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1.
Mol Cell ; 58(1): 35-46, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25728771

RESUMO

The ERK pathway not only upregulates growth-promoting genes, but also downregulates anti-proliferative and tumor-suppressive genes. In particular, ERK signaling contributes to repression of the E-cadherin gene during epithelial-mesenchymal transition (EMT). The CtBP transcriptional co-repressor is also involved in gene silencing of E-cadherin. However, the functional relationship between ERK signaling and CtBP is unknown. Here, we identified an ERK substrate, designated MCRIP1, which bridges ERK signaling and CtBP-mediated gene silencing. CtBP is recruited to promoter elements of target genes by interacting with the DNA-binding transcriptional repressor ZEB1. We found that MCRIP1 binds to CtBP, thereby competitively inhibiting CtBP-ZEB1 interaction. When phosphorylated by ERK, MCRIP1 dissociates from CtBP, allowing CtBP to interact with ZEB1. In this manner, the CtBP co-repressor complex is recruited to, and silences, the E-cadherin promoter by inducing chromatin modifications. Our findings reveal a molecular mechanism underlying ERK-induced epigenetic gene silencing during EMT and its dysregulation in cancer.


Assuntos
Oxirredutases do Álcool/genética , Caderinas/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Transição Epitelial-Mesenquimal/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Caderinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Inativação Gênica , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Plasmídeos/química , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Homeobox 1 de Ligação a E-box em Dedo de Zinco
2.
Glycobiology ; 32(3): 251-259, 2022 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-34735570

RESUMO

Modification of the domain architecture of galectins has been attempted to analyze their biological functions and to develop medical applications. Several types of galectin-1 repeat mutants were previously reported but, however, it was not clear whether the native structure of the wild type was retained. In this study, we determined the crystal structure of a galectin-1 tandem-repeat mutant with a short linker peptide, and compared the unfolding profiles of the wild type and mutant by chemical denaturation. The structure of the mutant was consistent with that of the dimer of the wild type, and both carbohydrate-binding sites were retained. The unfolding curve of the wild type with lactose suggested that the dimer dissociation and the tertiary structure unfolding was concomitant at micromolar protein concentrations. The midpoint denaturant concentration of the wild type was dependent on the protein concentration and lower than that of the mutant. Linking the two subunits significantly stabilized the tertiary structure. The mutant exhibited higher T-cell growth-inhibition activity and comparable hemagglutinating activity. Structural stabilization may prevent the oxidation of the internal cysteine residue.


Assuntos
Galectina 1 , Galectinas , Sítios de Ligação , Carboidratos/química , Galectina 1/metabolismo , Galectinas/metabolismo , Conformação Molecular
3.
Artigo em Inglês | MEDLINE | ID: mdl-32081418

RESUMO

The galectins are a family of ß-galactoside-specific animal lectins, and have attracted much attention as novel regulators of the immune system. Galectin-10 is well-expressed in eosinophils, and spontaneously forms Charcot-Leyden crystals (CLCs), during prolonged eosinophilic inflammatory reactions, which are frequently observed in eosinophilic diseases. Although biochemical and structural characterizations of galectin-10 have been done, its biological role and molecular mechanism are still unclear, and few X-ray structures of galectin-10 in complex with monosaccharides/oligosaccharides have been reported. Here, X-ray structures of galectin-10 in complexes with seven monosaccharides are presented with biochemical analyses to detect interactions of galectin-10 with monosaccharides/oligosaccharides. Galectin-10 forms a homo-dimer in the face-to-face orientation, and the monosaccharides bind to the carbohydrate recognition site composed of amino acid residues from two galectin-10 molecules of dimers, suggesting that galectin-10 dimer likely captures the monosaccharides in solution and in vivo. d-Glucose, d-allose, d-arabinose, and D-N-acetylgalactosamine bind to the interfaces between galectin-10 dimers in crystals, and they affect the stability of molecular packing in crystals, leading to easy-dissolving of CLCs, and/or inhibiting the formation of CLCs. These monosaccharides may serve as effectors of G10 to form CLCs in vivo.

