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1.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514372

RESUMO

Cellular regulation or signaling processes are mediated by many proteins which often have one or several intrinsically disordered regions (IDRs). These IDRs generally serve as binders to different proteins with high specificity. In many cases, IDRs undergo a disorder-to-order transition upon binding, following a mechanism between two possible pathways, the induced fit or the conformational selection. Since these mechanisms contribute differently to the kinetics of IDR associations, it is important to investigate them in order to gain insight into the physical factors that determine the biomolecular recognition process. The verprolin homology domain (V) of the Neural Wiskott-Aldrich Syndrome Protein (N-WASP), involved in the regulation of actin polymerization, is a typical example of IDR. It is composed of two WH2 motifs, each being able to bind one actin molecule. In this study, we investigated the early steps of the recognition process of actin by the WH2 motifs of N-WASP domain V. Using docking calculations and molecular dynamics simulations, our study shows that actin is first recognized by the N-WASP domain V regions which have the highest propensity to form transient α -helices. The WH2 motif consensus sequences "LKKV" subsequently bind to actin through large conformational changes of the disordered domain V.


Assuntos
Actinas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Probabilidade , Domínios Proteicos , Multimerização Proteica , Fatores de Tempo
2.
Immunol Cell Biol ; 97(4): 389-402, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30779216

RESUMO

Inherited defects in genes encoding for proteins that are involved in the assembly and dynamics of the actin skeleton have increasingly been identified in patients presenting with primary immunodeficiencies. In this review, we summarize a subset of the recently described conditions, emphasizing the clinical features as well as the immunophenotype and pathophysiology.


Assuntos
Citoesqueleto de Actina/genética , Proteínas do Citoesqueleto/metabolismo , Imunidade/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Humanos , /fisiopatologia , /terapia
3.
Mol Biol Cell ; 30(5): 579-590, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30601711

RESUMO

Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Músculo Esquelético/metabolismo , Animais , Desmina/metabolismo , Dinamina II/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/ultraestrutura , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Mutação/genética , Miopatias Congênitas Estruturais/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
4.
Nat Med ; 25(1): 130-140, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510251

RESUMO

In T lymphocytes, the Wiskott-Aldrich Syndrome protein (WASP) and WASP-interacting-protein (WIP) regulate T cell antigen receptor (TCR) signaling, but their role in lymphoma is largely unknown. Here we show that the expression of WASP and WIP is frequently low or absent in anaplastic large cell lymphoma (ALCL) compared to other T cell lymphomas. In anaplastic lymphoma kinase-positive (ALK+) ALCL, WASP and WIP expression is regulated by ALK oncogenic activity via its downstream mediators STAT3 and C/EBP-ß. ALK+ lymphomas were accelerated in WASP- and WIP-deficient mice. In the absence of WASP, active GTP-bound CDC42 was increased and the genetic deletion of one CDC42 allele was sufficient to impair lymphoma growth. WASP-deficient lymphoma showed increased mitogen-activated protein kinase (MAPK) pathway activation that could be exploited as a therapeutic vulnerability. Our findings demonstrate that WASP and WIP are tumor suppressors in T cell lymphoma and suggest that MAP-kinase kinase (MEK) inhibitors combined with ALK inhibitors could achieve a more potent therapeutic effect in ALK+ ALCL.


Assuntos
Linfoma de Células T/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Quinase do Linfoma Anaplásico/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteínas do Citoesqueleto/metabolismo , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Linfoma de Células T/enzimologia , Linfoma de Células T/patologia , Sistema de Sinalização das MAP Quinases , Camundongos , Ligação Proteica , Fator de Transcrição STAT3/metabolismo , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína cdc42 de Ligação ao GTP/metabolismo
5.
J Clin Invest ; 128(9): 4115-4131, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-30124469

RESUMO

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Neutropenia/genética , Neutropenia/metabolismo , Neutrófilos/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Feminino , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutropenia/congênito , Neutrófilos/ultraestrutura , Fagocitose , Fosforilação , Conformação Proteica , Proteína da Síndrome de Wiskott-Aldrich/química
6.
Proc Natl Acad Sci U S A ; 115(37): E8642-E8651, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30150414

