Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 32
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 7: 13305, 2016 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-27827364

RESUMO

Retromer is a membrane coat complex that is recruited to endosomes by the small GTPase Rab7 and sorting nexin 3. The timing of this interaction and consequent endosomal dynamics are thought to be regulated by the guanine nucleotide cycle of Rab7. Here we demonstrate that TBC1d5, a GTPase-activating protein (GAP) for Rab7, is a high-affinity ligand of the retromer cargo selective complex VPS26/VPS29/VPS35. The crystal structure of the TBC1d5 GAP domain bound to VPS29 and complementary biochemical and cellular data show that a loop from TBC1d5 binds to a conserved hydrophobic pocket on VPS29 opposite the VPS29-VPS35 interface. Additional data suggest that a distinct loop of the GAP domain may contact VPS35. Loss of TBC1d5 causes defective retromer-dependent trafficking of receptors. Our findings illustrate how retromer recruits a GAP, which is likely to be involved in the timing of Rab7 inactivation leading to membrane uncoating, with important consequences for receptor trafficking.


Assuntos
Endossomos/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Cristalografia por Raios X , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Transporte Proteico
2.
Mol Biol Cell ; 26(1): 91-103, 2015 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25355947

RESUMO

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.


Assuntos
Citoesqueleto de Actina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Movimento Celular , Cobre/metabolismo , ATPases Transportadoras de Cobre , Citoplasma/metabolismo , Endossomos/metabolismo , Células HEK293 , Células HeLa , Homeostase , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular
3.
J Cell Sci ; 128(2): 373-84, 2015 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-25431135

RESUMO

The pentameric WASH complex is best known for its role in regulating receptor trafficking from retromer-rich endosomal subdomains. FAM21 functions to stabilize the WASH complex through its N-terminal head domain and localizes it to endosomes by directly binding the retromer through its extended C-terminal tail. Herein, we used affinity purification combined with mass spectrometry to identify additional FAM21-interacting proteins. Surprisingly, multiple components of the nuclear factor κB (NF-κB) pathway were identified, including the p50 and p65 (RelA) NF-κB subunits. We show that FAM21 interacts with these components and regulates NF-κB-dependent gene transcription at the level of p65 chromatin binding. We further demonstrate that FAM21 contains a functional monopartite nuclear localization signal sequence (NLS) as well as a CRM1/exportin1-dependent nuclear export signal (NES), both of which work jointly with the N-terminal head domain and C-terminal retromer recruitment domain to regulate FAM21 cytosolic and nuclear subcellular localization. Finally, our findings indicate that FAM21 depletion sensitizes pancreatic cancer cells to gemcitabine and 5-fluorouracil. Thus, FAM21 not only functions as an integral component of the cytoplasmic WASH complex, but also modulates NF-κB gene transcription in the nucleus.


Assuntos
Proteínas dos Microfilamentos/metabolismo , NF-kappa B/genética , Neoplasias Pancreáticas/genética , Proteínas/genética , Fator de Transcrição RelA/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Citoplasma/metabolismo , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Proteínas dos Microfilamentos/genética , NF-kappa B/metabolismo , Sinais de Localização Nuclear/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Proteínas de Ligação a Fosfato , Ligação Proteica/genética , Fator de Transcrição RelA/genética , Gencitabina
4.
PLoS One ; 9(6): e98606, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24886983

RESUMO

Immature dendritic cells (DCs) maintain a highly dynamic pool of recycling MHCII that promotes sampling of environmental antigens for presentation to T helper cells. However, the molecular basis of MHCII recycling and the cellular machinery that orchestrates MHCII trafficking are incompletely understood. Using a mouse model we show that WASH, an actin regulatory protein that facilitates retromer function, is essential for MHCII recycling and efficient priming of T helper cells. We further demonstrate that WASH deficiency results in impaired MHCII surface levels, recycling, and an accumulation of polyubiquitinated MHCII complexes, which are subsequently slated for premature lysosomal degradation. Consequently, conditional deletion of the Wash gene in DCs impairs priming of both conventional and autoimmune T helper cells in vivo and attenuates disease progression in a model of experimental autoimmune encephalitis (EAE). Thus, we identify a novel mechanism in which DCs employ the evolutionarily conserved WASH and retromer complex for MHCII recycling in order to regulate T helper cell priming.