4.
Glycobiology ; 29(1): 22-35, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30383222

RESUMO

Galectin-9 is the most potent inducer of cell death in lymphomas and other malignant cell types among the members of the galectin family. We investigated the mechanism of galectin-9-induced cell death in PC-3 prostate cancer cells in comparison with in Jurkat T cells. Galectin-9 induced apoptotic cell death in Jurkat cells, as typically revealed by DNA ladder formation. On the other hand, DNA ladder formation and other features of apoptosis were not apparent in PC-3 cells undergoing galectin-9-induced death. Exogenous galectin-9 was endocytosed and destined to the lysosomal compartment in PC-3 cells. The internalized galectin-9 was resistant to detergent solubilization but was solubilized with lactose. Agents inhibiting actin filament dynamics abolished the internalization and cytocidal effect of galectin-9 in PC-3 but not Jurkat cells. Galectin-9 induced accumulation of ubiquitinated proteins, possibly heterogeneously ubiquitinated and/or monoubiquitinated proteins, in PC-3 cells. PYR-41, an inhibitor of the ubiquitin-activating E1 enzyme, suppressed the cytocidal effect of galectin-9. Although ubiquitination was upregulated also in Jurkat cells by galectin-9, PYR-41 was ineffective against galectin-9-induced cell death. Colocalization of ubiquitinated proteins and LAMP-1 was detectable in PC-3 cells treated with galectin-9. The ubiquitinated proteins were recovered in the insoluble fraction upon cell fractionation. In contrast, ubiquitinated proteins that accumulated after treatment with proteasome inhibitors did not co-localize with LAMP-1 and were mainly recovered in soluble fraction. The results suggest that atypical ubiquitination and accumulation of ubiquitinated proteins in lysosomes play a pivotal role in galectin-9-induced non-apoptotic death in PC-3 cells.


Assuntos
Endocitose/efeitos dos fármacos , Galectinas/farmacologia , Lisossomos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/metabolismo , Ubiquitinação/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Humanos , Células Jurkat , Lisossomos/genética , Lisossomos/patologia , Masculino , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
5.
Langmuir ; 34(16): 4762-4773, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29584443

RESUMO

A layered sodium silicate, octosilicate (Na2Si8O17· nH2O), was modified with an organosulfonic-acid moiety (sulfonated propyl (SPr) group, sulfonated phenethyl (SPhE) group, or sulfonated p-trifluoromethylphenyl (STFPh) group) for use as a host material to accommodate a cationic guest, tris(2,2'-bipyridine)ruthenium(II) cation ([Ru(bpy)3]2+). The organosulfonic-acid moiety was bound to the silicate layer via a reaction of an alkylammonium-exchanged octosilicate with a silane coupling agent, and subsequent treatment (oxidation or sulfonation) of the bound organosilyl groups; the surface densities of the organosulfonic-acid moiety were varied by controlling the added amount of silane coupling agents. Adsorption of [Ru(bpy)3]2+ onto surface-modified octosilicates was conducted to find that some surface-modified octosilicates successfully adsorbed [Ru(bpy)3]2+ in the interlayer space (intercalation), while other surface-modified octosiliates did not. In addition, the UV-vis absorption and the luminescence indicate that intercalated [Ru(bpy)3]2+ diffused in the interlayer and that the distribution of the time-averaged location varied depending on the kind and amount of the organosulfonic-acid moieties. Thus, the kind and surface density of the organosulfonic-acid moiety, which correlates to the interactions between the group and the guest species, the volume of free nanospace for adsorption and motion of guests, and the swelling properties, are the key factors not only for the intercalation ability but also for the dynamics of the guest in the interlayer space.

6.
J Biol Chem ; 291(2): 968-79, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26582205

RESUMO

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo- and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgE-lectin interaction.


Assuntos
Galectinas/metabolismo , Glicopeptídeos/isolamento & purificação , Imunoglobulina E/metabolismo , Oligossacarídeos/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Degranulação Celular , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Glicopeptídeos/metabolismo , Humanos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica , Ratos , Serina Endopeptidases/metabolismo , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tripsina/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
7.
Biochem Biophys Res Commun ; 490(4): 1287-1293, 2017 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-28687490

RESUMO

Galectin-9 (G9) is a tandem-repeat type ß-galactoside-specific animal lectin having N-terminal and C-terminal carbohydrate recognition domains (N-CRD and C-CRD, respectively) joined by a linker peptide that is involved in the immune system. G9 is divalent in glycan binding, and structural information about the spatial arrangement of the two CRDs is very important for elucidating its biological functions. As G9 is protease sensitive due to the long linker, the protease-resistant mutant form of G9 (G9Null) was developed by modification of the linker peptide, while retaining its biological functions. The X-ray structure of a mutant form of G9Null with the replacement of Arg221 by Ser (G9Null_R221S) having two CRDs was determined. The structure of G9Null_R221S was compact to associate the two CRDs in the back-to-back orientation with a large interface area, including hydrogen bonds and hydrophobic interactions. A metal ion was newly found in the galectin structure, possibly contributing to the stable structure of protein. The presented X-ray structure was thought to be one of the stable structures of G9, which likely occurs in solution. This was supported by structural comparisons with other tandem-repeated galectins and the analyses of protein thermostability by CD spectra measurements.


