RESUMO
To enter epithelial cells, the obligate intracellular pathogen Chlamydia pneumoniae secretes early effector proteins, which bind to and modulate the host-cell's plasma membrane and recruit several pivotal endocytic host proteins. Here, we present the high-resolution structure of an entry-related chlamydial effector protein, SemD. Co-crystallisation of SemD with its host binding partners demonstrates that SemD co-opts the Cdc42 binding site to activate the actin cytoskeleton regulator N-WASP, making active, GTP-bound Cdc42 superfluous. While SemD binds N-WASP much more strongly than Cdc42 does, it does not bind the Cdc42 effector protein FMNL2, indicating effector protein specificity. Furthermore, by identifying flexible and structured domains, we show that SemD can simultaneously interact with the membrane, the endocytic protein SNX9, and N-WASP. Here, we show at the structural level how a single effector protein can hijack central components of the host's endocytic system for efficient internalization.
Assuntos
Proteínas de Bactérias , Chlamydophila pneumoniae , Endocitose , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Proteína cdc42 de Ligação ao GTP/metabolismo , Membrana Celular/metabolismo , Chlamydophila pneumoniae/metabolismo , Cristalografia por Raios X , Células HeLa , Interações Hospedeiro-Patógeno , Mimetismo Molecular , Ligação Proteica , Nexinas de Classificação/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Animais , RatosRESUMO
Neurite outgrowth is a critical step in neural development, leading to the generation of neurite branches that allow individual neurons to make contacts with multiple neurons within the target region. Polyglutamine-binding protein 1 (PQBP1) is a highly conserved protein with a key role in neural development. Our recent mass spectrometric analysis showed that PQBP1 associates with neural Wiskott-Aldrich syndrome protein (N-WASP), an important actin polymerization-promoting factor involved in neurite outgrowth. Here, we report that the WW domain of PQBP1 directly interacts with the proline-rich domain of N-WASP. The disruption of this interaction leads to impaired neurite outgrowth and growth cone size. Furthermore, we demonstrate that PQBP1/N-WASP interaction is critical for the recruitment of N-WASP to the growth cone, but does not affect N-WASP protein levels or N-WASP-induced actin polymerization. Our results indicated that PQBP1 regulates neurite outgrowth by recruiting N-WASP to the growth cone, thus representing an alternative molecular mechanism via which PQBP1-mediates neurite outgrowth.
Assuntos
Crescimento Neuronal , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Humanos , Animais , Cones de Crescimento/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Actinas/metabolismo , Neuritos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células HEK293 , Camundongos , Ligação Proteica , RatosRESUMO
The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.
Assuntos
Proteínas de Bactérias , Fatores de Virulência , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Humanos , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/genética , Animais , Camundongos , Ligação Proteica , Brucella/metabolismo , Brucella/genética , Brucella/patogenicidade , Motivos de Aminoácidos , Actinas/metabolismo , Brucelose/microbiologia , Macrófagos/microbiologia , Interações Hospedeiro-PatógenoRESUMO
Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.
Assuntos
Caenorhabditis elegans , Proteína Neuronal da Síndrome de Wiskott-Aldrich , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Animais , Humanos , Caenorhabditis elegans/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Sinapsinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
Filopodia are thin, actin-rich projection from the plasma membrane that promote cancer cell invasion and migration. Sex-determining region Y-related high-mobility group-box 4 (SOX4) is a crucial transcription factor that plays a role in the development and metastasis of colorectal cancer (CRC). However, the involvement of SOX4 in cytoskeleton remodeling in CRC remains unknown. For the first time, we demonstrate that SOX4 is a potent regulator of filopodia formation in CRC cells. Overexpression of SOX4 protein enhances both migration and invasion ability of HCT116, and CACO2 cells, which is relevant to the metastasis. Furthermore, through phalloidin staining, cytoskeleton re-assembly was observed in SOX4-modified cell lines. Enhanced expression of SOX4 increased the number and length of filopodia on cell surface. In contrast, silencing SOX4 in SW620 cells with higher endogenous expression of SOX4, impeded the filopodia formation. Moreover, SOX4 was found to be positively regulating the expression of central regulators of actin cytoskeleton - N-Wiskott-Aldrich syndrome protein (N-WASP); WAVE2; Actin related proteins, ARP2 and ARP3. Inhibiting the N-WASP/ARP2/3 pathway diminishes the filopodia formation and the migration of CRC cells. These results indicate the crucial role of SOX4 in the regulation of filopodia formation mediated by N-WASP/ARP2/3 pathway in CRC cells.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Movimento Celular , Neoplasias Colorretais , Citoesqueleto , Pseudópodes , Fatores de Transcrição SOXC , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Humanos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/genética , Fatores de Transcrição SOXC/metabolismo , Fatores de Transcrição SOXC/genética , Movimento Celular/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Citoesqueleto/metabolismo , Pseudópodes/metabolismo , Células CACO-2 , Transdução de Sinais , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Células HCT116 , Citoesqueleto de Actina/metabolismoRESUMO
Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that ß1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of ß1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase ß1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.