Assuntos
Células Dendríticas/fisiologia , Antígenos de Histocompatibilidade Classe II/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Sequência de Bases , Primers do DNA , Encefalomielite Autoimune Experimental/imunologia , Ensaio de Imunoadsorção Enzimática , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ubiquitinação
5.
Mol Cell Biol ; 33(20): 3983-93, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23918802

RESUMO

Macroautophagy is an evolutionarily conserved cellular process involved in the clearance of proteins and organelles. Although the cytoplasmic machinery that orchestrates autophagy induction during starvation, hypoxia, or receptor stimulation has been widely studied, the key epigenetic events that initiate and maintain the autophagy process remain unknown. Here we show that the methyltransferase G9a coordinates the transcriptional activation of key regulators of autophagosome formation by remodeling the chromatin landscape. Pharmacological inhibition or RNA interference (RNAi)-mediated suppression of G9a induces LC3B expression and lipidation that is dependent on RNA synthesis, protein translation, and the methyltransferase activity of G9a. Under normal conditions, G9a associates with the LC3B, WIPI1, and DOR gene promoters, epigenetically repressing them. However, G9a and G9a-repressive histone marks are removed during starvation and receptor-stimulated activation of naive T cells, two physiological inducers of macroautophagy. Moreover, we show that the c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of autophagy gene expression during naive-T-cell activation. Together, these findings reveal that G9a directly represses genes known to participate in the autophagic process and that inhibition of G9a-mediated epigenetic repression represents an important regulatory mechanism during autophagy.


Assuntos
Autofagia/genética , Cromatina/metabolismo , Epigênese Genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Linfócitos T/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Montagem e Desmontagem da Cromatina , Fibroblastos/citologia , Fibroblastos/metabolismo , Glucose/deficiência , Células HeLa , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/genética , Fagossomos/metabolismo , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Ativação Transcricional
6.
Cell ; 152(5): 1051-64, 2013 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-23452853

RESUMO

Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Transporte Proteico , Proteínas/metabolismo , Actinas/metabolismo , Proteínas de Ligação a DNA/genética , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Complexo de Golgi/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/genética , Proteínas/genética , Interferência de RNA , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinação
7.
Mol Cell Biol ; 33(5): 958-73, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23275443

RESUMO

WASH is an Arp2/3 activator of the Wiskott-Aldrich syndrome protein superfamily that functions during endosomal trafficking processes in collaboration with the retromer and sorting nexins, but its in vivo function has not been examined. To elucidate the physiological role of WASH in T cells, we generated a WASH conditional knockout (WASHout) mouse model. Using CD4(Cre) deletion, we found that thymocyte development and naive T cell activation are unaltered in the absence of WASH. Surprisingly, despite normal T cell receptor (TCR) signaling and interleukin-2 production, WASHout T cells demonstrate significantly reduced proliferative potential and fail to effectively induce experimental autoimmune encephalomyelitis. Interestingly, after activation, WASHout T cells fail to maintain surface levels of TCR, CD28, and LFA-1. Moreover, the levels of the glucose transporter, GLUT1, are also reduced compared to wild-type T cells. We further demonstrate that the loss of surface expression of these receptors in WASHout cells results from aberrant accumulation within the collapsed endosomal compartment, ultimately leading to degradation within the lysosome. Subsequently, activated WASHout T cells experience reduced glucose uptake and metabolic output. Thus, we found that WASH is a newly recognized regulator of TCR, CD28, LFA-1, and GLUT1 endosome-to-membrane recycling. Aberrant trafficking of these key T cell proteins may potentially lead to attenuated proliferation and effector function.


