RESUMO
Antibodies are indispensable tools in protein engineering and structural biology. Antibodies suitable for structural studies should recognize the 3-dimensional (3D) conformations of target proteins. Generating such antibodies and characterizing their complexes with antigens take a significant amount of time and effort. Here, we show that we can expand the application of well-characterized antibodies by "transplanting" the epitopes that they recognize to proteins with completely different structures and sequences. Previously, several antibodies have been shown to recognize the alpha-helical conformation of antigenic peptides. We demonstrate that these antibodies can be made to bind to a variety of unrelated "off-target" proteins by modifying amino acids in the preexisting alpha helices of such proteins. Using X-ray crystallography, we determined the structures of the engineered protein-antibody complexes. All of the antibodies bound to the epitope-transplanted proteins, forming accurately predictable structures. Furthermore, we showed that binding of these antihelix antibodies to the engineered target proteins can modulate their catalytic activities by trapping them in selected functional states. Our method is simple and efficient, and it will have applications in protein X-ray crystallography, electron microscopy, and nanotechnology.
Assuntos
Epitopos/química , Proteínas/química , Anticorpos de Cadeia Única/química , Cristalografia por Raios X , Humanos , Conformação Proteica em alfa-HéliceRESUMO
Hemagglutinin (HA) displayed on a ferritin nano-cage has been shown to be effective in generating a potent immune response against a broad range of influenza infections. Here, we showed that conjugation of flagellin together with HA to the exterior surface of the ferritin cage greatly enhanced not only the humoral immune response in mice but also antigen-specific T cell responses that include Th1 cytokine secretion. The effect of flagellin remained essentially unchanged when the molar ratio of flagellin to HA was reduced from 1:1 to 1:3. Injection of the ferritin-HA-flagellin cage provided protection against lethal virus challenge in mice. We used a small immunoglobulin fragment VL12.3 as a convenient method for attaching HA and flagellin to the ferritin cage. This attachment method can be used for rapid screening of a variety of protein cages and nano-assemblies to identify the most suitable carrier and adjuvant proteins for the target antigen.
Assuntos
Adjuvantes Imunológicos/química , Ferritinas/química , Flagelina/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A/química , Salmonella typhimurium/química , Adjuvantes Imunológicos/farmacologia , Animais , Linhagem Celular , Feminino , Ferritinas/farmacologia , Flagelina/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/farmacologia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/químicaRESUMO
Toll-like receptor 2 (TLR2) initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. We have determined the crystal structures of TLR2-TLR6-diacylated lipopeptide, TLR2-lipoteichoic acid, and TLR2-PE-DTPA complexes. PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, is a synthetic phospholipid derivative. Two major factors contribute to the ligand specificity of TLR2-TLR1 or TLR2-TLR6 heterodimers. First, the lipid channel of TLR6 is blocked by two phenylalanines. Simultaneous mutation of these phenylalanines made TLR2-TLR6 fully responsive not only to diacylated but also to triacylated lipopeptides. Second, the hydrophobic dimerization interface of TLR2-TLR6 is increased by 80%, which compensates for the lack of amide lipid interaction between the lipopeptide and TLR2-TLR6. The structures of the TLR2-lipoteichoic acid and the TLR2-PE-DTPA complexes demonstrate that a precise interaction pattern of the head group is essential for a robust immune response by TLR2 heterodimers.