RESUMO

We used fluorescence spectroscopy and EM to determine how binding of ATP, nucleation-promoting factors, actin monomers, and actin filaments changes the conformation of Arp2/3 complex during the process that nucleates an actin filament branch. We mutated subunits of Schizosaccharomyces pombe Arp2/3 complex for labeling with fluorescent dyes at either the C termini of Arp2 and Arp3 or ArpC1 and ArpC3. We measured Förster resonance energy transfer (FRET) efficiency (ETeff) between the dyes in the presence of the various ligands. We also computed class averages from electron micrographs of negatively stained specimens. ATP binding made small conformational changes of the nucleotide-binding cleft of the Arp2 subunit. WASp-VCA, WASp-CA, and WASp-actin-VCA changed the ETeff between the dyes on the Arp2 and Arp3 subunits much more than between dyes on ArpC1 and ArpC3. Ensemble FRET detected an additional structural change that brought ArpC1 and ArpC3 closer together when Arp2/3 complex bound actin filaments. VCA binding to Arp2/3 complex causes a conformational change that favors binding to the side of an actin filament, which allows further changes required to nucleate a daughter filament.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Trifosfato de Adenosina/química , Transferência Ressonante de Energia de Fluorescência , Microscopia Eletrônica de Transmissão , Mutação , Ligação Proteica , Conformação Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteína da Síndrome de Wiskott-Aldrich/química
7.
J Exp Clin Cancer Res ; 37(1): 141, 2018 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-29986736

RESUMO

BACKGROUND: Metastasis is the leading cause of cancer mortality and is a major hurdle for lung cancer treatment. Invadopodia, which are cancer-specific protrusive structures, play a crucial role in the metastatic cascade through degradation of the basement membrane and surrounding stroma. Cortactin, a critical component of invadopodia, frequently used as an invadopodia marker, a universally important player in invadopodia function, and is frequently overexpressed in cancer, but the exact mechanism of regulation is not yet fully understood. METHODS: The expression level of CTTN in human non-small cell lung cancer (NSCLC) tissues was detected by qRT-PCR. Cell migration, invasion and invadopodia formation were assessed in vitro by wound-healing, transwell assay and immunofluorescence, respectively. The dual-luciferase reporter assay was used to identify the direct target of miR-182. RESULTS: Hepatocyte growth factor (HGF) and phorbol 12,13-dibutyrate (PDBu) can induce CTTN expression, motility, and invasion ability, as well as invadopodia formation in non-small cell lung cancer (NSCLC). Moreover, miR-182 suppressed metastasis and invadopodia formation by targeting CTTN in NSCLC. Our qRT-PCR results showed that CTTN expression was inversely correlated with miR-182 expression that suppressed invadopodia formation via suppression of the Cdc42/N-WASP pathway. Furthermore, miR-182 negatively regulated invadopodia function, and suppressed extracellular matrix(ECM) degradation in lung cancer cells by inhibiting cortactin. CONCLUSION: Collectively, our results demonstrated that miR-182 targeted CTTN gene in NSCLC and suppressed lung cancer invadopodia formation, and thus suppressed lung cancer metastasis. This suggests a therapeutic application of miR-182 in NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Cortactina/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , MicroRNAs/genética , Podossomos/genética , Regiões 3' não Traduzidas , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Interferência de RNA , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
8.
J Cell Sci ; 131(13)2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29853633

RESUMO

The cell wall integrity checkpoint monitors synthesis of cell wall materials during the Saccharomyces cerevisiae cell cycle. Upon perturbation of cell wall synthesis, the cell wall integrity checkpoint is activated, downregulating Clb2 transcription. Here, we identified genes involved in this checkpoint by genetic screening of deletion mutants. In addition to the previously identified dynactin complex, the Las17 complex, in particular the Bzz1 and Vrp1 components, plays a role in this checkpoint. We also revealed that the high osmolarity glycerol (HOG) and cell wall integrity mitogen-activated protein kinase (MAPK) signaling pathways are essential for checkpoint function. The defective checkpoint caused by the deficient dynactin and Las17 complexes was rescued by hyperactivation of the cell wall integrity MAPK pathway, but not by the activated form of Hog1, suggesting an order to these signaling pathways. Mutation of Fkh2, a transcription factor important for Clb2 expression, suppressed the checkpoint-defective phenotype of Las17, HOG MAPK and cell wall integrity MAPK mutations. These results provide genetic evidence that signaling from the cell surface regulates the downstream transcriptional machinery to activate the cell wall integrity checkpoint.


Assuntos
Parede Celular/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Parede Celular/genética , Ciclina B/genética , Ciclina B/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
9.
Nature ; 559(7712): 61-66, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29925947

RESUMO

DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Núcleo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo de DNA por Recombinação , Xenopus laevis/genética , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/metabolismo , Animais , Extratos Celulares , Cromatina/metabolismo , Reparo do DNA por Junção de Extremidades , Feminino , Movimento , Ligação Proteica , Transporte Proteico , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
10.
Nat Commun ; 9(1): 1779, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725003

RESUMO

Mutations in Wiskott-Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.