Assuntos
Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/imunologia , Animais , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Antígenos CD4/genética , Antígenos CD4/imunologia , Proliferação de Células , Células Cultivadas , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Deleção de Genes , Transportador de Glucose Tipo 1/imunologia , Transportador de Glucose Tipo 1/metabolismo , Glicólise , Interleucina-2/imunologia , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
8.
Mol Biol Cell ; 23(16): 3215-28, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22718907

RESUMO

The Arp2/3-activator Wiskott-Aldrich syndrome protein and Scar homologue (WASH) is suggested to regulate actin-dependent membrane scission during endosomal sorting, but its cellular roles have not been fully elucidated. To investigate WASH function, we generated tamoxifen-inducible WASH-knockout mouse embryonic fibroblasts (WASHout MEFs). Of interest, although EEA1(+) endosomes were enlarged, collapsed, and devoid of filamentous-actin and Arp2/3 in WASHout MEFs, we did not observe elongated membrane tubules emanating from these disorganized endomembranes. However, collapsed WASHout endosomes harbored segregated subdomains, containing either retromer cargo recognition complex-associated proteins or EEA1. In addition, we observed global collapse of LAMP1(+) lysosomes, with some lysosomal membrane domains associated with endosomes. Both epidermal growth factor receptor (EGFR) and transferrin receptor (TfnR) exhibited changes in steady-state cellular localization. EGFR was directed to the lysosomal compartment and exhibited reduced basal levels in WASHout MEFs. However, although TfnR was accumulated with collapsed endosomes, it recycled normally. Moreover, EGF stimulation led to efficient EGFR degradation within enlarged lysosomal structures. These results are consistent with the idea that discrete receptors differentially traffic via WASH-dependent and WASH-independent mechanisms and demonstrate that WASH-mediated F-actin is requisite for the integrity of both endosomal and lysosomal networks in mammalian cells.


Assuntos
Endossomos/metabolismo , Fibroblastos/metabolismo , Lisossomos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas de Transporte Vesicular/genética , Actinas/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Receptores ErbB/metabolismo , Fibroblastos/fisiologia , Fibroblastos/ultraestrutura , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Complexos Multiproteicos/metabolismo , Transporte Proteico , Proteólise , Receptores da Transferrina/metabolismo , Proteínas de Transporte Vesicular/metabolismo
9.
Mol Biol Cell ; 23(13): 2505-15, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22593205

RESUMO

The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.


Assuntos
Endossomos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Humanos , Membranas Intracelulares/metabolismo , Células Jurkat , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Via Secretória , Deleção de Sequência , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/química
10.
J Immunol ; 188(12): 6135-44, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22573807

RESUMO

The Ras GTPase-activating-like protein IQGAP1 is a multimodular scaffold that controls signaling and cytoskeletal regulation in fibroblasts and epithelial cells. However, the functional role of IQGAP1 in T cell development, activation, and cytoskeletal regulation has not been investigated. In this study, we show that IQGAP1 is dispensable for thymocyte development as well as microtubule organizing center polarization and cytolytic function in CD8(+) T cells. However, IQGAP1-deficient CD8(+) T cells as well as Jurkat T cells suppressed for IQGAP1 were hyperresponsive, displaying increased IL-2 and IFN-γ production, heightened LCK activation, and augmented global phosphorylation kinetics after TCR ligation. In addition, IQGAP1-deficient T cells exhibited increased TCR-mediated F-actin assembly and amplified F-actin velocities during spreading. Moreover, we found that discrete regions of IQGAP1 regulated cellular activation and F-actin accumulation. Taken together, our data suggest that IQGAP1 acts as a dual negative regulator in T cells, limiting both TCR-mediated activation kinetics and F-actin dynamics via distinct mechanisms.