Assuntos
Lipopeptídeos/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Acilação , Animais , Sítios de Ligação , Cristalografia por Raios X , Feiticeiras (Peixe) , Humanos , Ligantes , Lipopeptídeos/química , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Camundongos , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/imunologia , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Ácidos Teicoicos/química , Ácidos Teicoicos/imunologia , Ácidos Teicoicos/metabolismo , Receptor 1 Toll-Like/química , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/química , Receptor 6 Toll-Like/químicaRESUMO
The activity and morphology of mitochondria are maintained by dynamic fusion and fission processes regulated by a group of proteins residing in, or attached to, their inner and outer membranes. Hypoxia-induced gene domain protein-1a (Higd-1a)/HIMP1-a/HIG1, a mitochondrial inner membrane protein, plays a role in cell survival under hypoxic conditions. In the present study, we showed that Higd-1a depletion resulted in mitochondrial fission, depletion of mtDNA, disorganization of cristae, and growth retardation. We demonstrated that Higd-1a functions by specifically binding to Optic atrophy 1 (Opa1), a key element in fusion of the inner membrane. In the absence of Higd-1a, Opa1 was cleaved, resulting in the loss of its long isoforms and accumulation of small soluble forms. The small forms of Opa1 do not interact with Higd-1a, suggesting that a part of Opa1 in or proximal to the membrane is required for that interaction. Opa1 cleavage, mitochondrial fission, and cell death induced by dissipation of the mitochondrial membrane potential were significantly inhibited by ectopic expression of Higd-1a. Furthermore, growth inhibition due to Higd-1a depletion could be overcome by overexpression of a noncleavable form of Opa1. Collectively, our observations demonstrate that Higd-1a inhibits Opa1 cleavage and is required for mitochondrial fusion by virtue of its interaction with Opa1.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Neoplasias/metabolismo , Trifosfato de Adenosina/metabolismo , Western Blotting , GTP Fosfo-Hidrolases/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Potencial da Membrana Mitocondrial/genética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/genética , Proteínas de Neoplasias/genética , Ligação Proteica , Interferência de RNARESUMO
The lipopolysaccharide (LPS) of Gram negative bacteria is a well-known inducer of the innate immune response. Toll-like receptor (TLR) 4 and myeloid differentiation factor 2 (MD-2) form a heterodimer that recognizes a common 'pattern' in structurally diverse LPS molecules. To understand the ligand specificity and receptor activation mechanism of the TLR4-MD-2-LPS complex we determined its crystal structure. LPS binding induced the formation of an m-shaped receptor multimer composed of two copies of the TLR4-MD-2-LPS complex arranged symmetrically. LPS interacts with a large hydrophobic pocket in MD-2 and directly bridges the two components of the multimer. Five of the six lipid chains of LPS are buried deep inside the pocket and the remaining chain is exposed to the surface of MD-2, forming a hydrophobic interaction with the conserved phenylalanines of TLR4. The F126 loop of MD-2 undergoes localized structural change and supports this core hydrophobic interface by making hydrophilic interactions with TLR4. Comparison with the structures of tetra-acylated antagonists bound to MD-2 indicates that two other lipid chains in LPS displace the phosphorylated glucosamine backbone by approximately 5 A towards the solvent area. This structural shift allows phosphate groups of LPS to contribute to receptor multimerization by forming ionic interactions with a cluster of positively charged residues in TLR4 and MD-2. The TLR4-MD-2-LPS structure illustrates the remarkable versatility of the ligand recognition mechanisms employed by the TLR family, which is essential for defence against diverse microbial infection.
Assuntos
Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito/química , Antígeno 96 de Linfócito/imunologia , Receptor 4 Toll-Like/química , Receptor 4 Toll-Like/imunologia , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Relação Estrutura-AtividadeRESUMO
Sialyllactose (SL) is a functional human milk oligosaccharide essential for immune support, brain development, intestinal maturation, and antiviral defense. However, despite its established health benefits, the effect of SL on exercise performance and muscle mass in mice remains unknown. Here, we aimed to investigate, for the first time, the effects of 6'-SL on muscle functions. Seven-week-old male C57BL/6J mice were administered 100 mg/kg 6'-SL for 12 weeks, after which exhaustive treadmill performance was conducted. Moreover, muscle strength was examined by grip strength, and muscle phenotype characteristics such as muscle mass, muscle fiber size, and muscle protein expression were also examined. The administration of 6'-SL significantly improved exhaustive treadmill performance metrics, including distance and exhaustion time. Grip strength was also increased by 6'-SL administration. Additionally, 6'-SL increased muscle mass in both the gastrocnemius (GAS) and soleus. 6'-SL administration led to an increase in the minimum Feret's diameter and the protein expression of total myosin heavy chain in the GAS muscle. In conclusion, 6'-SL administration in vivo led to increased running distance and time by increasing muscle mass and strength. These findings collectively indicate that 6'-SL is a potential agent for improving muscle health and exercise performance.