Assuntos
Integrina beta1 , Metástase Neoplásica , Fator de Resposta Sérica , Proteínas Ativadoras de ras GTPase , Humanos , Integrina beta1/metabolismo , Integrina beta1/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas Ativadoras de ras GTPase/genética , Linhagem Celular Tumoral , Fator de Resposta Sérica/metabolismo , Masculino , Feminino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/genética , Animais , Transativadores/metabolismo , Adesão Celular , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Camundongos , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Regulação Neoplásica da Expressão Gênica , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
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
Human WASP and N-WASP are homologous proteins that require the binding of multiple regulators, including the acidic lipid PIP2 and the small GTPase Cdc42, to relieve autoinhibition before they can stimulate the initiation of actin polymerization. Autoinhibition involves intramolecular binding of the C-terminal acidic and central motifs to an upstream basic region and GTPase binding domain. Little is known about how a single intrinsically disordered protein, WASP or N-WASP, binds multiple regulators to achieve full activation. Here we used molecular dynamics simulations to characterize the binding of WASP and N-WASP with PIP2 and Cdc42. In the absence of Cdc42, both WASP and N-WASP strongly associate with PIP2-containing membranes, through their basic region and also possibly through a tail portion of the N-terminal WH1 domain. The basic region also participates in Cdc42 binding, especially for WASP; consequently Cdc42 binding significantly compromises the ability of the basic region in WASP, but not N-WASP, to bind PIP2. PIP2 binding to the WASP basic region is restored only when Cdc42 is prenylated at the C-terminus and tethered to the membrane. This distinction in the activation of WASP and N-WASP may contribute to their different functional roles.
Assuntos
Prenilação de Proteína , Proteína da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Ligação ao GTP , Humanos , Actinas/química , Actinas/metabolismo , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/metabolismo , Ligação Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Polimerização , Simulação de Dinâmica MolecularRESUMO
During neural development, the actin filament network must be precisely regulated to form elaborate neurite structures. N-WASP tightly controls actin polymerization dynamics by activating an actin nucleator Arp2/3. However, the importance of N-WASP-Arp2/3 signaling in the assembly of neurite architecture in vivo has not been clarified. Here, we demonstrate that N-WASP-Arp2/3 signaling plays a crucial role in the maturation of cerebellar Purkinje cell (PC) dendrites in vivo in mice. N-WASP was expressed and activated in developing PCs. Inhibition of Arp2/3 and N-WASP from the beginning of dendrite formation severely disrupted the establishment of a single stem dendrite, which is a characteristic basic structure of PC dendrites. Inhibition of Arp2/3 after stem dendrite formation resulted in hypoplasia of the PC dendritic tree. Cdc42, an upstream activator of N-WASP, is required for N-WASP-Arp2/3 signaling-mediated PC dendrite maturation. In addition, overactivation of N-WASP is also detrimental to dendrite formation in PCs. These findings reveal that proper activation of N-WASP-Arp2/3 signaling is crucial for multiple steps of PC dendrite maturation in vivo.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Células de Purkinje , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Animais , Camundongos , Citoesqueleto de Actina/metabolismo , Dendritos/metabolismo , Neurogênese/genética , Células de Purkinje/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismoRESUMO
A key event at the onset of development is the activation of a contractile actomyosin cortex during the oocyte-to-embryo transition1-3. Here we report on the discovery that, in Caenorhabditis elegans oocytes, actomyosin cortex activation is supported by the emergence of thousands of short-lived protein condensates rich in F-actin, N-WASP and the ARP2/3 complex4-8 that form an active micro-emulsion. A phase portrait analysis of the dynamics of individual cortical condensates reveals that condensates initially grow and then transition to disassembly before dissolving completely. We find that, in contrast to condensate growth through diffusion9, the growth dynamics of cortical condensates are chemically driven. Notably, the associated chemical reactions obey mass action kinetics that govern both composition and size. We suggest that the resultant condensate dynamic instability10 suppresses coarsening of the active micro-emulsion11, ensures reaction kinetics that are independent of condensate size and prevents runaway F-actin nucleation during the formation of the first cortical actin meshwork.