Assuntos
Citoesqueleto de Actina/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Transdução de Sinais/imunologia , Proteínas Ativadoras de ras GTPase/metabolismo , Actinas/imunologia , Actinas/metabolismo , Animais , Western Blotting , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/imunologia , Citoesqueleto/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas Ativadoras de ras GTPase/imunologia
11.
Mol Biol Cell ; 23(12): 2352-61, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22513087

RESUMO

Wiskott-Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-[D/E](3-10)-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.


Assuntos
Actinas/metabolismo , Endossomos/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Bovinos , Eletroforese em Gel de Poliacrilamida , Células HeLa , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas dos Microfilamentos/genética , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Ligação Proteica , Transporte Proteico , Sequências Repetitivas de Aminoácidos/genética , Proteínas de Transporte Vesicular/genética
12.
J Cell Sci ; 124(Pt 18): 3118-26, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21868368

RESUMO

Formin-like 1 (FMNL1) is a member of the formin family of actin nucleators, and is one of the few formins for which in vitro activities have been well characterized. However, the functional roles of this mammalian formin remain ill-defined. In particular, it is unclear how the unique in vitro biochemical properties of FMNL1 relate to its regulation of cellular processes. Here, we demonstrate that FMNL1 depletion caused a dramatic increase in cellular F-actin content, which resulted in Golgi complex fragmentation. Moreover, increased F-actin and maintenance of Golgi structure were distinctly regulated by the gamma isoform of FMNL1, which required binding to actin. Importantly, in addition to Golgi fragmentation, increased F-actin content in the absence of FMNL1 also led to cation-independent mannose 6-phosphate receptor dispersal, lysosomal enlargement and missorting of cathepsin D. Taken together, our data support a model in which FMNL1 regulates cellular F-actin levels required to maintain structural integrity of the Golgi complex and lysosomes.


Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/patologia , Receptor IGF Tipo 2/metabolismo , Catepsina D/metabolismo , Proteínas do Citoesqueleto/genética , Citoesqueleto , Forminas , Complexo de Golgi/patologia , Células HeLa , Homeostase , Humanos , Células Jurkat , Lisossomos/efeitos dos fármacos , Estrutura Molecular , Ligação Proteica , Isoformas de Proteínas/genética , Transporte Proteico , RNA Interferente Pequeno/genética , Transdução de Sinais
13.
J Immunol ; 186(8): 4805-18, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21398607

RESUMO

Dendritic cells (DCs) are professional APCs that reside in peripheral tissues and survey the body for pathogens. Upon activation by inflammatory signals, DCs undergo a maturation process and migrate to lymphoid organs, where they present pathogen-derived Ags to T cells. DC migration depends on tight regulation of the actin cytoskeleton to permit rapid adaptation to environmental cues. We investigated the role of hematopoietic lineage cell-specific protein 1 (HS1), the hematopoietic homolog of cortactin, in regulating the actin cytoskeleton of murine DCs. HS1 localized to lamellipodial protrusions and podosomes, actin-rich structures associated with adhesion and migration. DCs from HS1(-/-) mice showed aberrant lamellipodial dynamics. Moreover, although these cells formed recognizable podosomes, their podosome arrays were loosely packed and improperly localized within the cell. HS1 interacts with Wiskott-Aldrich syndrome protein (WASp), another key actin-regulatory protein, through mutual binding to WASp-interacting protein. Comparative analysis of DCs deficient for HS1, WASp or both proteins revealed unique roles for these proteins in regulating podosomes with WASp being essential for podosome formation and with HS1 ensuring efficient array organization. WASp recruitment to podosome cores was independent of HS1, whereas HS1 recruitment required Src homology 3 domain-dependent interactions with the WASp/WASp-interacting protein heterodimer. In migration assays, the phenotypes of HS1- and WASp-deficient DCs were related, but distinct. WASp(-/y) DCs migrating in a chemokine gradient showed a large decrease in velocity and diminished directional persistence. In contrast, HS1(-/-) DCs migrated faster than wild-type cells, but directional persistence was significantly reduced. These studies show that HS1 functions in concert with WASp to fine-tune DC cytoarchitecture and direct cell migration.


