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1.
Nat Commun ; 12(1): 6287, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34725372

RESUMO

Angiopoietin (Angpt)-Tie receptor 2 (Tie2) plays key roles in vascular development and homeostasis as well as pathological vascular remodeling. Therefore, Tie2-agonistic antibody and engineered Angpt1 variants have been developed as potential therapeutics for ischemic and inflammatory vascular diseases. However, their underlying mechanisms for Tie2 clustering and activation remain elusive and the poor manufacturability and stability of Angpt1 variants limit their clinical application. Here, we develop a human Tie2-agonistic antibody (hTAAB), which targets the membrane proximal fibronectin type III domain of Tie2 distinct from the Angpt-binding site. Our Tie2/hTAAB complex structures reveal that hTAAB tethers the preformed Tie2 homodimers into polygonal assemblies through specific binding to Tie2 Fn3 domain. Notably, the polygonal Tie2 clustering induced by hTAAB is critical for Tie2 activation and are resistant to antagonism by Angpt2. Our results provide insight into the molecular mechanism of Tie2 clustering and activation mediated by hTAAB, and the structure-based humanization of hTAAB creates a potential clinical application.


Assuntos
Anticorpos Monoclonais/química , Receptor TIE-2/química , Angiopoietina-2/química , Angiopoietina-2/genética , Angiopoietina-2/imunologia , Animais , Anticorpos Monoclonais/imunologia , Dimerização , Fibronectinas/química , Fibronectinas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Receptor TIE-2/agonistas , Receptor TIE-2/genética , Receptor TIE-2/imunologia , Remodelação Vascular
2.
Exp Mol Med ; 51(7): 1-14, 2019 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-31358736

RESUMO

During ligand-mediated receptor endocytosis, the small GTPase Rab5 functions in vesicle fusion and trafficking. Rab5 activation is known to require interactions with its guanine nucleotide-exchange factors (GEFs); however, the mechanism regulating Rab5 interactions with GEFs remains unclear. Here, we show that the SH3-adapter protein SPIN90 participates in the activation of Rab5 through the recruitment of both Rab5 and its GEF, Gapex5, to endosomal membranes during epidermal growth factor (EGF)-mediated endocytosis. SPIN90 strongly interacts with the inactive Rab5/GDI2 complex through its C-terminus. In response to EGF signaling, extracellular signal-regulated kinase (ERK)-mediated phosphorylation of SPIN90 at Thr-242 enables SPIN90 to bind Gapex5 through its N-terminal SH3 domain. Gapex5 is a determinant of Rab5 membrane targeting, while SPIN90 mediates the interaction between Gapex5 and Rab5 in a phosphorylation-dependent manner. Collectively, our findings suggest that SPIN90, as an adaptor protein, simultaneously binds inactive Rab5 and Gapex5, thereby altering their spatial proximity and facilitating Rab5 activation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Musculares/metabolismo , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Endocitose/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Musculares/genética , Fosforilação , Ligação Proteica , Proteínas rab5 de Ligação ao GTP/genética , Domínios de Homologia de src
3.
Nat Commun ; 8: 15090, 2017 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-28489072

RESUMO

Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.


Assuntos
Anticorpos Monoclonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Citosol/metabolismo , Imunoglobulina G/farmacologia , Neoplasias/genética , Proteínas ras/genética , Animais , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HL-60 , Células HT29 , Células HeLa , Humanos , Células K562 , Células MCF-7 , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Células NIH 3T3 , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/antagonistas & inibidores , Proteínas ras/metabolismo
5.
Cancer Cell ; 30(6): 953-967, 2016 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-27960088

RESUMO

A destabilized tumor vasculature leads to limited drug delivery, hypoxia, detrimental tumor microenvironment, and even metastasis. We performed a side-by-side comparison of ABTAA (Ang2-Binding and Tie2-Activating Antibody) and ABA (Ang2-Blocking Antibody) in mice with orthotopically implanted glioma, with subcutaneously implanted Lewis lung carcinoma, and with spontaneous mammary cancer. We found that Tie2 activation induced tumor vascular normalization, leading to enhanced blood perfusion and chemotherapeutic drug delivery, markedly lessened lactate acidosis, and reduced tumor growth and metastasis. Moreover, ABTAA favorably altered the immune cell profile within tumors. Together, our findings establish that simultaneous Tie2 activation and Ang2 inhibition form a powerful therapeutic strategy to elicit a favorable tumor microenvironment and enhanced delivery of a chemotherapeutic agent into tumors.