Assuntos
Actomiosina , Condensados Biomoleculares , Caenorhabditis elegans , Oócitos , Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Actomiosina/química , Actomiosina/metabolismo , Animais , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Emulsões/química , Emulsões/metabolismo , Oócitos/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
BACKGROUND: The number of neutrophils is significantly reduced in myelodysplastic syndrome (MDS), but the molecular basis remains unclear. We recently found that miR-34a was significantly increased in MDS neutrophils. Therefore, this study aims to clarify the effects of aberrant miR-34a expression on neutrophil counts. METHODS: miR-34a mimics/inhibitor transfection were performed in neutrophil-like differentiated HL60 (dHL60) cells, and a FACSCalibur flow cytometer was used to measure ROS production and apoptosis. In addition, the Cdc42-WASP-Arp2/3 pathway inhibitor (ML141) and activator (CN02) treated the dHL60 cells, and then ROS production, apoptosis and related proteins expression were detected. And, luciferase reporter assay to verify the relationship of miR-34a and the Cdc42-WASP-Arp2/3 pathway. RESULTS: overexpression of miR-34a could induce ROS production and apoptosis, decrease the expression levels of DOCK8, p-WASP, WASP, Arp2, Arp3, and increase F-actin's expression. Meanwhile, knockdown of miR-34a could decrease ROS production and apoptosis, increase the expression of DOCK8, p-WASP, WASP, Arp2, Arp3, and decrease F-actin's expression. Immunofluorescence staining showed aberrant miR-34a and Cdc42-WASP-Arp2/3 pathway could induce F-actin membrane transfer. Luciferase reporter assay indicated that DOCK8 was a direct target gene of miR-34a. CONCLUSION: These data indicates miR-34a may induce neutrophil apoptosis by regulating Cdc42-WASP-Arp2/3 pathway-mediated F-actin remodeling and ROS production.
Assuntos
Actinas , MicroRNAs , Actinas/genética , Actinas/metabolismo , Apoptose/genética , MicroRNAs/genética , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Insulin resistance (IR) attributed by the deficiency of lipophagy, is an abnormal state of downregulation of insulin-mediated glucose uptake and use into the liver. Chromosome 9 open reading frame 72 (C9orf72) variously modulates autophagy. We investigated the role and the downstream pathway of C9orf72 in hepatic IR. We found that C9orf72 knockdown alleviated hepatic IR by lipophagy promotion in T2DM mice and in IR-challenged hepatocytes in vitro. C9orf72 interacted with and activated cell division cycle 42 (Cdc42) protein in IR-challenged hepatocytes, Which in turn, inhibits lipophagy by promoting neural Wiskott-Aldrich syndrome protein (N-WASP) expression and activation. C9orf72 inhibited lipophagy by activating the Cdc42/N-WASP axis to facilitate hepatic IR; therefore, the knockdown of C9orf72 may be potentially therapeutic for the treatment of IR.