Assuntos
Quimiotaxia/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Actinas/genética , Actinas/metabolismo , Animais , Apresentação de Antígeno/imunologia , Western Blotting , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Movimento Celular/imunologia , Células Cultivadas , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Fator Estimulador de Colônias de Granulócitos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Ligação Proteica , Pseudópodes/imunologia , Pseudópodes/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
14.
Nature ; 468(7323): 533-8, 2010 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21107423

RESUMO

Members of the Wiskott-Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.


Assuntos
Actinas/metabolismo , Modelos Moleculares , Família de Proteínas da Síndrome de Wiskott-Aldrich/química , Animais , Células HeLa , Humanos , Insetos/citologia , Fosforilação , Estrutura Quaternária de Proteína , Proteínas rac1 de Ligação ao GTP/metabolismo
15.
Proc Natl Acad Sci U S A ; 107(23): 10442-7, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20498093

RESUMO

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.


Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Bovinos , Células HeLa , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/ultraestrutura , Microscopia Eletrônica , Mutação , Ligação Proteica , Interferência de RNA , Coelhos , Família de Proteínas da Síndrome de Wiskott-Aldrich/ultraestrutura
16.
J Biol Chem ; 285(2): 888-902, 2010 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-19887445

RESUMO

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/metabolismo , Carcinógenos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Ligante Fas/farmacologia , Células HL-60 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células Jurkat , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia
17.
Dev Cell ; 17(5): 699-711, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19922874

RESUMO

The Arp2/3 complex regulates endocytosis, sorting, and trafficking, yet the Arp2/3-stimulating factors orchestrating these distinct events remain ill defined. WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homolog) is an Arp2/3 activator with unknown function that was duplicated during primate evolution. We demonstrate that WASH associates with tubulin and localizes to early endosomal subdomains, which are enriched in Arp2/3, F-actin, and retromer components. Although WASH localized with activated receptors, it was not essential for endocytosis. However, WASH did regulate retromer-mediated retrograde CI-MPR trafficking, which required its association with endosomes, Arp2/3-directed F-actin regulation, and tubulin interaction. Moreover, WASH exists in a multiprotein complex containing FAM21, which links WASH to endosomes and is required for WASH-dependent retromer-mediated sorting. Significantly, without WASH, retromer tubulation was exaggerated, supporting a model wherein WASH links retromer-mediated cargo containing tubules to microtubules for Golgi-directed trafficking and generates F-actin-driven force for tubule scission.


Assuntos
Proteínas de Transporte/metabolismo , Endocitose , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Humanos , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Transporte Proteico , Tubulina (Proteína)/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética
18.
J Immunol ; 183(11): 7352-61, 2009 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-19917685

RESUMO

Productive T cell activation requires efficient reorganization of the actin cytoskeleton. We showed previously that the actin-regulatory protein, hematopoietic lineage cell-specific protein 1 (HS1), is required for the stabilization of F-actin and Vav1 at the immunological synapse and for efficient calcium responses. The Tec family kinase IL-2-inducible T cell kinase (Itk) regulates similar aspects of T cell activation, suggesting that these proteins act in the same pathway. Using video microscopy, we show that T cells lacking Itk or HS1 exhibited similar defects in actin responses, extending unstable lamellipodial protrusions upon TCR stimulation. HS1 and Itk could be coimmunoprecipitated from T cell lysates, and GST-pulldown studies showed that Itk's Src homology 2 domain binds directly to two phosphotyrosines in HS1. In the absence of Itk, or in T cells overexpressing an Itk Src homology 2 domain mutant, HS1 failed to localize to the immunological synapse, indicating that Itk serves to recruit HS1 to sites of TCR engagement. Because Itk is required for phospholipase C (PLC)gamma1 phosphorylation and calcium store release, we examined the calcium signaling pathway in HS1(-/-) T cells in greater detail. In response to TCR engagement, T cells lacking HS1 exhibited diminished calcium store release, but TCR-dependent PLCgamma1 phosphorylation was intact, indicating that HS1's role in calcium signaling is distinct from that of Itk. HS1-deficient T cells exhibited defective cytoskeletal association of PLCgamma1 and altered formation of PLCgamma1 microclusters. We conclude that HS1 functions as an effector of Itk in the T cell actin-regulatory pathway, and directs the spatial organization of PLCgamma1 signaling complexes.


