospectivos , Lactente , Adulto Jovem , Biomarcadores , Transplante de Células-Tronco Hematopoéticas , Índice de Gravidade de Doença , Proteína da Síndrome de Wiskott-Aldrich/genética , Seguimentos , Pessoa de Meia-Idade , Prognóstico , Taxa de SobrevidaRESUMO
The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-ß stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-ß, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.
Assuntos
Diferenciação Celular , Proliferação de Células , Linfócitos T Reguladores , Timo , Proteína da Síndrome de Wiskott-Aldrich , Animais , Linfócitos T Reguladores/imunologia , Diferenciação Celular/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Camundongos , Timo/imunologia , Timo/citologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Interleucina-2/metabolismo , Interleucina-2/imunologia , Mutação , Fator de Crescimento Transformador beta/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Subunidade alfa de Receptor de Interleucina-2/genética , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Proteínas de Saccharomyces cerevisiae , Proteína da Síndrome de Wiskott-Aldrich , Animais , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Sítios de Ligação , Mamíferos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Long noncoding RNA MYLK antisense RNA 1 (MYLK-AS1) is the crux in multiple diseases. Therefore, the purpose of this study was to investigate the possible mechanism of MYLK-AS1. A total of 62 colon cancer (CC) specimens and paired adjacent normal tissues were collected, and the expression of MYLK-AS1, microRNA (miR)-101-5p/cell division cycle 42 (CDC42) was detected. CC cell lines were transfected with MYLK-AS1, miR-101-5p, CDC42-related plasmids, and the biological functions and markers of epithelial-mesenchymal transition (EMT) were analyzed. The binding relationship between MYLK-AS1, miR-101-5p, and CDC42 was evaluated. In CC tissues and cell lines, MYLK-AS1 and CDC42 were highly expressed, and miR-101-5p was lowly expressed. Inhibition of MYLK-AS1 or upregulation of miR-101-5p can inhibit CC cell growth and EMT. miR-101-5p inhibited CDC42/N-wasp axis activation in CC cells by targeting CDC42. Knockdown of CDC42 or upregulation of miR-101-5p partially reversed the effects caused by upregulation of MYLK-AS1. MYLK-AS1, which is significantly upregulated in CC, may be a molecular sponge for miR-101-5p, and MYLK-AS1 promotes the activation of the CDC42/N-wasp axis in CC cells by targeting CDC42 through miR-101-5p, which in turn promotes tumor development. MYLK-AS1 may be a potential biomarker and target for CC therapy.
Assuntos
Neoplasias do Colo , MicroRNAs , RNA Longo não Codificante , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Transição Epitelial-Mesenquimal/genética , Neoplasias do Colo/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Proteínas de Ligação ao Cálcio/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismoRESUMO
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.
Assuntos
Neoplasias Hematológicas , Neoplasias , Síndrome de Wiskott-Aldrich , Humanos , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/terapia , Neoplasias Hematológicas/genética , Biologia Molecular , Actinas/metabolismoRESUMO
Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer which is the deadliest type of cancer for both men and women. Previous studies already showed that cell-intrinsic loss of WASp causes B cell tolerance and WASp deficiency in T helper (TH) cells is linked to negative effects on cytokine gene transcription necessary for TH1 differentiation. In the current study, we investigated the molecular mechanisms involved in WASp-mediated epigenetic regulation of B cell differentiation during NSCLC. Our ChIP-qPCR data suggest the less percentage enrichment of the B cell differentiating factors (Ikaros, Pax5, PU.1, BATF) and WASp across the WAS gene in the B cells of NSCLC patients in comparison with normal healthy donors and overexpression of WASp showed the reverse effects. WASp-depleted B cells while co-culturing with respective PBMCs isolated from normal healthy donors and NSCLC patients, we observed upregulation of TH2-, TH17-, and Treg-specific cytokines (IL4, ILI7A, IL10) & transcription factors (GATA3, RORC, FOXP3) and downregulation of TH1-specific cytokine (IFNγ) & transcription factor (TBX21). Our study showed that the overexpression of WASp resulted into upregulation of B cell differentiating factors, tumor suppressor protein (p53), histone methylation marker (H3K4me3) with concomitant downregulation of tumor-promoting factors (Notch 1, ß-Catenin, DNAPKcs) and histone deacetylation marker (HDAC2) and increase in percentage cytotoxicity of NSCLC-specific cells (A549). Successful overexpression of WASp not only helps in epigenetic regulation of B cell differentiation but also supports tumor suppression in NSCLC. Thus, WASp can be targeted for therapeutic intervention of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Proteína da Síndrome de Wiskott-Aldrich , Feminino , Humanos , Masculino , Carcinoma Pulmonar de Células não Pequenas/genética , Diferenciação Celular/genética , Citocinas/metabolismo , Epigênese Genética , Histonas/metabolismo , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Linfócitos B/metabolismoRESUMO
Cortactin coactivates Arp2/3 complex synergistically with WASP-family nucleation-promoting factors (NPFs) and stabilizes branched networks by linking Arp2/3 complex to F-actin. It is poorly understood how cortactin performs these functions. We describe the 2.89 Å resolution cryo-EM structure of cortactin's N-terminal domain (Cort1-76) bound to Arp2/3 complex. Cortactin binds Arp2/3 complex through an inverted Acidic domain (D20-V29), which targets the same site on Arp3 as the Acidic domain of NPFs but with opposite polarity. Sequences N- and C-terminal to cortactin's Acidic domain do not increase its affinity for Arp2/3 complex but contribute toward coactivation with NPFs. Coactivation further increases with NPF dimerization and for longer cortactin constructs with stronger binding to F-actin. The results suggest that cortactin contributes to Arp2/3 complex coactivation with NPFs in two ways, by helping recruit the complex to F-actin and by stabilizing the short-pitch (active) conformation, which are both byproducts of cortactin's core function in branch stabilization.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Cortactina , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Cortactina/metabolismo , Actinas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismoRESUMO
Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.