Assuntos
Galactosídeos/química , Galectinas/química , Metais/química , Mutação , Adenoviridae/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Galectinas/genética , Galectinas/metabolismo , Expressão Gênica , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Toxascaris/química
8.
Biosci Biotechnol Biochem ; 81(11): 2098-2104, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28958189

RESUMO

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.


Assuntos
Elastina/química , Elastina/metabolismo , Galectinas/metabolismo , Proteoglicanos Pequenos Ricos em Leucina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Espaço Extracelular/metabolismo , Solubilidade , Suínos , Água/química
9.
Chem Pharm Bull (Tokyo) ; 64(7): 924-9, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27373647

RESUMO

Three new acylated triterpene saponins, acernikoenosides A-C (1-3), were isolated from the stem bark of Acer nikoense, together with a known sterol glucoside. Their structures were elucidated on the basis of extensive spectroscopic analyses. This study provided the first example of triterpene saponins isolated from this plant. The anti-genotoxic activity of 1, 3 and 4 against ultraviolet irradiation was evaluated by comet assay.


Assuntos
Aceraceae/química , Antimutagênicos/farmacologia , Casca de Planta/química , Caules de Planta/química , Saponinas/farmacologia , Triterpenos/farmacologia , Acilação , Antimutagênicos/química , Antimutagênicos/isolamento & purificação , Células Cultivadas , Humanos , Estrutura Molecular , Saponinas/química , Saponinas/isolamento & purificação , Triterpenos/química , Triterpenos/isolamento & purificação , Raios Ultravioleta
10.
Biochim Biophys Acta ; 1840(6): 1892-901, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24462947

RESUMO

BACKGROUND: There is a continuous demand for new immunosuppressive agents for organ transplantation. Galectin-9, a member of the galactoside-binding animal lectin family, has been shown to suppress pathogenic T-cell responses in autoimmune disease models and experimental allograft transplantation. In this study, an attempt has been made to develop new collagen matrices, which can cause local, contact-dependent immune suppression, using galectin-9 and collagen-binding galectin-9 fusion proteins as active ingredients. METHODS: Galectin-9 and galectin-9 fusion proteins having collagen-binding domains (CBDs) derived from bacterial collagenases and a collagen-binding peptide (CBP) were tested for their ability to bind to collagen matrices, and to induce Jurkat cell death in solution and in the collagen-bound state. RESULTS: Galectin-9-CBD fusion proteins exhibited collagen-binding activity comparable to or lower than that of the respective CBDs, while their cytocidal activity toward Jurkat cells in solution was 80~10% that of galectin-9. Galectin-9 itself exhibited oligosaccharide-dependent collagen-binding activity. The growth of Jurkat cells cultured on collagen membranes treated with galectin-9 was inhibited by~90%. The effect was dependent on direct cell-to-membrane contact. Galectin-9-CBD/CBP fusion proteins bound to collagen membranes via CBD/CBP moieties showed a low or negligible effect on Jurkat cell growth. CONCLUSIONS: Among the proteins tested, galectin-9 exhibited the highest cytocidal effect on Jurkat cells in the collagen-bound state. The effect was not due to galectin-9 released into the culture medium but was dependent on direct cell-to-membrane contact. GENERAL SIGNIFICANCE: The study demonstrates the possible use of galectin-9-modified collagen matrices for local, contact-dependent immune suppression in transplantation.


Assuntos
Colágeno/metabolismo , Galectinas/farmacologia , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Galectinas/química , Humanos , Imunossupressores/farmacologia , Células Jurkat , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/farmacologia
11.
Glycobiology ; 25(7): 792-803, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25804418

RESUMO

Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer-tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer-dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galß1-3GalNAc and not Galß1-4GlcNAc, while xgalectin-Ib preferred Galß1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.


Assuntos
Galectinas/metabolismo , Pele/metabolismo , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Configuração de Carboidratos , Cristalografia por Raios X , Galectinas/química , Modelos Moleculares
12.
Plant Cell Physiol ; 55(1): e1, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24092884

RESUMO

The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.