Assuntos
Autofagia , Proteína C9orf72/metabolismo , Técnicas de Silenciamento de Genes , Resistência à Insulina , Fígado/metabolismo , Fígado/patologia , Animais , Diabetes Mellitus Tipo 2/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Obesos , Ligação Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Actin-based, finger-like cell protrusions such as microvilli and filopodia play important roles in epithelial cells. Several proteins have been identified to regulate cell protrusion formation, which helps us to learn about the underlying mechanism of this process. FCH domain and double SH3 domains containing protein 2 (FCHSD2) belongs to the FCH and Bin-Amphiphysin-Rvs (F-BAR) protein family, containing an N-terminal F-BAR domain, two SH3 domains, and a C-terminal PDZ domain-binding interface (PBI). Previously, we found that FCHSD2 interacts with WASP/N-WASP and stimulates ARP2/3-mediated actin polymerization in vitro. In the present work, we show that FCHSD2 promotes the formation of apical and lateral cell protrusions in cultured cells. Our data suggest that FCHSD2 cooperates with CDC42 and N-WASP in regulating apical cell protrusion formation. In line with this, biochemical studies reveal that FCHSD2 and CDC42 simultaneously bind to N-WASP, forming a protein complex. Interestingly, the F-BAR domain of FCHSD2 induces lateral cell protrusion formation independently of N-WASP. Furthermore, we show that the ability of FCHSD2 to induce cell protrusion formation requires its plasma membrane-binding ability. In summary, our present work suggests that FCHSD2 cooperates with CDC42 and N-WASP to regulate cell protrusion formation in a membrane-dependent manner.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Pseudópodes/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Ligação ProteicaRESUMO
Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Melanócitos/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/genética , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/química , Actinas/genética , Animais , Sítios de Ligação , Bovinos , Citoesqueleto/ultraestrutura , Gelsolina/química , Gelsolina/genética , Gelsolina/metabolismo , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Melanócitos/citologia , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Modelos Moleculares , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citologia , Timo/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/química , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Macrothrombocytopenia is a common pathology of missense mutations in genes regulating actin dynamics. Takenouchi-Kosaki syndrome (TKS) harboring the c.191A > G, Tyr64Cys (Y64C) variant in Cdc42 exhibits a variety of clinical manifestations, including immunological and hematological anomalies. In the present study, we investigated the functional abnormalities of the Y64C mutant in HEK293 cells and elucidated the mechanism of macrothrombocytopenia, one of the symptoms of TKS patients, by monitoring the production of platelet-like particles (PLP) using MEG-01 cells. We found that the Y64C mutant was concentrated at the membrane compartment due to impaired binding to Rho-GDI and more active than the wild-type. The Y64C mutant also had lower association with its effectors Pak1/2 and N-WASP. Y64C mutant-expressing MEG-01 cells demonstrated short cytoplasmic protrusions with aberrant F-actin and microtubules, and reduced PLP production. This suggested that the Y64C mutant facilitates its activity and membrane localization, resulting in impaired F-actin dynamics for proplatelet extension, which is necessary for platelet production. Furthermore, such dysfunction was ameliorated by either suppression of Cdc42 activity or prenylation using chemical inhibitors. Our study may lead to pharmacological treatments for TKS patients.