Assuntos
Eczema , Transplante de Células-Tronco Hematopoéticas , Síndrome de Wiskott-Aldrich , Humanos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Proteína da Síndrome de Wiskott-Aldrich/genética , Células-Tronco Hematopoéticas/metabolismo , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Terapia Genética/métodos , Eczema/etiologia , Eczema/metabolismo , Eczema/terapiaRESUMO
The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency caused by a mutation in the WAS gene. This leads to altered or absent WAS protein (WASp) expression and function resulting in thrombocytopenia, eczema, recurrent infections, and autoimmunity. In T cells, WASp is required for immune synapse formation. Patients with WAS show reduced numbers of peripheral blood T lymphocytes and an altered T-cell receptor repertoire. In vitro, their peripheral T cells show decreased proliferation and cytokine production upon aCD3/aCD28 stimulation. It is unclear whether these T-cell defects are acquired during peripheral activation or are, in part, generated during thymic development. Here, we assessed the role of WASp during T-cell differentiation using artificial thymic organoid cultures and in the thymus of humanized mice. Although CRISPR/Cas9 WAS knockout hematopoietic stem and progenitor cells (HSPCs) rearranged the T-cell receptor and differentiated to T-cell receptor (TCR)+ CD4+ CD8+ double-positive (DP) cells similar to wild-type HSPCs, a partial defect in the generation of CD8 single-positive (SP) cells was observed, suggesting that WASp is involved in their positive selection. TCR repertoire analysis of the DP and CD8+ SP population, however, showed a polyclonal repertoire with no bias toward autoreactivity. To our knowledge, this is the first study of the role of WASp in human T-cell differentiation and on TCR repertoire generation.
Assuntos
Proteína da Síndrome de Wiskott-Aldrich , Síndrome de Wiskott-Aldrich , Humanos , Animais , Camundongos , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Linhagem da Célula , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Diferenciação CelularAssuntos
Dermatite , Transplante de Células-Tronco Hematopoéticas , Síndrome de Wiskott-Aldrich , Humanos , Síndrome de Wiskott-Aldrich/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Anticorpos Monoclonais Humanizados/uso terapêutico , Proteína da Síndrome de Wiskott-AldrichRESUMO
Background: Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Results: Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. Conclusion: We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.
Assuntos
Diacilglicerol Quinase , Proteína da Síndrome de Wiskott-Aldrich , Humanos , Diacilglicerol Quinase/genética , Diglicerídeos , Interleucina-2 , Receptores de Antígenos de Linfócitos TRESUMO
BACKGROUND: Wiskott-Aldrich syndrome is an Inborn Error of Immunity characterized by thrombocytopenia, small platelets, severe eczema, recurrent infections, tendency to autoimmune diseases and neoplasms. The diagnosis of the syndrome can be difficult, especially when platelets are of normal size. CASE REPORT: A three-year-old male patient was referred to a specialized sector of university hospital for presenting acute otitis media that progressed to sepsis by Haemophilus influenzae. At one month of age, he had been diagnosed with autoimmune thrombocytopenia, and splenectomy was performed at two years of age. During follow-up, three hospitalizations were necessary: an infection by Streptococcus pneumoniae, which progressed to sepsis; one due to exacerbation of eczema, isolating S. epidermidis; another due to fever of undetermined origin. The tests showed normal number of platelets after splenectomy, platelets always with normal size. At age four, tests were performed: IgE 3128 Ku/L; IgA, IgG, and normal anti-polysaccharide antibodies; decreased IgM; decrease CD19, TCD4, naïve T and B; increased TCD8; normal NK. A diagnostic hypothesis of "probable" WAS was made. Genetic research has identified the c.295C>T mutation in the WAS gene. CONCLUSIONS: The case reported expressed a new mutation in the SWA gene, characterized by clinical manifestations of the mild phenotype of Wiskott-Aldrich syndrome, with thrombocytopenia, platelets of normal size, and X-linked inheritance. It is important to establish the early diagnosis and treatment to offer a better quality of life in these patients.
