RESUMO
While the shelterin complex guards and coordinates the mechanism of telomere regulation, deregulation of this process is tightly linked to malignant transformation and cancer. Here, we present the novel finding of a germline stop-gain variant (p.Q199*) in the shelterin complex gene POT1, which was identified in a child with acute myeloid leukemia. We show that the cells overexpressing the mutated POT1 display increased DNA damage and chromosomal instabilities compared to the wildtype counterpart. Protein and mRNA expression analyses in the primary patient cells further confirm that, physiologically, the variant leads to a nonfunctional POT1 allele in the patient. Subsequent telomere length measurements in the primary cells carrying heterozygous POT1 p.Q199* as well as POT1 knockdown AML cells revealed telomeric elongation as the main functional effect. These results show a connection between POT1 p.Q199* and telomeric dysregulation and highlight POT1 germline deficiency as a predisposition to myeloid malignancies in childhood.
Assuntos
Predisposição Genética para Doença/genética , Leucemia Mieloide Aguda/genética , Transtornos Mieloproliferativos/genética , Complexo Shelterina/genética , Proteínas de Ligação a Telômeros/genética , Adulto , Dano ao DNA/genética , Células Germinativas , Mutação em Linhagem Germinativa/genética , Células HEK293 , Humanos , Células Mieloides , RNA Mensageiro/genética , Telômero/genética , Adulto JovemRESUMO
Asthma and allergies are major health concerns in which Ig isotype E plays a pivotal role. Ag-bound IgE drives mast cells and basophils into exocytosis, thereby promoting allergic and potentially anaphylactic reactions. The importance of tightly regulated IgE production is underscored by severe immunological conditions in humans with elevated IgE levels. Cytokines direct IgH class-switching to a particular isotype by initiation of germline transcription (GLT) from isotype-specific intronic (I) promoters. The switch to IgE depends on IL-4, which stimulates GLT of the Iε promoter, but is specifically and strongly impaired in Swap-70(-/-) mice. Although early events in IL-4 signal transduction (i.e., activation of the JAK/STAT6 pathway) do not require SWAP-70, SWAP-70 deficiency results in impaired Iε GLT. The affinity of STAT6 to chromatin is reduced in absence of SWAP-70. Chromatin immunoprecipitation revealed that SWAP-70 binds to Iε and is required for association of STAT6 with Iε. BCL6, known to antagonize STAT6 particularly at Iε, is increased on Iε in absence of SWAP-70. Other promoters bound by BCL6 and STAT6 were found unaffected. We conclude that SWAP-70 controls IgE production through regulation of the antagonistic STAT6 and BCL6 occupancy of Iε. The identification of this mechanism opens new avenues to inhibit allergic reactions triggered by IgE.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Switching de Imunoglobulina/imunologia , Imunoglobulina E/biossíntese , Proteínas Nucleares/metabolismo , Fator de Transcrição STAT6/metabolismo , Células 3T3 , Animais , Linfócitos B/imunologia , Células Cultivadas , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Hipersensibilidade/imunologia , Switching de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias épsilon de Imunoglobulina/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais/imunologia , Transcrição GênicaRESUMO
Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.
Assuntos
Actinas , Neoplasias da Mama , Neoplasias Pulmonares , Proteínas dos Microfilamentos , Metástase Neoplásica , Animais , Actinas/metabolismo , Camundongos , Humanos , Feminino , Linhagem Celular Tumoral , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Movimento Celular/genética , Citoesqueleto de Actina/metabolismo , Proliferação de Células/genética , Adesão Celular/genética , Ligação ProteicaRESUMO
B-cell migration into and within lymphoid tissues is not only central to the humoral immune response but also for the development of malignancies and autoimmunity. We previously demonstrated that SWAP-70, an F-actin-binding, Rho GTPase-interacting protein strongly expressed in activated B cells, is necessary for normal B-cell migration in vivo. SWAP-70 regulates integrin-mediated adhesion and cell attachment. Here we show that upon B-cell activation, SWAP-70 is extensively posttranslationally modified and becomes tyrosine phosphorylated by SYK at position 517. This phosphorylation inhibits binding of SWAP-70 to F-actin. Phospho-site mutants of SWAP-70 disrupt B-cell polarization in a dominant-negative fashion in vitro and impair migration in vivo. After CXCL12 stimulation of B cells SYK becomes activated and SWAP-70 is phosphorylated in a SYK-dependent manner. Use of the highly specific SYK inhibitor BAY61-3606 showed SYK activity is necessary for normal chemotaxis and B-cell polarization in vitro and for entry of B cells into lymph nodes in vivo. These findings demonstrate a novel requirement for SYK in migration and polarization of naive recirculating B cells and show that SWAP-70 is an important target of SYK in this pathway.
Assuntos
Actinas/metabolismo , Linfócitos B/metabolismo , Movimento Celular , Proteínas de Ligação a DNA/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/fisiologia , Proteínas Tirosina Quinases/metabolismo , Tirosina/metabolismo , Animais , Linfócitos B/citologia , Western Blotting , Citometria de Fluxo , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Camundongos , Antígenos de Histocompatibilidade Menor , Fosforilação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/isolamento & purificação , Quinase SykRESUMO
The migration of B cells into secondary lymphoid organs is required for the generation of an effective immune response. Here we analyzed the involvement of SWAP-70, a Rac-interacting protein involved in actin rearrangement, in B cell entry into lymph nodes. We noted reduced migration of Swap70-/- B cells into lymph nodes in vivo. Swap70-/- B cells rolled and adhered, yet accumulated in lymph node high endothelial venules. This defect was not due to impaired integrin expression or chemotaxis. Instead, Swap70-/- B cells aberrantly regulated integrin-mediated adhesion. During attachment, Swap70-/- B cells showed defective polarization and did not form uropods or stabilize lamellipodia at a defined region. Thus, SWAP-70 selectively regulates processes essential for B cell entry into lymph nodes.