Assuntos
Fator Estimulador de Colônias de Granulócitos/imunologia , Sinapses Imunológicas/imunologia , Fosfolipase C gama/imunologia , Proteínas Tirosina Quinases/imunologia , Linfócitos T/imunologia , Actinas/metabolismo , Animais , Western Blotting , Sinalização do Cálcio/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Sinapses Imunológicas/metabolismo , Imunoprecipitação , Células Jurkat , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia de Fluorescência , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Pseudópodes/metabolismo , Pseudópodes/patologia , Interferência de RNA , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Transfecção
19.
J Immunol ; 181(10): 6995-7001, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981119

RESUMO

NK cells are innate immune cells that can eliminate their targets through granule release. In this study, we describe a specialized role for the large GTPase Dynamin 2 (Dyn2) in the regulation of these secretory events leading to cell-mediated cytotoxicity. By modulating the expression of Dyn2 using small interfering RNA or by inhibiting its activity using a pharmacological agent, we determined that Dyn2 does not regulate conjugate formation, proximal signaling, or granule polarization. In contrast, during cell-mediated killing, Dyn2 localizes with lytic granules and polarizes to the NK cell-target interface where it regulates the final fusion of lytic granules with the plasma membrane. These findings identify a novel role for Dyn2 in the exocytic events required for effective NK cell-mediated cytotoxicity.


Assuntos
Citotoxicidade Imunológica , Dinamina II/imunologia , Exocitose/imunologia , Células Matadoras Naturais/imunologia , Vesículas Secretórias/imunologia , Dinamina II/metabolismo , Imunofluorescência , Humanos , Células Matadoras Naturais/metabolismo , Lisossomos/imunologia , Lisossomos/metabolismo , Transporte Proteico/imunologia , Vesículas Secretórias/metabolismo
20.
Adv Immunol ; 97: 1-64, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18501768

RESUMO

More than a quarter of a century has passed since the observation that T cells rapidly polarize their actin and microtubule cytoskeletal systems toward antigen-presenting cells during activation. Since this initial discovery, several receptors on T cells (e.g., T cell receptor [TCR], co-receptors, integrins, and chemokine receptors) have been identified to regulate these two cytoskeletal networks through complex signaling pathways, which are still being elucidated. There is now an undeniable body of biochemical, pharmacological, and genetic evidence indicating that regulators of actin and microtubule dynamics are crucial for T cell activation and effector functions. In fact, the actin cytoskeleton participates in the initial clustering of TCR-major histocompatibility complex or peptide complexes, formation and stabilization of the immune synapse, integrin-mediated adhesion, and receptor sequestration, whereas both the actin and microtubule cytoskeletons regulate the establishment of cell polarity, cell migration, and directed secretion of cytokines and cytolytic granules. Over the past several years, we have begun to more thoroughly understand the contributions of specific actin-regulatory and actin-nucleating proteins that govern these processes. Herein, we discuss our current understanding of how activating receptors on T lymphocytes regulate the actin and microtubule cytoskeletons, and how in turn, these distinct but integrated cytoskeletal networks coordinate T cell immune responses.


Assuntos
Citoesqueleto/fisiologia , Ativação Linfocitária , Linfócitos T/fisiologia , Actinas/fisiologia , Animais , Movimento Celular/fisiologia , Humanos , Integrinas/fisiologia , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/fisiologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...