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1.
Carbohydr Polym ; 176: 381-391, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28927622

RESUMO

The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation.

2.
Plant Sci ; 263: 219-225, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28818378

RESUMO

Nitrogen (N) is a major macronutrient that is essential for plant growth. It is important for us to understand the key genes that are involved in the regulation of N utilization. In this study, we focused on a GARP-type transcription factor known as NSR1/MYR2, which has been reported to be induced under N-deficient conditions. Our results demonstrated that NSR1/MYR2 has a transcriptional repression activity and is specifically expressed in vascular tissues, especially in phloem throughout the plant under daily light-dark cycle regulation. The overexpression of NSR1/MYR2 delays nutrient starvation- and dark-triggered senescence in the mature leaves of excised whole aerial parts of Arabidopsis plants. Furthermore, the expression of asparagine synthetase 1 (ASN1), which plays an important role in N remobilization and reallocation, i.e. N reutilization, in Arabidopsis, is negatively regulated by NSR1/MYR2, since the expressions of NSR1/MYR2 and ASN1 were reciprocally regulated during the light-dark cycle and ASN1 expression was down-regulated in overexpressors of NSR1/MYR2 and up-regulated in T-DNA insertion mutants of NSR1/MYR2. Therefore, the present results suggest that NSR1/MYR2 plays a role in N reutilization as a negative regulator through controlling ASN1 expression.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Aspartato-Amônia Ligase/metabolismo , Nitrogênio/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/economia , Proteínas de Arabidopsis/genética , Aspartato-Amônia Ligase/genética , Transporte Biológico , Floema/metabolismo , Fotoperíodo , Folhas de Planta/metabolismo , Fatores de Transcrição/genética
3.
Plant Physiol ; 172(3): 1612-1624, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27600813

RESUMO

Xylem vessels, the water-conducting cells in vascular plants, undergo characteristic secondary wall deposition and programmed cell death. These processes are regulated by the VASCULAR-RELATED NAC-DOMAIN (VND) transcription factors. Here, to identify changes in metabolism that occur during protoxylem vessel element differentiation, we subjected tobacco (Nicotiana tabacum) BY-2 suspension culture cells carrying an inducible VND7 system to liquid chromatography-mass spectrometry-based wide-target metabolome analysis and transcriptome analysis. Time-course data for 128 metabolites showed dynamic changes in metabolites related to amino acid biosynthesis. The concentration of glyceraldehyde 3-phosphate, an important intermediate of the glycolysis pathway, immediately decreased in the initial stages of cell differentiation. As cell differentiation progressed, specific amino acids accumulated, including the shikimate-related amino acids and the translocatable nitrogen-rich amino acid arginine. Transcriptome data indicated that cell differentiation involved the active up-regulation of genes encoding the enzymes catalyzing fructose 6-phosphate biosynthesis from glyceraldehyde 3-phosphate, phosphoenolpyruvate biosynthesis from oxaloacetate, and phenylalanine biosynthesis, which includes shikimate pathway enzymes. Concomitantly, active changes in the amount of fructose 6-phosphate and phosphoenolpyruvate were detected during cell differentiation. Taken together, our results show that protoxylem vessel element differentiation is associated with changes in primary metabolism, which could facilitate the production of polysaccharides and lignin monomers and, thus, promote the formation of the secondary cell wall. Also, these metabolic shifts correlate with the active transcriptional regulation of specific enzyme genes. Therefore, our observations indicate that primary metabolism is actively regulated during protoxylem vessel element differentiation to alter the cell's metabolic activity for the biosynthesis of secondary wall polymers.


Assuntos
Vias Biossintéticas , Parede Celular/metabolismo , Polímeros/metabolismo , Tabaco/metabolismo , Xilema/metabolismo , Aminoácidos/metabolismo , Diferenciação Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glicólise , Lignina/metabolismo , Metaboloma , Metabolômica , Análise de Componente Principal , Ácido Chiquímico/metabolismo , Tabaco/citologia
4.
Front Plant Sci ; 6: 288, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25999964

RESUMO

Plant cells biosynthesize primary cell walls (PCW) in all cells and produce secondary cell walls (SCWs) in specific cell types that conduct water and/or provide mechanical support, such as xylem vessels and fibers. The characteristic mechanical stiffness, chemical recalcitrance, and hydrophobic nature of SCWs result from the organization of SCW-specific biopolymers, i.e., highly ordered cellulose, hemicellulose, and lignin. Synthesis of these SCW-specific biopolymers requires SCW-specific enzymes that are regulated by SCW-specific transcription factors. In this review, we summarize our current knowledge of the transcriptional regulation of SCW formation in plant cells. Advances in research on SCW biosynthesis during the past decade have expanded our understanding of the transcriptional regulation of SCW formation, particularly the functions of the NAC and MYB transcription factors. Focusing on the NAC-MYB-based transcriptional network, we discuss the regulatory systems that evolved in land plants to modify the cell wall to serve as a key component of structures that conduct water and provide mechanical support.