Assuntos
Colite/imunologia , Homeostase , Inflamação/imunologia , Interleucina-10/fisiologia , Mucosa Intestinal/metabolismo , Macrófagos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Colite/prevenção & controle , Deleção de Genes , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Imunidade nas Mucosas , Interleucina-10/metabolismo , Interleucina-1beta/fisiologia , Interleucina-23/fisiologia , Mucosa Intestinal/imunologia , Macrófagos/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
11.
Sci Signal ; 11(528)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29717064

RESUMO

Glycerol monolaurate (GML) is a monoglyceride with potent antimicrobial properties that suppresses T cell receptor (TCR)-induced signaling and T cell effector function. Actin rearrangement is needed for the interaction of T cells with antigen-presenting cells and for migration to sites of infection. Because of the critical role actin rearrangement plays in T cell effector function, we analyzed the effect of GML on the rearrangement of the actin cytoskeleton after TCR activation. We found that GML-treated human T cells were less adherent than untreated T cells and did not form actin ring structures but instead developed numerous inappropriate actin-mediated filopodia. The formation of these filopodia was not due to disruption of TCR-proximal regulators of actin or microtubule polymerization. Instead, total internal reflection fluorescence microscopy demonstrated mislocalization of actin nucleation protein Arp2 microclusters, but not those containing the adaptor proteins SLP-76 and WASp, or the actin nucleation protein ARPC3, which are necessary for TCR-induced actin rearrangement. Additionally, SLP-76 microclusters colocalized with WASp and WAVE microclusters but not with LAT. Together, our data suggest that GML alters actin cytoskeletal rearrangements and identify diverse functions for GML as a T cell-suppressive agent.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Lauratos/farmacologia , Proteínas de Membrana/metabolismo , Monoglicerídeos/farmacologia , Fosfoproteínas/metabolismo , Pseudópodes/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Ativação Linfocitária/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Pseudópodes/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tensoativos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
12.
J Leukoc Biol ; 103(5): 867-883, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29656494

RESUMO

The uptake of cholesterol carried by low-density lipoprotein (LDL) is tightly controlled in the body. Macrophages are not well suited to counteract the cellular consequences of excess cholesterol leading to their transformation into "foam cells," an early step in vascular plaque formation. We have uncovered and characterized a novel mechanism involving phospholipase D (PLD) in foam cell formation. Utilizing bone marrow-derived macrophages from genetically PLD deficient mice, we demonstrate that PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL (Agg-Ox-LDL), which was phenocopied with a PLD2-selective inhibitor. We also report a role for PLD2 in coupling Agg-oxLDL phagocytosis with WASP, Grb2, and Actin. Further, the clearance of LDL particles is mediated by both CD36 and PLD2, via mutual dependence on each other. In the absence of PLD2, CD36 does not engage in Agg-Ox-LDL removal and when CD36 is blocked, PLD2 cannot form protein-protein heterocomplexes with WASP or Actin. These results translated into humans using a GEO database of microarray expression data from atheroma plaques versus normal adjacent carotid tissue and observed higher values for NFkB, PLD2 (but not PLD1), WASP, and Grb2 in the atheroma plaques. Human atherectomy specimens confirmed high presence of PLD2 (mRNA and protein) as well as phospho-WASP in diseased arteries. Thus, PLD2 interacts in macrophages with Actin, Grb2, and WASP during phagocytosis of Agg-Ox-LDL in the presence of CD36 during their transformation into "foam cells." Thus, this study provides new molecular targets to counteract vascular plaque formation and atherogenesis.


Assuntos
Antígenos CD36/metabolismo , Células Espumosas/patologia , Lipoproteínas LDL/metabolismo , Fagocitose , Fosfolipase D/fisiologia , Placa Aterosclerótica/patologia , Animais , Antígenos CD36/genética , Células Cultivadas , Colesterol/metabolismo , Feminino , Células Espumosas/metabolismo , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica/genética , Placa Aterosclerótica/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
13.
Int J Mol Sci ; 19(2)2018 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-29382077

RESUMO

A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott-Aldrich-Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP.


Assuntos
Fatores de Despolimerização de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Cones de Crescimento/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Gânglios Espinais/citologia , Cones de Crescimento/efeitos dos fármacos , Quinases Lim/metabolismo , Transporte Proteico , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
14.
Proc Natl Acad Sci U S A ; 115(7): E1409-E1418, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29386393

RESUMO

Arp2/3 complex nucleates branched actin filaments important for cellular motility and endocytosis. WASP family proteins are Arp2/3 complex activators that play multiple roles in branching nucleation, but little is known about the structural bases of these WASP functions, owing to an incomplete understanding of how WASP binds Arp2/3 complex. Recent data show WASP binds two sites, and biochemical and structural studies led to models in which the WASP C segment engages the barbed ends of the Arp3 and Arp2 subunits while the WASP A segment binds the back side of the complex on Arp3. However, electron microscopy reconstructions showed density for WASP inconsistent with these models on the opposite (front) side of Arp2/3 complex. Here we use chemical cross-linking and mass spectrometry (XL-MS) along with computational docking and structure-based mutational analysis to map the two WASP binding sites on the complex. Our data corroborate the barbed end and back side binding models and show one WASP binding site on Arp3, on the back side of the complex, and a second site on the bottom of the complex, spanning Arp2 and ARPC1. The XL-MS-identified cross-links rule out the front side binding model and show that the A segment of WASP binds along the bottom side of the ARPC1 subunit, instead of at the Arp2/ARPC1 interface, as suggested by FRET experiments. The identified binding sites support the Arp3 tail release model to explain WASP-mediated activating conformational changes in Arp2/3 complex and provide insight into the roles of WASP in branching nucleation.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Sequência de Aminoácidos , Sítios de Ligação , Ligação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência , Proteína da Síndrome de Wiskott-Aldrich/química
15.
ACS Chem Biol ; 13(1): 100-109, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29215267