Assuntos
Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trombocitopenia/metabolismo , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Alquil e Aril Transferases/antagonistas & inibidores , Benzamidas/farmacologia , Plaquetas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HEK293 , Humanos , Mutação , Prenilação de Proteína/efeitos dos fármacos , Pirazóis/farmacologia , Transdução de Sinais/genética , Sulfonamidas/farmacologia , Síndrome , Trombocitopenia/genética , Trombopoese/efeitos dos fármacos , Trombopoese/genética , Transfecção , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismoRESUMO
Actin cytoskeletal reorganization plays an important role in regulating smooth muscle contraction, which is essential for the modulation of various physiological functions including airway tone. The adapter protein Abi1 (Abelson interactor 1) participates in the control of smooth muscle contraction. The mechanisms by which Abi1 coordinates smooth muscle function are not fully understood. Here, we found that contractile stimulation elicited Abi1 acetylation in human airway smooth muscle (HASM) cells. Mutagenesis analysis identified lysine-416 (K416) as a major acetylation site. Replacement of K416 with Q (glutamine) enhanced the interaction of Abi1 with neuronal Wiskott-Aldrich syndrome protein (N-WASP), an important actin-regulatory protein. Moreover, the expression of K416Q Abi1 promoted actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19 and vimentin phosphorylation at Ser-56. Furthermore, p300 is a lysine acetyltransferase that catalyzes acetylation of histone and non-histone proteins in various cell types. Here, we discovered that a portion of p300 was localized in the cytoplasm of HASM cells. Knockdown of p300 reduced the agonist-induced Abi1 acetylation in HASM cells and in mouse airway smooth muscle tissues. Smooth muscle conditional knockout of p300 inhibited actin polymerization and the contraction of airway smooth muscle tissues without affecting myosin light chain phosphorylation and vimentin phosphorylation. Together, our results suggest that contractile stimulation induces Abi1 acetylation via p300 in smooth muscle. Acetylation at K416 promotes the coupling of Abi1 with N-WASP, which facilitates actin polymerization and smooth muscle contraction. This is a novel acetylation-dependent regulation of the actin cytoskeleton in smooth muscle.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Acetilação , Animais , Células Cultivadas , Proteína p300 Associada a E1A/metabolismo , Humanos , Lisina Acetiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Cadeias Leves de Miosina/metabolismo , Fosforilação/fisiologia , Transdução de Sinais/fisiologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms ß-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Materiais Biocompatíveis/metabolismo , Proteínas Oncogênicas/metabolismo , Peptídeos/metabolismo , Actinas/metabolismo , Animais , Materiais Biocompatíveis/química , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Biossíntese de Peptídeos Independentes de Ácido Nucleico , Peptídeos/química , Conformação Proteica em Folha beta , Domínios Proteicos , Multimerização Proteica , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
BACKGROUND: Knowledge of genetic determinants in Parkinson's disease is still limited. Familial forms of the disease continue to provide a rich resource to capture the genetic spectrum in disease pathogenesis, and this approach is exploited in this study. METHODS: Informative members from a three-generation family of Indian ethnicity manifesting a likely autosomal recessive mode of inheritance of Parkinson's disease were used for whole exome sequencing. Variant data analysis and in vitro functional characterisation of variant(s) segregating with the phenotype were carried out in HEK-293 and SH-SY5Y cells using gene constructs of interest. RESULTS: Two compound heterozygous variants, a rare missense (c.1139C > T:p.P380L) and a novel splice variant (c.1456 + 2 delTAGA, intron10) in Wiskott-Aldrich syndrome like gene (WASL, 7q31), both predicted to be deleterious were shared among the proband and two affected siblings. WASL, a gene not previously linked to a human Mendelian disorder is known to regulate actin polymerisation via Arp2/3 complex. Based on exon trapping assay using pSPL3 vector in HEK-293 cells, the splice variant showed skipping of exon10. Characterisation of the missense variant in SH-SY5Y cells demonstrated: i) significant alterations in neurite length and number; ii) decreased reactive oxygen species tolerance in mutation carrying cells on Tetrabutylphosphonium hydroxide induction and iii) increase in alpha-synuclein protein. Screening for WASL variants in two independent PD cohorts identified four individuals with heterozygous but none with biallelic variants. CONCLUSION: WASL, with demonstrated functional relevance in neurons may be yet another strong candidate gene for autosomal recessive PD encouraging assessment of its contribution across populations.
Assuntos
Doença de Parkinson/genética , Doença de Parkinson/fisiopatologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Idade de Início , Idoso , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Índia , Linhagem , Sequenciamento do ExomaRESUMO
Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process.IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.
Assuntos
Actinas/genética , Proteínas de Bactérias/genética , Chlamydia trachomatis/genética , Citoesqueleto/metabolismo , Chaperonas Moleculares/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/metabolismo , Citoesqueleto/microbiologia , Citoesqueleto/ultraestrutura , Células Epiteliais/microbiologia , Regulação da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Chaperonas Moleculares/metabolismo , Polimerização , Transdução de Sinais , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.