Assuntos
Bases de Dados como Assunto , Organelas/ultraestrutura , Células Vegetais/ultraestrutura , Pesquisa , Interface Usuário-Computador
13.
J Virol ; 87(17): 9633-42, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23804634

RESUMO

Most viruses possess strategies to circumvent host immune responses. The measles virus (MV) nonstructural C protein suppresses the interferon response, thereby allowing efficient viral growth, but its detailed mechanism has been unknown. We identified Shc Src homology 2 domain-binding protein 1 (SHCBP1) as one of the host proteins interacting with the C protein. Knockdown of SHCBP1 using a short-hairpin RNA greatly reduced MV growth. SHCBP1 was found to be required for viral RNA synthesis in the minigenome assay and to bind to the MV phosphoprotein, a subunit of the viral RNA polymerase. A stretch of 12 amino acid residues in the C protein were sufficient for SHCBP1 binding, and the peptide containing these 12 residues could suppress MV RNA synthesis, like the full-length C protein. The central region of SHCBP1 was found to bind to the C protein, as well as the phosphoprotein, but the two viral proteins did not compete for SHCBP1 binding. Our results indicate that the C protein modulates MV RNA polymerase activity by binding to the host protein SHCBP1. SHCBP1 may be exploited as a target of antiviral compounds.


Assuntos
RNA Polimerases Dirigidas por DNA/fisiologia , Vírus do Sarampo/fisiologia , Vírus do Sarampo/patogenicidade , Proteínas Adaptadoras da Sinalização Shc/fisiologia , Proteínas não Estruturais Virais/fisiologia , Linhagem Celular , Técnicas de Silenciamento de Genes , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Vírus do Sarampo/genética , Modelos Biológicos , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Adaptadoras da Sinalização Shc/antagonistas & inibidores , Proteínas Adaptadoras da Sinalização Shc/genética , Técnicas do Sistema de Duplo-Híbrido , Proteínas não Estruturais Virais/genética
14.
J Biochem ; 175(6): 629-641, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38299728

RESUMO

Proper regulation of cellular response to environmental stress is crucial for maintaining biological homeostasis and is achieved by the balance between cell death processes, such as the formation of the pyroptosis-inducing NLRP3 inflammasome, and pro-survival processes, such as stress granule (SG) assembly. However, the functional interplay between these two stress-responsive organelles remains elusive. Here, we identified DHX33, a viral RNA sensor for the NLRP3 inflammasome, as a SG component, and the SG-nucleating protein G3BP as an NLRP3 inflammasome component. We also found that a decrease in intracellular potassium (K+) concentration, a key 'common' step in NLRP3 inflammasome activation, markedly inhibited SG assembly. Therefore, when macrophages are exposed to stress stimuli with the potential to induce both SGs and the NLRP3 inflammasome, such as cytoplasmic poly(I:C) stimulation, they preferentially form the NLRP3 inflammasome but avoid SG assembly by sequestering G3BP into the inflammasome and by inducing a reduction in intracellular K+ levels. Thus, under such conditions, DHX33 is primarily utilized as a viral RNA sensor for the inflammasome. Our data reveal the functional crosstalk between NLRP3 inflammasome-mediated pyroptosis and SG-mediated cell survival pathways and delineate a molecular mechanism that regulates cell-fate decisions and anti-viral innate immunity under stress.


Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Grânulos de Estresse , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Inflamassomos/metabolismo , Humanos , Grânulos de Estresse/metabolismo , Camundongos , Animais , Potássio/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Piroptose , RNA Helicases/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Poli I-C/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , DNA Helicases
15.
Glycobiology ; 23(8): 920-5, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23507964

RESUMO

We previously developed a stable form of galectin-9, an immunomodulatory animal lectin with a truncated linker peptide (G9Null), to overcome the protease sensitivity of wild-type galectin-9. G9Null is highly resistant to proteolysis, while the modification marginally improved the low solubility of the wild-type protein. To increase its solubility, we further modified the remaining linker region of G9Null. A 10-amino acid deletion with a single amino acid substitution resulted in an ∼400% increase in solubility and yield without an adverse effect on its biological activity. This mutant protein might be useful for large-scale recombinant production needed for evaluation of the therapeutic potential of galectin-9.