Assuntos
Camundongos Endogâmicos C57BL , Força Muscular , Músculo Esquelético , Condicionamento Físico Animal , Animais , Masculino , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Força Muscular/efeitos dos fármacos , Condicionamento Físico Animal/fisiologia , Camundongos , Lactose/análogos & derivados , Lactose/farmacologia , Cadeias Pesadas de Miosina/metabolismo , Proteínas Musculares/metabolismoRESUMO
Tumor-associated macrophages (TAM) are involved in tumor progression, metastasis, and immunosuppression. Because TAMs are highly plastic and could alter their phenotypes to proinflammatory M1 in response to environmental stimuli, reeducating TAMs has emerged as a promising approach to overcoming the challenges of solid cancer treatment. This study investigated the effect of IL9 on macrophage M1 polarization and verified its antitumor potential to retrain TAMs and promote chemokine secretion. We demonstrated that IL9 stimulated macrophage proliferation and polarized them toward the proinflammatory M1 phenotype in an IFNγ-dependent manner. Tumor-localized IL9 also polarized TAMs toward M1 in vivo and made them release CCL3/4 and CXCL9/10 to recruit antitumor immune cells, including T and natural killer cells, into the tumor microenvironment. Furthermore, peritoneal treatment with recombinant IL9 delayed the growth of macrophage-enriched B16F10 melanoma and 4T1 breast cancer in syngeneic mice, although IL9 treatment did not reduce tumor growth in the absence of macrophage enrichment. These results demonstrate the efficacy of IL9 in macrophage polarization to trigger antitumor immunity. Significance: These findings clarified the effect of IL9 on macrophage M1 polarization and verified its antitumor potential through retraining TAMs and chemokine secretion.
Assuntos
Interleucina-9 , Melanoma , Camundongos , Animais , Interleucina-9/farmacologia , Macrófagos , Melanoma/patologia , Ativação de Macrófagos , Quimiocinas/farmacologia , Microambiente TumoralRESUMO
CD40 is a tumor necrosis factor receptor (TNFR) family protein that plays an important role in B cell development. CD154/CD40L is the physiological ligand of CD40. We have determined the crystal structure of the CD40-CD154 complex at 3.5 Å resolution. The binding site of CD40 is located in a crevice formed between two CD154 subunits. Charge complementarity plays a critical role in the CD40-CD154 interaction. Some of the missense mutations found in hereditary hyper-IgM syndrome can be mapped to the CD40-CD154 interface. The CD40 interaction area of one of the CD154 subunits is twice as large as that of the other subunit forming the binding crevice. This is because cysteine-rich domain 3 (CRD3) of CD40 has a disulfide bridge in an unusual position that alters the direction of the ladder-like structure of CD40. The Ser(132) loop of CD154 is not involved in CD40 binding but its substitution significantly reduces p38- and ERK-dependent signaling by CD40, whereas JNK-dependent signaling is not affected. These findings suggest that ligand-induced di- or trimerization is necessary but not sufficient for complete activation of CD40.