5.
J Neurosci ; 35(11): 4776-87, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25788693

RESUMO

During brain development, Reelin exerts a variety of effects in a context-dependent manner, whereas its underlying molecular mechanisms remain poorly understood. We previously showed that the C-terminal region (CTR) of Reelin is required for efficient induction of phosphorylation of Dab1, an essential adaptor protein for canonical Reelin signaling. However, the physiological significance of the Reelin CTR in vivo remains unexplored. To dissect out Reelin functions, we made a knock-in (KI) mouse in which the Reelin CTR is deleted. The amount of Dab1, an indication of canonical Reelin signaling strength, is increased in the KI mouse, indicating that the CTR is necessary for efficient induction of Dab1 phosphorylation in vivo. Formation of layer structures during embryonic development is normal in the KI mouse. Intriguingly, the marginal zone (MZ) of the cerebral cortex becomes narrower at postnatal stages because upper-layer neurons invade the MZ and their apical dendrites are misoriented and poorly branched. Furthermore, Reelin undergoes proteolytic cleavage by proprotein convertases at a site located 6 residues from the C terminus, and it was suggested that this cleavage abrogates the Reelin binding to the neuronal cell membrane. Results from ectopic expression of mutant Reelin proteins in utero suggest that the dendrite development and maintenance of the MZ require Reelin protein with an intact CTR. These results provide a novel model regarding Reelin functions involving its CTR, which is not required for neuronal migration during embryonic stages but is required for the development and maintenance of the MZ in the postnatal cerebral cortex.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Células COS , Cercopithecus aethiops , Técnicas de Introdução de Genes/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteólise
6.
Plant Cell Physiol ; 56(2): 242-54, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25265867

RESUMO

The secondary cell walls of xylem cells, including vessel elements, provide mechanical strength and contribute to the conduction of water and minerals. VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a NAC-domain transcription factor that regulates the expression of genes required for xylem vessel element formation. Transient expression assays using 68 transcription factors that are expressed during xylem vessel differentiation showed that 14 transcription factors, including VND1-VND7, are putative positive regulators of VND7 expression. Electrophoretic mobility shift assays revealed that all seven VND proteins bound to the VND7 promoter region at its SMBE/TERE motif, indicating that VND7 is a direct target of all of the VND transcription factors. Overexpression of VND1-VND5, GATA12 and ANAC075, newly identified transcription factors that function upstream of VND7, resulted in ectopic xylem vessel element formation. These data suggest that VND7 transcription is a regulatory target of multiple classes of transcription factors.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Diferenciação Celular , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/metabolismo , Xilema/citologia , Xilema/genética , Arabidopsis/citologia , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Ensaios Enzimáticos , Redes Reguladoras de Genes , Genes de Plantas , Luciferases/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Motivos de Nucleotídeos/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fatores de Transcrição/genética , Regulação para Cima
7.
Science ; 343(6178): 1505-8, 2014 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-24652936

RESUMO

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.


Assuntos
Adaptação Fisiológica/genética , Arabidopsis/fisiologia , Bryopsida/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Transativadores/fisiologia , Água/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Bryopsida/genética , Loci Gênicos , Genoma de Planta , Dados de Sequência Molecular , Proteínas de Plantas/genética , Caules de Planta/crescimento & desenvolvimento , Transativadores/genética , Transcrição Genética
8.
Plant Cell Environ ; 35(11): 2031-44, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22574770

RESUMO

Rice internodes are vital for supporting high-yield panicles, which are controlled by various factors such as cell division, cell elongation and cell wall biosynthesis. Therefore, formation and regulation of the internode cell-producing intercalary meristem (IM) are important for determining the shape of internodes. To understand the regulation of internode development, we analysed a rice dwarf mutant, dwarf 50 (d50). Previously, we reported that parenchyma cells in the elongated internodes of d50 ectopically deposit cell wall phenolics. In this study, we revealed that D50 encodes putative inositol polyphosphate 5-phosphatase (5PTase), which may be involved in phosphoinositide signalling required for many essential cellular functions, such as cytoskeleton organization, endocytosis and vesicular trafficking in eukaryotes. Analysis of the rice genome revealed 20 putative 5PTases including D50. The d50 mutation induced abnormally oriented cell division, irregular deposition of cell wall pectins and thick actin bundles in the parenchyma cells of the IM, resulting in abnormally organized cell files of the internode parenchyma and dwarf phenotype. Our results suggest that the putative 5PTase, encoded by D50, is essential for IM formation, including the direction of cell division, deposition of cell wall pectins and control of actin organization.