Assuntos
Substituição de Aminoácidos , Galectinas/genética , Animais , Galectinas/biossíntese , Galectinas/química , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solubilidade
16.
Anticancer Res ; 43(3): 1121-1130, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36854515

RESUMO

BACKGROUND/AIM: Oral 5-fluorouracil (5-FU)-based prodrugs, used in cancer chemotherapeutic regimens, exhibit large inter- and intra-patient variability in plasma 5-FU concentrations, contributing to treatment failure. Although dosage determination criteria according to plasma drug concentrations are required, the relationship between pharmacokinetics and drug response after multiple oral 5-FU derivative administrations remain unknown. MATERIALS AND METHODS: We evaluated the pharmacokinetics and pharmacodynamics/toxicodynamics of uracil-tegafur (UFT) after multiple administrations in colorectal cancer (CRC) model rats, and applied a pharmacometric approach to describe the time-course alterations of plasma 5-FU concentrations and tumor shrinkage. CRC was induced in rats using 1,2-dimethylhydrazine and dextran sulfate sodium. UFT (30 mg/kg as tegafur) was administered to CRC rats for 14 days. RESULTS: Plasma 5-FU exposure levels increased with the dosing time, and large variations were observed in tumor 5-FU concentrations (32.0-125.8% with coefficient of variation). Although severe hematological toxicities were not observed, a weak correlation was observed between blood platelet count and the plasma 5-FU concentration (r=0.439, p=0.176). A simple pharmacokinetic-pharmacodynamic model was developed comprising of a small number of parameters and successfully describing plasma 5-FU levels and tumor shrinkage after multiple UFT administrations. CONCLUSION: A pharmacometric model approach can help establish the dose-determination criteria based on plasma 5-FU concentration of UFT-based regimens, and contribute to improvement of clinical outcomes.


Assuntos
Neoplasias Colorretais , Tegafur , Animais , Ratos , Uracila/farmacologia , Fluoruracila/farmacologia , Administração Oral , Neoplasias Colorretais/tratamento farmacológico
17.
Curr Biol ; 33(10): 1967-1981.e8, 2023 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-37119817

RESUMO

Cytoplasmic stress granules (SGs) are phase-separated membrane-less organelles that form in response to various stress stimuli. SGs are mainly composed of non-canonical stalled 48S preinitiation complexes. In addition, many other proteins also accumulate into SGs, but the list is still incomplete. SG assembly suppresses apoptosis and promotes cell survival under stress. Furthermore, hyperformation of SGs is frequently observed in various human cancers and accelerates tumor development and progression by reducing stress-induced damage of cancer cells. Therefore, they are of clinical importance. However, the precise mechanism underlying SG-mediated inhibition of apoptosis remains ill-defined. Here, using a proximity-labeling proteomic approach, we comprehensively analyzed SG-resident proteins and identified the executioner caspases, caspase-3 and -7, as SG components. We demonstrate that accumulation of caspase-3/7 into SGs is mediated by evolutionarily conserved amino acid residues within their large catalytic domains and inhibits caspase activities and consequent apoptosis induced by various stresses. Expression of an SG-localization-deficient caspase-3 mutant in cells largely counteracted the anti-apoptotic effect of SGs, whereas enforced relocalization of the caspase-3 mutant to SGs restored it. Thus, SG-mediated sequestration of executioner caspases is a mechanism underlying the broad cytoprotective function of SGs. Furthermore, using a mouse xenograft tumor model, we show that this mechanism prevents cancer cells from apoptosis in tumor tissues, thereby promoting cancer progression. Our results reveal the functional crosstalk between SG-mediated cell survival and caspase-mediated cell death signaling pathways and delineate a molecular mechanism that dictates cell-fate decisions under stress and promotes tumorigenesis.


Assuntos
Caspases , Proteômica , Humanos , Caspase 3/metabolismo , Caspase 3/farmacologia , Caspases/metabolismo , Caspases/farmacologia , Grânulos de Estresse , Grânulos Citoplasmáticos/metabolismo , Apoptose , Estresse Fisiológico
18.
Biochim Biophys Acta ; 1810(7): 643-51, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21514365