Assuntos
Antígenos CD40 , Ligante de CD40 , Mutação de Sentido Incorreto , Transdução de Sinais/fisiologia , Animais , Sítios de Ligação , Antígenos CD40/química , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ligante de CD40/química , Ligante de CD40/genética , Ligante de CD40/metabolismo , Cristalografia por Raios X , Dissulfetos , Células HEK293 , Humanos , Síndrome de Imunodeficiência com Hiper-IgM/genética , Síndrome de Imunodeficiência com Hiper-IgM/metabolismo , MAP Quinase Quinase 4/química , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-AtividadeRESUMO
Higd-1a (hypoxia induced gene domain family-1a) is a mitochondrial inner membrane protein with a conformation of N-terminal outside-C-terminal outside and loop inside. There are four Higd genes, Higd-1a, -1b, -1c and -2a, in the mouse. Higd-1a and -2a are expressed primarily in the brain, heart, kidney and leukocytes. HIF (hypoxia-inducible factor) overexpression induced the endogenous expression and promoter activity of Higd-1a. Mutation of the HRE (hypoxia-response element) site at -32bp in the Higd-1a promoter reduced the promoter activity, suggesting that transcription of Higd-1a is regulated by binding of the transcription factor HIF to the HRE. Higd-1a promoted cell survival under hypoxia. RAW264.7 cells stably transfected with Higd-1a underwent less apoptosis than control cells in a hypoxic condition, and hypoxia-induced apoptosis was strongly enhanced when endogenous Higd-1a was silenced by siRNA. The survival effect of Higd-1a was completely abolished by deletion of the 26 N-terminal amino acids, and we showed that Higd-1a increased survival by inhibiting cytochrome C release and reducing the activities of caspases. However, expression of Bcl-2, Bax, Bad, and BNIP3 and translocation of AIF were unaffected under the same conditions. Higd-2a also enhanced cell survival under hypoxia. Cells transfected with Higd-2a underwent less apoptosis than control cells in hypoxic conditions, and hypoxia-induced apoptosis increased when endogenous Higd-2a was depleted. Together these observations indicate that Higd-1a is induced by hypoxia in a HIF-dependent manner and its anti-apoptotic effect results from inhibiting cytochrome C release and reducing caspase activities.
Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Sequência de Aminoácidos , Animais , Western Blotting , Hipóxia Celular , Células Cultivadas , DNA Mitocondrial/genética , Ativação Enzimática , Citometria de Fluxo , Células HeLa , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
This study reports the use of the BacMam system to deliver and express self-assembling IL-15 and IL-15Rα genes to murine B16F10 melanoma and CT26 colon cancer cells. BacMam-based IL-15 and IL-15Rα were well-expressed and assembled to form the biologically functional IL-15:IL-15Rα complex. Immunization with this IL-15:IL-15Rα cancer vaccine delayed tumor growth in mice by inducing effector memory CD4+ and CD8+ cells and effector NK cells which are tumor-infiltrating. It caused strong antitumor immune responses of CD8+ effector cells in a tumor-antigen specific manner both in vitro and in vivo and significantly attenuated Treg cells which a control virus-infected cancer vaccine could induce. Post-treatment with this cancer vaccine after a live cancer cell injection also prominently delayed the growth of the tumor. Collectively, we demonstrate a vaccine platform consisting of BacMam virus-infected B16F10 or CT26 cancer cells that secrete IL-15:IL-15Rα. This study is the first demonstration of a functionally competent soluble IL-15:IL-15Rα complex-related cancer vaccine using a baculovirus system and advocates that the BacMam system can be used as a secure and rapid method of producing a protective and therapeutic cancer vaccine.
RESUMO
IL-9 has been reported to play dual roles in the pathogenesis of autoimmune disorders and cancers. The collaboration of IL-9 with microenvironmental factors including the broader cytokine milieu and other cellular components may provide important keys to explain its conflicting effects in chronic conditions. In this review, we summarize recent findings on the cellular sources of, and immunological responders to IL-9, in order to interpret the role of IL-9 in the regulation of immune responses. This knowledge will provide new perspectives to improve clinical benefits and limit adverse effects of IL-9 when treating pathologic conditions.