Assuntos
Anticorpos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Glioma/tratamento farmacológico , Receptor TIE-2/metabolismo , Ribonuclease Pancreático/antagonistas & inibidores , Animais , Anticorpos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Neoplasias da Mama/irrigação sanguínea , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Sinergismo Farmacológico , Feminino , Glioma/irrigação sanguínea , Humanos , Camundongos , Transplante de Neoplasias , Ligação Proteica/efeitos dos fármacos , Temozolomida , Resultado do Tratamento , Microambiente Tumoral/efeitos dos fármacos
6.
Biochem Biophys Res Commun ; 467(4): 771-7, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26482850

RESUMO

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 µM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.


Assuntos
Anticorpos Monoclonais/metabolismo , Bioensaio/métodos , Citosol/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina G/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/genética , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293/metabolismo , Células HeLa/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Espectrometria de Fluorescência/métodos
7.
J Control Release ; 216: 56-68, 2015 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-26260451

RESUMO

Neuropilin-1 (NRP1) receptor, involved in vascular endothelial growth factor (VEGF)-mediated vascular permeability and tumor angiogenesis, is targeted by peptides that bind to its VEGF-binding site. However, these peptides also cross-react with the structurally related receptor, NRP2. Here, we describe an immunoglobulin Fc-fused peptide, Fc-TPP11, which specifically binds to the VEGF-binding site of NRP1 with approximately 2nM affinity, but negligibly to that of NRP2. Fc-TPP11 triggered NRP1-dependent signaling, enhanced vascular permeability via vascular endothelial (VE)-cadherin downregulation, and increased paracellular permeability via E-cadherin downregulation in tumor tissues. Fc-TPP11 also significantly enhanced the tumor penetration of co-injected anti-cancer drug, doxorubicin, leading to the improved in vivo anti-tumor efficacy. Fc-TPP11 was easily adapted to the full-length anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) cetuximab (Erbitux), cetuximab-TPP11, exhibiting more than 2-fold improved tumor penetration than the parent cetuximab. Fc-TPP11 exhibited a similar whole-body half-life to that of intact Fc in tumor bearing mice. In addition to the tumor-penetrating activity, Fc-TPP11 suppressed VEGF-dependent angiogenesis by blocking VEGF binding to NRP1, thereby inhibiting tumor growth without promoting metastasis in the mouse model. Our results show that NRP1-specific, high-affinity binding of Fc-TPP11, is useful to validate NRP1 signaling, independent of NRP2. Thus, Fc-TPP11 can be used as a tumor penetration-promoting agent with anti-angiogenic activity or directly adapted to mAb-TPP11 format for more potent anti-cancer antibody therapy.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Neoplasias Experimentais/tratamento farmacológico , Neuropilina-1/química , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacocinética , Animais , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacocinética , Sítios de Ligação/efeitos dos fármacos , Caderinas/biossíntese , Cetuximab/farmacologia , Regulação para Baixo/efeitos dos fármacos , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Humanos , Fragmentos Fc das Imunoglobulinas/química , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/metabolismo , Permeabilidade , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
8.
MAbs ; 6(6): 1402-14, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25484049

RESUMO

Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were co-expressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira®) and bevacizumab (Avastin®), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.


Assuntos
Anticorpos Monoclonais/metabolismo , Citosol/metabolismo , Endocitose , Imunoglobulina G/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Células CHO , Linhagem Celular Tumoral , Sobrevivência Celular , Cricetinae , Cricetulus , Células HT29 , Células HeLa , Humanos , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Células K562 , Células MCF-7 , Microscopia Confocal , Dados de Sequência Molecular , Engenharia de Proteínas/métodos , Transporte Proteico , Homologia de Sequência de Aminoácidos , Imagem com Lapso de Tempo/métodos
9.
Cell Mol Life Sci ; 70(22): 4369-83, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23765104