RESUMO

BACKGROUND: Galectins form a large family of animal lectins, individual members having variously divergent carbohydrate-recognition domains (CRDs) responsible for extensive physiological phenomena. Sugar-binding affinities of galectins were previously investigated by us using frontal affinity chromatography (FAC) with a relatively small set (i.e., 41) of oligosaccharides. However, total understanding of a consensus rule for galectin-recognition saccharides is still hampered by the lack of fundamental knowledge about their sugar-binding specificity toward a much larger panel of oligosaccharides in terms of dissociation constant (K(d)). METHODS: In the present study, we extended a FAC analysis from a more systematic viewpoint by using 142 fluorescent-labeled oligosaccharides, initially with focus on functional human galectins-1-9. Binding characteristics were further validated with 11 non-human galectins and 13 non-galectin Gal/GalNAc-binding lectins belonging to different families. RESULTS: An empirical [Galß-equatorial] rule for galectin-recognition disaccharides was first derived by our present research and previous works by others. However, this rule was not valid for a recently reported nematode disaccharide, "Galß1-4-L-Fuc" [Butschi et al. PLoS Pathog, 2010; 6(1):e1000717], because this glycosidic linkage was directed to 'axial' 4-OH of L-Fuc. After careful reconsideration of the structural data, we reached an ultimate rule of galectin-recognition disaccharides, which all of the galectins so far identified fulfilled, i.e., under the re-defined configuration "Galß-(syn)-gauche". The rule also worked perfectly for differentiation of galectins from other types of lectins. GENERAL SIGNIFICANCE: The present attempt should provide a basis to solve the riddle of the glyco-code as well as to develop therapeutic inhibitors mimicking galectin ligands.


Assuntos
Cromatografia de Afinidade/métodos , Dissacarídeos/química , Galectinas/química , Conformação Molecular , Sítios de Ligação , Ligação Competitiva , Cromatografia de Afinidade/instrumentação , Dissacarídeos/metabolismo , Galectinas/genética , Galectinas/metabolismo , Humanos , Estrutura Molecular , Oligossacarídeos/química , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
19.
Langmuir ; 28(19): 7505-11, 2012 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-22530924

RESUMO

The surface modification of a layered alkali silicate, octosilicate (Na(2)Si(8)O(17)·nH2O), with a sulfonic acid group was conducted. The sulfonic acid group was attached to the silicate layer by the reaction of octosilicate with phenethyl(dichloro)methylsilane and the subsequent sulfonation of the attached phenethyl groups with chlorosulfonic acid. The modified octosilicate is a solid acid as indicated by the intercalation of dodecylamine. A systematic expansion of the interlayer space was observed by the ion exchange with a series of alkyltrimethylammonium ions to show the variation of the layer charge density.

20.
J Biol Chem ; 285(27): 20882-90, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20435897

RESUMO

Measles virus (MV), an enveloped negative-strand RNA virus, remains a major cause of morbidity and mortality in developing countries. MV predominantly infects immune cells by using signaling lymphocyte activation molecule (SLAM; also called CD150) as a receptor, but it also infects polarized epithelial cells, forming tight junctions in a SLAM-independent manner. Although the ability of MV to infect polarized epithelial cells is thought to be important for its transmission, the epithelial cell receptor for MV has not been identified. A transcriptional repressor, Snail, induces epithelial-mesenchymal transition (EMT), in which epithelial cells lose epithelial cell phenotypes, such as adherens and tight junctions. In this study, EMT was induced by expressing Snail in a lung adenocarcinoma cell line, II-18, which is highly susceptible to wild-type MV. Snail-expressing II-18 cells lost adherens and tight junctions. Microarray analysis confirmed the induction of EMT in II-18 cells and suggested a novel function of Snail in protein degradation and distribution. Importantly, wild-type MV no longer entered EMT-induced II-18 cells, suggesting that the epithelial cell receptor is down-regulated by the induction of EMT. Other polarized cell lines, NCI-H358 and HT-29, also lost susceptibility to wild-type MV when EMT was induced. However, the complete formation of tight junctions rather reduced MV entry into HT-29 cells. Taken together, these data suggest that the unidentified epithelial cell receptor for MV is involved in the formation of epithelial intercellular junctions.


Assuntos
Células Epiteliais/citologia , Vírus do Sarampo/patogenicidade , Sarampo/prevenção & controle , Mesoderma/citologia , Animais , Linhagem Celular , Suscetibilidade a Doenças , Células Epiteliais/fisiologia , Células Epiteliais/virologia , Citometria de Fluxo , Predisposição Genética para Doença , Vetores Genéticos , Genoma Viral , Humanos , Ativação Linfocitária , Macaca mulatta/virologia , Sarampo/imunologia , Sarampo/transmissão , Sarampo/veterinária , Vírus do Sarampo/genética , Proteína Cofatora de Membrana/fisiologia , Mesoderma/fisiologia , Mesoderma/virologia , Análise de Sequência com Séries de Oligonucleotídeos , Plasmídeos , Receptores Virais/fisiologia , Junções Íntimas/fisiologia , Eliminação de Partículas Virais
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