RESUMO
Interleukin-9 (IL-9) is well known for its role in allergic inflammation. For cancer, both pro- and anti-tumor effects of IL-9 were controversially reported, but the impact of IL-9 on tumor metastasis has not yet been clarified. In this study, IL-9 was expressed as a secretory form (sIL-9) and a membrane-bound form (mbIL-9) on B16F10 melanoma cells. The mbIL-9 was engineered as a chimeric protein with the transmembrane and cytoplasmic region of TNF-α. The effect of either mbIL-9 or sIL-9 expressing cells were analyzed on the metastasis capability of the cancer cells. After three weeks of tumor implantation into C57BL/6 mice through the tail vein, the number of tumor modules in lungs injected with IL-9 expressing B16F10 was 5-fold less than that of control groups. The percentages of CD4+ T cells, CD8+ T cells, NK cells, and M1 macrophages considerably increased in the lungs of the mice injected with IL-9 expressing cells. Among them, the M1 macrophage subset was the most significantly enhanced. Furthermore, peritoneal macrophages, which were stimulated with either sIL-9 or mbIL-9 expressing transfectant, exerted higher anti-tumor cytotoxicity compared with that of the mock control. The IL-9-stimulated peritoneal macrophages were highly polarized to M1 phenotype. Stimulation of RAW264.7 macrophages with sIL-9 or mbIL-9 expressing cells also significantly increased the cytotoxicity of those macrophages against wild-type B16F10 cells. These results clearly demonstrate that IL-9 can induce an anti-metastasis effect by enhancing the polarization and proliferation of M1 macrophages.
Assuntos
Interleucina-9/metabolismo , Neoplasias Pulmonares/imunologia , Macrófagos/imunologia , Melanoma/imunologia , Neoplasias Experimentais/imunologia , Animais , Citocinas/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Ativação de Macrófagos , Melanoma/patologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias Experimentais/patologia , Células Th1/imunologiaRESUMO
Sepsis is the leading cause of death in critically ill patients. Today, around 60% of all cases of sepsis are caused by Gram-negative bacteria. The cell wall component lipopoly-saccharide (LPS) is the main initiator of the cascade of cellular reactions in Gram-negative infections. The core receptors for LPS are toll-like receptor 4 (TLR4), MD-2 and CD14. Attempts have been made to antagonize the toxic effect of endotoxin using monoclonal antibodies against CD14 and synthetic lipopolysaccharides but there is as yet no effective treatment for septic syndrome. Here, we describe an inhibitory effect of a phosphatidylethanolamine derivative, PE-DTPA (phosphatidylethanolamine diethyl-enetriaminepentaacetate) on LPS recognition. PE-DTPA bound strongly to CD14 (K ( d ), 9.52 x 10(-8) M). It dose dependency inhibited LPS-mediated activation of human myeloid cells, mouse macrophage cells and human whole blood as measured by the production of tumor necrosis factor-a (TNF-alpha) and nitric oxide, whereas other phospho-lipids including phosphatidylserine and phosphatidylethanolamine had little effect. PE-DTPA also inhibited transcription dependent on NF-kappaB activation when it was added together with LPS, and it rescued LPS-primed mice from septic death. These results suggest that PE-DTPA is a potent antagonist of LPS, and that it acts by competing for binding to CD14.
Assuntos
Lipopolissacarídeos/farmacologia , Ácido Pentético/farmacologia , Fosfatidiletanolaminas/farmacologia , Sepse/prevenção & controle , Adulto , Animais , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Ácido Pentético/química , Fosfatidiletanolaminas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sepse/induzido quimicamente , Sepse/metabolismo , Ressonância de Plasmônio de Superfície , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Interleukin (IL)-15 is an essential immune-modulator with high potential for use in cancer treatment. Natural IL-15 has a low biological potency because of its short half-life and difficulties in mass-production. IL-15Rα, a member of the IL-15 receptor complex, is famous for its high affinity to IL-15 and its ability to lengthen the half-life of IL-15. We have double-transfected IL-15 and its truncated receptor IL-15Rα into CT26 colon cancer cells to target them for intracellular assembly. The secreted IL-15:IL-15Rα complexes were confirmed in ELISA and Co-IP experiments. IL-15:IL15Rα secreting clones showed a higher anti-tumor effect than IL-15 secreting clones. Furthermore, we also evaluated the vaccine and therapeutic efficacy of the whole cancercell vaccine using mitomycin C (MMC)-treated IL-15:IL15Rα secreting CT26 clones. Three sets of experiments were evaluated; (1) therapeutics, (2) vaccination, and (3) longterm protection. Wild-type CT26-bearing mice treated with a single dose of MMC-inactivated secreted IL-15:IL-15Rα clones prolonged survival compared to the control group. Survival of MMC-inactivated IL-15:IL-15Rα clone-vaccinated mice (without any further adjuvant) exceeded up to 100%. This protection effect even lasted for at least three months after the immunization. Secreted IL-15:IL-15Rα clones challenging trigger anti-tumor response via CD4+ T, CD8+ T, and natural killer (NK) cell-dependent cytotoxicity. Our result suggested that cell-based vaccine secreting IL-15:IL-15Rα, may offer the new tools for immunotherapy to treat cancer.