RESUMO

Actin plays a fundamental role in the regulation of spine morphology (both shrinkage and enlargement) upon synaptic activation. In particular, actin depolymerization is crucial for the spine shrinkage in NMDAR-mediated synaptic depression. Here, we define the role of SPIN90 phosphorylation/dephosphorylation in regulating actin depolymerization via modulation of cofilin activity. When neurons were treated with NMDA, SPIN90 was dephosphorylated by STEP61 (striatal-enriched protein tyrosine phosphatase) and translocated from the spines to the dendritic shafts. In addition, phosphorylated SPIN90 bound cofilin and then inhibited cofilin activity, suggesting that SPIN90 dephosphorylation is a prerequisite step for releasing cofilin so that cofilin can adequately sever actin filaments into monomeric form. We found that SPIN90 YE, a phosphomimetic mutant, remained in the spines after NMDAR activation where it bound cofilin, thereby effectively preventing actin depolymerization. This led to inhibition of the activity-dependent redistribution of cortactin and drebrin A, as well as of the morphological changes in the spines that underlie synaptic plasticity. These findings indicate that NMDA-induced SPIN90 dephosphorylation and translocation initiates cofilin-mediated actin dynamics and spine shrinkage within dendritic spines, thereby modulating synaptic activity.


Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cofilina 1/metabolismo , Hipocampo/metabolismo , Proteínas Musculares/metabolismo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Espinhas Dendríticas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Musculares/genética , Mutação , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Ratos , Transfecção
10.
PLoS One ; 8(1): e54276, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23342115

RESUMO

The correct rearrangement of postsynaptic components in dendritic spines is important for driving changes of spine structure and synaptic function. SPIN90 plays an essential role in many cellular processes including actin polymerization, endocytosis, growth cone formation and dendritic spine morphogenesis. Here, we demonstrate that SPIN90, which is a binding partner of PSD95 and Shank in spines, is targeted to synapses and leads to enhanced synaptic activity in neurons. We show, using in vitro and in vivo kinase assays, that SPIN90 is tyrosine phosphorylated by Src kinase. SPIN90 that was tyrosine-phosphorylated by Src was targeted to dendritic spines in cultured hippocampal neurons. Moreover, a SPIN90 phospho-deficient mutant was unable to accumulate at dendritic spines whereas SPIN90 WT and a phospho-mimicking mutant were localized at spines and bound PSD95 and Shank with increased efficiency. Consistent with these findings, hippocampal neurons that overexpressed SPIN90 WT or a phospho-mimicking mutant had enlarged spine heads, leading to enhanced postsynaptic function in terms of both amplitude and frequency. Together, our findings show that SPIN90 modulates synaptic activity in neurons as a result of its phosphorylation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Coluna Vertebral/metabolismo , Sinapses/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Eletrofisiologia , Hipocampo/citologia , Humanos , Imunoprecipitação , Fosforilação
11.
PLoS One ; 7(4): e34514, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22496823

RESUMO

Synaptic plasticity is an important feature of neurons essential for learning and memory. Postsynaptic organization and composition are dynamically remodeled in response to diverse synaptic inputs during synaptic plasticity. During this process, the dynamics and localization of postsynaptic proteins are also precisely regulated. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family. Overexpression of NESH is associated with reduced cell motility and tumor metastasis. Strong evidence of a close relationship between NESH and the actin cytoskeleton has been documented. Although earlier studies have shown that NESH is prominently expressed in the brain, its function and characteristics are yet to be established. Data from the present investigation suggest that synaptic localization of NESH in hippocampal neurons is regulated in an F-actin-dependent manner. The dynamic fraction of NESH in the dendritic spine was analyzed using FRAP (fluorescence recovery after photobleaching). Interestingly, F-actin stabilization and disruption significantly affected the mobile fraction of NESH, possibly through altered interactions of NESH with the F-actin. In addition, NESH was synaptically targeted from the dendritic shaft to spine after induction of chemical LTP (long-term potentiation) and the translocation was dependent on F-actin. Our data collectively support the significance of the F-actin cytoskeleton in synaptic targeting of NESH as well as its dynamics.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Espinhas Dendríticas/metabolismo , Hipocampo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Feminino , Recuperação de Fluorescência Após Fotodegradação , Hipocampo/embriologia , Processamento de Imagem Assistida por Computador , Potenciação de Longa Duração , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , Neurônios/citologia , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Sinapses/fisiologia
12.
PLoS One ; 7(4): e34677, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22485184

RESUMO

BACKGROUND: Dendritic spines are small membranous protrusions on the neuronal dendrites that receive synaptic input from axon terminals. Despite their importance for integrating the enormous information flow in the brain, the molecular mechanisms regulating spine morphogenesis are not well understood. NESH/Abi-3 is a member of the Abl interactor (Abi) protein family, and its overexpression is known to reduce cell motility and tumor metastasis. NESH is prominently expressed in the brain, but its function there remains unknown. METHODOLOGY/PRINCIPAL FINDINGS: NESH was strongly expressed in the hippocampus and moderately expressed in the cerebral cortex, cerebellum and striatum, where it co-localized with the postsynaptic proteins PSD95, SPIN90 and F-actin in dendritic spines. Overexpression of NESH reduced numbers of mushroom-type spines and synapse density but increased thin, filopodia-like spines and had no effect on spine density. siRNA knockdown of NESH also reduced mushroom spine numbers and inhibited synapse formation but it increased spine density. The N-terminal region of NESH co-sedimented with filamentous actin (F-actin), which is an essential component of dendritic spines, suggesting this interaction is important for the maturation of dendritic spines. CONCLUSIONS/SIGNIFICANCE: NESH is a novel F-actin binding protein that likely plays important roles in the regulation of dendritic spine morphogenesis and synapse formation.


Assuntos
Espinhas Dendríticas/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Forma Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/citologia , Humanos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo
13.
Exp Cell Res ; 317(16): 2276-87, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21763308

RESUMO

SPIN90 is an F-actin binding protein thought to play important roles in regulating cytoskeletal dynamics. It is known that SPIN90 is expressed during the early stages of neuronal development, but details of its localization and function in growth cones have not been fully investigated. Our immunocytochemical data show that SPIN90 is enriched throughout growth cones and neuronal shafts in young hippocampal neurons. We also found that its localization correlates with and depends upon the presence of F-actin. Detailed observation of primary cultures of hippocampal neurons revealed that SPIN90 knockout reduces both growth cone areas and in the numbers of filopodia, as compared to wild-type neurons. In addition, total neurite length, the combined lengths of the longest (axonal) and shorter (dendritic) neurites, was smaller in SPIN90 knockout neurons than wild-type neurons. Finally, Cdc42 activity was down-regulated in SPIN90 knockout neurons. Taken together, our findings suggest that SPIN90 plays critical roles in controlling growth cone dynamics and neurite outgrowth.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cones de Crescimento/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/fisiologia , Células Cultivadas , Citocalasina D/farmacologia , Embrião de Mamíferos/citologia , Feminino , Cones de Crescimento/patologia , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/genética , Neuritos/patologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pseudópodes/metabolismo , Pseudópodes/patologia , Ratos , Ratos Endogâmicos , Tiazolidinas/farmacologia , Tubulina (Proteína)/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
14.
PLoS One ; 6(5): e20125, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21625594

RESUMO

BACKGROUND: Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas. CONCLUSIONS/SIGNIFICANCE: Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.


Assuntos
Proteína Substrato Associada a Crk/fisiologia , Dinaminas/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Animais , Western Blotting , Endocitose , Imunofluorescência , Humanos , Fosforilação
15.
PLoS One ; 5(12): e15645, 2010 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-21187975

RESUMO

BACKGROUND: The nuclear inclusion a (NIa) protease of turnip mosaic virus (TuMV) is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12)-His-His-Gln(15), near the presumptive α-secretase cleavage site of the amyloid-ß (Aß) peptide led us to hypothesize that NIa could possess activity against Aß. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aß can be degraded by NIa in vitro. The specific cleavage of Aß was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aß. Moreover, lentiviral-mediated expression of NIa in APP(sw)/PS1 transgenic mice significantly reduced the levels of Aß and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aß in the cytoplasm could be a novel strategy to control the levels of Aß, plaque formation, and the associated cell death.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Brassica napus/virologia , Endopeptidases/química , Regulação Enzimológica da Expressão Gênica , Vírus do Mosaico/enzimologia , Proteínas Virais/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Citoplasma/metabolismo , Progressão da Doença , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Vírus do Mosaico/genética , Neurônios/citologia , Ratos , Frações Subcelulares/metabolismo , Especificidade por Substrato
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