RESUMO

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/ß-fold and central two-winged ß-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Sítios de Ligação , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Epitopos , Transferência Ressonante de Energia de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Jurkat , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Imagem Molecular/métodos , Complexos Multiproteicos , Mutação , Domínios Proteicos , Dobramento de Proteína , Ubiquitinação , Proteína da Síndrome de Wiskott-Aldrich/genética
16.
Immunology ; 153(1): 42-50, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28771705

RESUMO

The T-cell receptor (TCR)-CD3 complex, expressed on T cells, determines the outcome of a T-cell response. It consists of the TCR-αß heterodimer and the non-covalently associated signalling dimers of CD3εγ, CD3εδ and CD3ζζ. TCR-αß binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR-CD3 complex upon antigen binding to TCR-αß have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP-70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.


Assuntos
Proteínas de Transporte/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Complexo CD3/química , Complexo CD3/genética , Complexo CD3/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo
17.
Nat Commun ; 8(1): 2210, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29263363

RESUMO

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.


Assuntos
Actinas/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Células Endoteliais/metabolismo , Junções Intercelulares/metabolismo , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Actinas/efeitos dos fármacos , Antígenos CD/efeitos dos fármacos , Caderinas/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Adesão Celular , Movimento Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular , Células Endoteliais da Veia Umbilical Humana , Humanos , Junções Intercelulares/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Modelos Cardiovasculares , Cadeias Leves de Miosina/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Remodelação Vascular , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo
18.
Nat Commun ; 8(1): 1576, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-29146903

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Autofagia/imunologia , Inflamassomos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Animais , Autofagia/genética , Carga Bacteriana/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Escherichia coli Enteropatogênica/imunologia , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nigericina/farmacologia , Septinas/metabolismo , Shigella flexneri/imunologia , Células THP-1 , Receptor 4 Toll-Like/imunologia , Síndrome de Wiskott-Aldrich/metabolismo
19.
Cell Death Dis ; 8(10): e3114, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022901

RESUMO

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Azacitidina/uso terapêutico , Carcinogênese/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/genética
20.
mBio ; 8(5)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874467

RESUMO

Many fundamental biological discoveries have been made in Caenorhabditis elegans The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for Orsay virus infection, we designed a forward genetic screen that utilizes a virally induced green fluorescent protein (GFP) reporter. Following chemical mutagenesis, two Viro (virus induced reporter off) mutants that failed to express GFP were mapped to sid-3, a nonreceptor tyrosine kinase, and B0280.13 (renamed viro-2), an ortholog of human Wiskott-Aldrich syndrome protein (WASP). Both mutants yielded Orsay virus RNA levels comparable to that of the residual input virus, suggesting that they are not permissive for Orsay virus replication. In addition, we demonstrated that both genes affect an early prereplication stage of Orsay virus infection. Furthermore, it is known that the human ortholog of SID-3, activated CDC42-associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that VIRO-2 may be a substrate for SID-3 in C. elegans A targeted RNA interference (RNAi) knockdown screen further identified the C. elegans gene nck-1, which has a human ortholog that interacts with TNK2 and WASP, as required for Orsay virus infection. Thus, genetic screening in C. elegans identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.IMPORTANCE Orsay virus is the only known virus capable of naturally infecting the model organism Caenorhabditis elegans, which shares many evolutionarily conserved genes with humans. We exploited the robust genetic tractability of C. elegans to identify three host genes, sid-3, viro-2, and nck-1, which are essential for Orsay virus infection. Mutant animals that lack these three genes are highly defective in viral replication. Strikingly, the human orthologs of these three genes, activated CDC42-associated kinase (TNK2), Wiskott-Aldrich syndrome protein (WASP), and noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) are part of a known signaling pathway in mammals. These results suggest that TNK2, WASP, and NCK1 may play important roles in mammalian virus infection.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/virologia , Evolução Molecular , Nodaviridae/fisiologia , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Fluorescência Verde/genética , Interações Hospedeiro-Patógeno , Mutagênese , Nodaviridae/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Viral/genética , Viroses , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
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