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias do Colo/terapia , Subunidade alfa de Receptor de Interleucina-15/imunologia , Interleucina-15/imunologia , Animais , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/prevenção & controle , Feminino , Memória Imunológica , Imunoterapia , Interleucina-15/genética , Interleucina-15/metabolismo , Subunidade alfa de Receptor de Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Higd-1a/HIMP1-a/HIG1, a mitochondrial inner membrane protein, promotes cell survival under low glucose and hypoxic conditions. We previously reported that it interacts with Opa1, a factor involved in mitochondrial fusion, to regulate mitochondrial homeostasis. In the present study, we found that depletion of Higd-1a inhibited the proliferation of pancreatic cancer cells in vitro and in mice xenografts. Higd-1a knockdown did not itself lead to cell death but it caused cell cycle arrest through induction of p27KIP1 and hypo-phosphorylation of RB protein. Knockdown of Higd-1a also induced cellular senescence as shown by increased granularity and SA-ß-galactosidase activity. We further showed that the mitochondrial stress induced by Higd-1a led to reduced ERK phosphorylation. Inhibition of the ERK pathway with U0126 induced p27KIP1 expression in the pancreatic cancer cells, confirming that the cell cycle retardation was the result of inhibition of the ERK pathway. Array analysis of human pancreatic cancers revealed that expression of Higd-1a was significantly elevated in pancreatic cancer tissues compared to normal tissue. Collectively, our results demonstrate that Higd-1a plays an important role in the proliferation of pancreatic cancer cells by regulating the pERK/p27KIP1/pRB signaling pathway.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/patologia , Proteína do Retinoblastoma/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Pontos de Checagem do Ciclo Celular , Movimento Celular , Inibidor de Quinase Dependente de Ciclina p27/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Nus , Proteínas Mitocondriais/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Prognóstico , Proteína do Retinoblastoma/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IL-9 is a known T cell growth factor with pleiotropic immunological functions, especially in parasite infection and colitis. However, its role in tumor growth is controversial. In this study, we generated tumor clones expressing the membrane-bound form of IL-9 (MB-IL-9) and investigated their influences on immune system. MB-IL-9 tumor clones showed reduced tumorigenicity but shortened survival accompanied with severe body weight loss in mice. MB-IL-9 expression on tumor cells had no effect on cell proliferation or major histocompatibility complex class I expression in vitro. MB-IL-9 tumor clones were effective in amplifying CD4+ and CD8+ T cells and increasing cytotoxic activity against CT26 cells in vivo. We also observed a prominent reduction in body weights and survival period of mice injected intraperitoneally with MB-IL-9 clones compared with control groups. Ratios of IL-17 to interferon (IFN)-γ in serum level and tumor mass were higher in mice implanted with MB-IL-9 tumor clones than those observed in mice implanted with control cells. These results indicate that the ectopic expression of the MB-IL-9 on tumor cells exerts an immune-stimulatory effect with toxicity. To exploit its benefits as a tumor vaccine, a strategy to control the toxicity of MB-IL-9 tumor clones should be developed.
RESUMO
TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants (KD) of LPS for immobilized CD14 and MD-2 were 8.7 microM, and 2.3 microM, respectively. The association rate constant (Kon) of LPS for MD-2 was 5.61 x 10(3) M-1S-1, and the dissociation rate constant (Koff) was 1.28 10 2 S 1, revealing slow association and fast dissociation with an affinity constant KD of 2.33 x 10-6 M at 25 degreesC. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex.