ANTECEDENTES: El síndrome de Wiskott-Aldrich es un error innato de la inmunidad, distinguido por trombocitopenia, plaquetas pequeñas, eccema severo, infecciones recurrentes, y susceptibilidad a enfermedades autoinmunes y neoplasias. El diagnóstico es difícil de establecer, especialmente cuando las plaquetas son de tamaño normal. REPORTE DE CASO: Paciente masculino de 3 años, enviado al Hospital Universitario da Santa Casa de São Paulo, Brasil, por otitis media aguda, con evolución a sepsis por Haemophilus influenzae. Al mes de edad fue diagnosticado con trombocitopenia autoinmune, y a los 2 años se llevó a cabo explenectomía. Durante el seguimiento requirió tres hospitalizaciones: una por infección por Streptococcus pneumoniae, que evolucionó a sepsis; otra por exacerbación de eccema, aislándose S. epidermidis, y la última por fiebre de origen indeterminado. Las pruebas de laboratorio informaron: concentración de plaquetas dentro de los valores de referencia después de la esplenectomía, y de tamaño normal. A los 4 años se efectuaron nuevas pruebas, que reportaron: IgE 3128 kU/L; IgA, IgG y anticuerpos anti-polisacáridos normales; disminución de IgM y de CD19, TCD4, T y B vírgenes; aumento de TCD8; NK normales. Se sospechó el diagnóstico de síndrome de Wiskott-Aldrich. Mediante estudios de genética se identificó la mutación c.295C>T en el gen WAS. CONCLUSIONES: El caso aquí expuesto expresó una nueva mutación en el gen SWA, caracterizado por manifestaciones clínicas de fenotipo leve del síndrome de Wiskott-Aldrich, con trombocitopenia, plaquetas de tamaño normal y herencia ligada al cromosoma X. Es importante establecer el diagnóstico y tratamiento oportunos para ofrecer una mejor calidad de vida en estos pacientes.
Assuntos
Eczema , Sepse , Trombocitopenia , Síndrome de Wiskott-Aldrich , Humanos , Masculino , Mutação , Qualidade de Vida , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Pré-EscolarRESUMO
The higher-order assembly of Bin-amphiphysin-Rvs (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, into lattice on the membrane is essential for the formation of subcellular structures. However, the regulation of their ordered assembly has not been elucidated. Here, we show that the higher ordered assembly of growth-arrested specific 7 (GAS7), an F-BAR domain protein, is regulated by the multivalent scaffold proteins of Wiskott-Aldrich syndrome protein (WASP)/neural WASP, that commonly binds to the BAR domain superfamily proteins, together with WISH, Nck, the activated small guanosine triphosphatase Cdc42, and a membrane-anchored phagocytic receptor. The assembly kinetics by fluorescence resonance energy transfer monitoring indicated that the GAS7 assembly on liposomes started within seconds and was further increased by the presence of these proteins. The regulated GAS7 assembly was abolished by Wiskott-Aldrich syndrome mutations both in vitro and in cellular phagocytosis. Therefore, Cdc42 and the scaffold proteins that commonly bind to the BAR domain superfamily proteins promoted GAS7 assembly.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteína da Síndrome de Wiskott-Aldrich , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Actinas/metabolismoRESUMO
Wiskott-Aldrich syndrome is an X-linked recessive primary immune-deficiency disorder very rarely reported from black African children. A 12-year old boy with recurrent sinopulmonary and diarrheal infections, eczema, thrombocytopenia, and low platelet volume was found by whole genome sequencing to harbor a predicted pathogenic c.1205dupC (p.Pro403Alafs*92) variant of a mutation in the WAS gene - confirming the diagnosis. This case report summarizes his presentation and management and provides a useful summary of the diagnosis and the responsible novel genetic mutation.
Assuntos
Eczema , Trombocitopenia , Síndrome de Wiskott-Aldrich , Masculino , Criança , Humanos , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Mutação/genéticaRESUMO
The actin-rich cortex plays a fundamental role in many cellular processes. Its architecture and molecular composition vary across cell types and physiological states. The full complement of actin assembly factors driving cortex formation and how their activities are spatiotemporally regulated remain to be fully elucidated. Using Dictyostelium as a model for polarized and rapidly migrating cells, we show that GxcM, a RhoGEF localized specifically in the rear of migrating cells, functions together with F-BAR protein Fbp17, a small GTPase RacC, and the actin nucleation-promoting factor WASP to coordinately promote Arp2/3 complex-mediated cortical actin assembly. Overactivation of this signaling cascade leads to excessive actin polymerization in the rear cortex, whereas its disruption causes defects in cortical integrity and function. Therefore, apart from its well-defined role in the formation of the protrusions at the cell front, the Arp2/3 complex-based actin carries out a previously unappreciated function in building the rear cortical subcompartment in rapidly migrating cells.
Assuntos
Actinas , Dictyostelium , Proteínas de Protozoários , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismoRESUMO
The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease.