RESUMO
Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer's patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.
Assuntos
Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Células Apresentadoras de Antígenos/metabolismo , Toxinas de Bacillus thuringiensis , Cristalização , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Proteínas de Fluorescência Verde/metabolismo , Luciferases/metabolismo , Medições Luminescentes , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Nódulos Linfáticos Agregados/metabolismo , Células RAW 264.7 , Proteínas Recombinantes de Fusão/metabolismoRESUMO
SHIP and SHIP-2 are inositol phosphatases that regulate FcγR-mediated phagocytosis through catalytic as well as non-catalytic mechanisms. In this study we have used two-dimensional fluorescence difference gel electrophoresis (DIGE) analysis to identify downstream signaling proteins that uniquely associate with SHIP or SHIP-2 upon FcγR clustering in human monocytes. We identified LyGDI as a binding partner of SHIP, associating inducibly with the SHIP/Grb2/Shc complex. Immunodepletion and competition experiments with recombinant SHIP domains revealed that Grb2 and the proline-rich domain of SHIP were necessary for SHIP-LyGDI association. Functional studies in primary human monocytes showed that LyGDI sequesters Rac in the cytosol, preventing it from localizing to the membrane. Consistent with this, suppression of LyGDI expression resulted in significantly enhanced FcγR-mediated phagocytosis.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Fagocitose , Monoéster Fosfórico Hidrolases/metabolismo , Receptores de IgG/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Proteína Adaptadora GRB2/metabolismo , Humanos , Inositol Polifosfato 5-Fosfatases , Monócitos/citologia , Monócitos/metabolismo , Monoéster Fosfórico Hidrolases/química , Prolina , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
The antitumor effects of therapeutic mAbs may depend on immune effector cells that express FcRs for IgG. IL-12 is a cytokine that stimulates IFN-γ production from NK cells and T cells. We hypothesized that coadministration of IL-12 with a murine anti-HER2/neu mAb (4D5) would enhance the FcR-dependent immune mechanisms that contribute to its antitumor activity. Thrice-weekly therapy with IL-12 (1 µg) and 4D5 (1 mg/kg) significantly suppressed the growth of a murine colon adenocarcinoma that was engineered to express human HER2 (CT-26(HER2/neu)) in BALB/c mice compared with the result of therapy with IL-12, 4D5, or PBS alone. Combination therapy was associated with increased circulating levels of IFN-γ, monokine induced by IFN-γ, and RANTES. Experiments with IFN-γ-deficient mice demonstrated that this cytokine was necessary for the observed antitumor effects of therapy with IL-12 plus 4D5. Immune cell depletion experiments showed that NK cells (but not CD4(+) or CD8(+) T cells) mediated the antitumor effects of this treatment combination. Therapy of HER2/neu-positive tumors with trastuzumab plus IL-12 induced tumor necrosis but did not affect tumor proliferation, apoptosis, vascularity, or lymphocyte infiltration. In vitro experiments with CT-26(HER2/neu) tumor cells revealed that IFN-γ induced an intracellular signal but did not inhibit cellular proliferation or induce apoptosis. Taken together, these data suggest that tumor regression in response to trastuzumab plus IL-12 is mediated through NK cell IFN-γ production and provide a rationale for the coadministration of NK cell-activating cytokines with therapeutic mAbs.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias do Colo/terapia , Interferon gama/biossíntese , Interleucina-12/uso terapêutico , Células Matadoras Naturais/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais Humanizados , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Testes Imunológicos de Citotoxicidade , Feminino , Interferon gama/fisiologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Receptor ErbB-2/imunologia , Trastuzumab , Regulação para Cima/imunologiaRESUMO
Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcgammaRIIIa) that mediates interferon (IFN)-gamma production in response to antibody (Ab)-coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-gamma as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-gamma production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal-regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-gamma production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-gamma production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-gamma production. Confocal microscopy and cellular fractionation experiments revealed that FcgammaRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-gamma production. These data suggest that dual recruitment of FcgammaRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-gamma production.
Assuntos
Interferon gama/biossíntese , Células Matadoras Naturais/enzimologia , Células Matadoras Naturais/imunologia , Microdomínios da Membrana/enzimologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptores de IgG/imunologia , Receptores de Interleucina-12/imunologia , Colesterol/deficiência , Ativação Enzimática/efeitos dos fármacos , Humanos , Imunoglobulina G/farmacologia , Interleucina-12/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição STAT4/metabolismo , Quinase Syk , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We report the labeling (internalization) of skeletal myoblasts (SMs) with a novel class of oxygen-sensing paramagnetic spin probe for noninvasive tracking and in situ monitoring of oxygenation in stem cell therapy using electron paramagnetic resonance (EPR) spectroscopy. SM cells were isolated from thigh muscle biopsies of mice and propagated in culture. Labeling of SM cells with the probe was achieved by coincubating the cells with submicron-sized (270 +/- 120 nm) particulates of the probe in culture for 48 h. The labeling had no significant effect on the viability or proliferation of the cells. The SM cells labeled with the probe were transplanted in the infarcted region of mouse hearts. The engraftment of the transplanted cells in the infarct region was verified by using MY-32 staining for skeletal myocytes. The in situ Po(2) in the heart was determined noninvasively and repeatedly for 4 wk after transplantation. The results showed significant enhancement of myocardial oxygenation at the site of cell transplant compared with untreated control. In conclusion, labeling of SM cells with the oxygen-sensing spin probe offers a unique opportunity for the noninvasive monitoring of transplanted cells as well as in situ tissue Po(2) in infarcted mouse hearts.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Coração/fisiologia , Coração/fisiopatologia , Músculo Esquelético/fisiologia , Infarto do Miocárdio/fisiopatologia , Oxigênio/análise , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Espectroscopia de Ressonância de Spin Eletrônica , Citometria de Fluxo , Camundongos , Modelos Animais , Músculo Esquelético/citologia , Miocárdio/citologiaRESUMO
Cellular stress leads to a change in distribution of RNA-binding proteins. HuR, a member of the ELAV/Hu family of RNA-binding proteins, is nuclear in distribution and following heat shock is found in large cytoplasmic stress granules where translation is inhibited. HuD, another ELAV/Hu RNA-binding protein, stabilizes the GAP-43 mRNA in response to nerve growth factor (NGF) stimulation in PC12 cells. We were interested in determining the nuclear distribution of HuD and if neurotrophic stimulation induced changes in the distribution of HuD. In PC12 cells, we found, as expected, that HuR translocates from the nucleus to the cytoplasm in response to heat shock. In response to heat shock, HuD forms large cytoplasmic stress granules, consistent with a role for HuD in the cessation of translation. In unstimulated cells, HuD is distributed in small granules in the cytoplasm and is consistently present at low levels in the nucleus. Stimulation of PC12 cells with NGF induces neuronal differentiation including outgrowth of neurites and increased levels of GAP-43 protein, whereas HuD remains localized in small cytoplasm granules and is still present in the nucleus. These results suggest that, following neurotrophic stimulation, the lack of changes in HuD distribution are due to continued steady state of HuD nuclear shuttling in PC12 cells, or that HuD is not normally shuttled from the nucleus in response to NGF.
Assuntos
Proteínas ELAV/metabolismo , Resposta ao Choque Térmico , Fator de Crescimento Neural/fisiologia , Animais , Diferenciação Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Proteínas ELAV/biossíntese , Proteínas ELAV/genética , Fator de Crescimento Neural/farmacologia , Neuritos/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Células PC12 , Transporte Proteico , RNA Mensageiro/metabolismo , RatosRESUMO
Human T-cell leukemia virus type 1 (HTLV-1) Gag is targeted to the plasma membrane for particle assembly and release. How HTLV-1 Gag targeting occurs is not well understood. The PPPY and PTAP motifs were previously shown to be involved in HTLV-1 particle release with PTAP playing a more subtle role in virus budding. These L domains function through the interaction with host cellular proteins normally involved in multivesicular body (MVB) morphogenesis. The plasma membrane pathway rather than the MVB pathway was found to be the primary pathway for HTLV-1 particle release in HeLa cells. Intriguingly, disruption of the PTAP motif led to a defect in the targeting of Gag from the plasma membrane to CD63-positive MVBs. Particles or particle buds were observed to be associated with MVBs by electron microscopy, implying that Gag targeting to the MVB resulted in particle budding. Blocking clathrin-dependent endocytosis was found not to influence localization of the HTLV-1 Gag PTAP mutant, indicating that Gag did not reach the MVBs through clathrin-dependent endocytosis. Our observations imply that the interaction between Gag and TSG101 is not required for Gag targeting to the MVB. Overexpression of dynamitin p50 increased particle release, suggesting that there was an increase in the intracellular transport of MVBs to the cell periphery by the utilization of the dynein-dynactin motor complex. Intriguingly, virus particle release with this mutant was reduced by 20-fold compared to that of wild type in HeLa cells, which is in marked contrast to the less-than-twofold defect observed for particle production of the HTLV-1 Gag PTAP mutant from 293T cells. These results indicate that the role of the PTAP motif in L domain function is cell type dependent.
Assuntos
Produtos do Gene gag/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Vírion/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Antígenos CD/metabolismo , Antígenos CD/ultraestrutura , Autoantígenos , Membrana Celular/metabolismo , Membrana Celular/virologia , Complexo Dinactina , Técnica Indireta de Fluorescência para Anticorpo , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene gag/ultraestrutura , Marcadores Genéticos , Células HeLa , Vírus Linfotrópico T Tipo 1 Humano/química , Vírus Linfotrópico T Tipo 1 Humano/genética , Vírus Linfotrópico T Tipo 1 Humano/ultraestrutura , Humanos , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Plasmídeos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Transfecção , Transferrina/metabolismo , Proteínas de Transporte Vesicular , Vírion/ultraestrutura , Montagem de VírusRESUMO
A new field of gene transfer is emerging as a simple, effective means to drive the expression foreign genes in cells: ultrasound-mediated gene transfer or sonoporation. We report here that sonoporation is an effective means of gene transfer for cultured neurons, a cell type that has been difficult to transfect. Neuronal cell types that are effectively sonoporated include chick retinal neurons, chick dorsal forebrain, chick optic tectum, PC12 cells, rat cerebellar neurons and mouse hippocampal neurons. Depending on the type of cell and conditions of sonoporation the transfection efficacy was as high as 20%. Sonoporation of plasmid DNA was effective for cells adherent to a substrate and for free-floating cells that were freshly dissociated. In the free-floating preparations, between 60 and 95% of the cells that were transfected were neuronal, as much as 90% higher than that observed for other methods of gene transfer including adenovirus and lipid-based transfection methods. We conclude that sonoporation is a simple, effective and inexpensive means by which to preferentially transfect DNA into neuronal cells.
Assuntos
Eletroporação/métodos , Técnicas de Transferência de Genes , Neurônios/metabolismo , Transfecção/métodos , Ultrassom , Animais , Morte Celular , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Embrião de Galinha , Galinhas/metabolismo , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Imuno-Histoquímica , Camundongos , Neurônios/citologia , Plasmídeos/administração & dosagem , Plasmídeos/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Ratos , Retina/citologia , Retina/metabolismoRESUMO
The neuron-specific ELAV/Hu family member, HuD, interacts with and stabilizes GAP-43 mRNA in developing neurons, and leads to increased levels of GAP-43 protein. As GAP-43 protein is enriched in growth cones, it is of interest to determine if HuD and GAP-43 mRNA are associated in developing growth cones. HuD granules in growth cones are found in the central domain that is rich in microtubules and ribosomes, in the peripheral domain with its actin network, and in filopodia. This distribution of HuD granules in growth cones is dependent on actin filaments but not on microtubules. GAP-43 mRNA is localized in granules found in both the central and peripheral domains, but not in filopodia. Ribosomes were extensively colocalized with HuD and GAP-43 mRNA granules in the central domain, consistent with a role in the control of GAP-43 mRNA stability in the growth cone. Together, these results demonstrate that many of the components necessary for GAP-43 mRNA translation/stabilization are present within growth cones.
Assuntos
Proteína GAP-43/fisiologia , Cones de Crescimento/fisiologia , Proteínas do Tecido Nervoso/fisiologia , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/fisiologia , Ribossomos/fisiologia , Animais , Proteínas ELAV , Proteína GAP-43/biossíntese , Proteína GAP-43/genética , Cones de Crescimento/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Células PC12 , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Ratos , Ribossomos/efeitos dos fármacos , Ribossomos/genéticaRESUMO
The regulation of receptor tyrosine kinases (RTKs) is important in several cellular events, including proliferation, differentiation, and apoptosis. Gangliosides are sialic acid-containing glycosphingolipids that can regulate RTK activity. The addition of ganglioside GM1 to the medium of Swiss 3T3 fibroblasts inhibits both platelet-derived growth factor (PDGF)-mediated tyrosine phosphorylation of PDGF receptor beta (PDGFRbeta) and receptor-mediated endocytosis. However, GM1 did not affect PDGF-mediated receptor phosphorylation, neuritogenesis, or endocytosis in PC12 cells stably transfected with the gene for PDGFRbeta. The ability of GM1 to modulate PDGFRbeta in 3T3 cells but not in transfected PC12 cells indicates a cell context-dependent response. We hypothesized that this inhibition of PDGFRbeta by GM1 must map to one or more domains of the receptor. Thus, a chimeric receptor was created that possessed the extracellular and transmembrane domains of the nerve growth factor (NGF) receptor TrkA and the cytoplasmic domain of PDGFRbeta (TTbeta). In 3T3 cells transfected with the TTbeta construct, GM1 did not inhibit NGF-induced tyrosine phosphorylation of the chimeric receptor or of Erk1/2 in this cell line. GM1 still inhibited PDGF-mediated tyrosine phosphorylation of endogenous PDGFRbeta and of Erk1/2 in Swiss TTbeta cells. Thus, the cytoplasmic domain of PDGFRbeta is not required for GM1-dependent inhibition of PDGFRbeta in 3T3 cells. This suggests that the inhibition of PDGFRbeta by GM1 in Swiss 3T3 fibroblasts maps to either the extracellular and/or transmembrane domain of PDGFRbeta.