Assuntos
Receptores de Lipopolissacarídeos/química , Lipopolissacarídeos/química , Antígeno 96 de Linfócito/química , Receptor 4 Toll-Like/química , Animais , Cinética , Camundongos , Ligação Proteica , Proteínas Recombinantes/química , SolubilidadeRESUMO
Generating artificial protein assemblies with complex shapes requires a method for connecting protein components with stable and predictable structures. Currently available methods for creating rigid protein assemblies rely on either complicated calculations or extensive trial and error. We describe a simple and efficient method for connecting two proteins via a fused alpha helix that is formed by joining two preexisting helices into a single extended helix. Because the end-to-end ligation of helices does not guarantee the formation of a continuous helix, we superimposed 1-2 turns of pairs of connecting helices by using a molecular graphics program. Then, we chose amino acids from the two natural sequences that would stabilize the connecting helix. This "shared helix method" is highly efficient. All the designed proteins that could be produced in Escherichia coli were readily crystallized and had the expected fusion structures. To prove the usefulness of this method, we produced two novel repeat proteins by assembling several copies of natural or artificial proteins with alpha helices at both termini. Their crystal structures demonstrated the successful assembly of the repeating units with the intended curved shapes. We propose that this method could dramatically expand the available repertoire of natural repeat proteins.
Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/genética , Aminoácidos/genética , Cristalografia por Raios X , Expressão Gênica , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Conformação Proteica em alfa-HéliceRESUMO
Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a mitochondrial pro-apoptotic protein that has a single Bcl-2 homology 3 (BH3) domain and a COOH-terminal transmembrane (TM) domain. Al-though it belongs to the Bcl-2 family and can hetero-dimerize with Bcl-2, its pro-apoptotic activity is distinct from those of other members of the Bcl-2 family. For example, cell death mediated by BNIP3 is independent of caspases and shows several characteristics of necrosis. Furthermore, the TM domain, but not the BH3 domain, is required for dimerization, mitochondrial targeting and pro-apoptotic activity. BNIP3 plays an important role in hypoxia-induced death of normal and malignant cells. Its expression is markedly increased in the hypoxic regions of some solid tumors and appears to be regulated by hypoxia-inducible fac-tor (HIF), which binds to a site on the BNIP3 promoter. Silencing, followed by methylation, of the BNIP3 gene occurs in a significant proportion of can-cer cases, especially in pancreatic cancers. BNIP3 also has a role in the death of cardiac myocytes in ischemia. Further studies of BNIP3 should provide insight into hypoxic cell death and may contribute to im-proved treatment of cancers and cardiovascular diseases.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Morte Celular , Hipóxia Celular , HumanosRESUMO
Interleukin-17A is a member of the IL-17 family, and is known as CTLA8 in the mouse. It is produced by T lymphocytes and NK cells and has proinflammatory roles, inducing cytokine and chemokine production. However, its role in tumor biology remains controversial. We investigated the effects of locally produced IL-17A by transferring the gene encoding it into CT26 colon cancer cells, either in a secretory or a membrane-bound form. Expression of the membrane-bound form on CT26 cells dramatically enhanced their proliferation in vitro. The enhanced growth was shown to be due to an increased rate of cell cycle progression: after synchronizing cells by adding and withdrawing colcemid, the rate of cell cycle progression in the cells expressing the membrane-bound form of IL-17A was much faster than that of the control cells. Both secretory and membrane-bound IL-17A induced the expression of Sca-1 in the cancer cells. When tumor clones were grafted into syngeneic BALB/c mice, the tumor clones expressing the membrane-bound form IL-17A grew rapidly; those expressing the secretory form also grew faster than the wild type CT26 cells, but slower than the clones expressing the membrane-bound form. These results indicate that IL-17A promotes tumorigenicity by enhancing cell cycle progression. This finding should be considered in treating tumors and immune-related diseases.