Assuntos
Meristema/genética , Oryza/enzimologia , Monoéster Fosfórico Hidrolases/fisiologia , Proteínas de Plantas/fisiologia , Actinas/metabolismo , Actinas/ultraestrutura , Sequência de Aminoácidos , Divisão Celular/genética , Forma Celular , Parede Celular/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Inositol Polifosfato 5-Fosfatases , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Pectinas/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Alinhamento de Sequência
9.
Plant Physiol ; 153(3): 906-14, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20488898

RESUMO

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the protein's activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula x tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Transdiferenciação Celular/genética , Técnicas Genéticas , Xilema/citologia , Xilema/genética , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Transdiferenciação Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Dexametasona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas , Populus/citologia , Populus/efeitos dos fármacos , Populus/genética , Biossíntese de Proteínas/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tabaco/citologia , Tabaco/efeitos dos fármacos , Tabaco/metabolismo , Regulação para Cima/efeitos dos fármacos , Xilema/efeitos dos fármacos
10.
Planta ; 232(1): 257-70, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20424856

RESUMO

The plant secondary cell wall is a highly ordered structure composed of various polysaccharides, phenolic components and proteins. Its coordinated regulation of a number of complex metabolic pathways and assembly has not been resolved. To understand the molecular mechanisms that regulate secondary cell wall synthesis, we isolated a novel rice mutant, cell wall architecture1 (cwa1), that exhibits an irregular thickening pattern in the secondary cell wall of sclerenchyma, as well as culm brittleness and reduced cellulose content in mature internodes. Light and transmission electron microscopy revealed that the cwa1 mutant plant has regions of local aggregation in the secondary cell walls of the cortical fibers in its internodes, showing uneven thickness. Ultraviolet microscopic observation indicated that localization of cell wall phenolic components was perturbed and that these components abundantly deposited at the aggregated cell wall regions in sclerenchyma. Therefore, regulation of deposition and assembly of secondary cell wall materials, i.e. phenolic components, appear to be disturbed by mutation of the cwa1 gene. Genetic analysis showed that cwa1 is allelic to brittle culm1 (bc1), which encodes the glycosylphosphatidylinositol-anchored COBRA-like protein specifically in plants. BC1 is known as a regulator that controls the culm mechanical strength and cellulose content in the secondary cell walls of sclerenchyma, but the precise function of BC1 has not been resolved. Our results suggest that CWA1/BC1 has an essential role in assembling cell wall constituents at their appropriate sites, thereby enabling synthesis of solid and flexible internodes in rice.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Parede Celular/metabolismo , Genes de Plantas , Mutação , Oryza/genética , Sequência de Bases , Clonagem Molecular , Primers do DNA , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão
11.
J Neurosci Res ; 87(14): 3043-53, 2009 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-19530167

RESUMO

Reelin is a secreted glycoprotein that plays pivotal roles in the development and function of the brain, but how it activates downstream intracellular signaling is not fully understood. We have recently reported that the highly conserved C-terminal region (CTR) of Reelin is required for its full signaling activity, although the underlying mechanism remains unknown. During biochemical study of Reelin, we serendipitously found that one commercially available anti-Reelin antibody G20 can bind to CTR-lacking mutant Reelin proteins, but not wild-type Reelin, on Western blotting. The G20 epitope resides in the last 19 residues of Reelin-repeat 8 (RR8), and neither posttranslational modification nor proteolysis can explain this effect. Furthermore, when an unrelated sequence, such as FLAG-tag, is inserted between RR8 and CTR, the reactivity of the corresponding antibody greatly decreases. These results suggest that RR8 and CTR form a tight structure that makes the surrounding sequence inaccessible to an antibody. Taking advantage of this phenomenon, we show the existence of CTR-lacking Reelin isoform in vivo for the first time and estimate its contribution to the total amount of secreted Reelin. Importantly, the extent to which Reelin mutants react with G20 is inversely correlated with their signaling activity, indicating that the CTR-induced structural change of RR8 is a prerequisite for downstream signaling activation, presumably via binding to a certain neuronal membrane molecule(s).


Assuntos
Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/metabolismo , Sítios de Ligação/genética , Western Blotting , Células COS , Linhagem Celular , Cercopithecus aethiops , Eletroforese em Gel de Poliacrilamida , Humanos , Imuno-Histoquímica , Imunoprecipitação , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
12.
Biochem Biophys Res Commun ; 380(1): 93-7, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19166810

RESUMO

Reelin is a secreted glycoprotein essential for normal brain development and function. In the extracellular milieu, Reelin is subject to specific cleavage at two (N-t and C-t) sites. The N-t cleavage of Reelin is implicated in psychiatric and Alzheimer's diseases, but the molecular mechanism and physiological significance of this cleavage are not completely understood. Particularly, whether the N-t cleavage affects the signaling activity of Reelin remains controversial. Here, we show that the protease in charge of the N-t cleavage of Reelin requires the activity of certain proprotein convertase family for maturation and has strong affinity for heparin. By taking advantage of these observations, we for the first time succeeded in obtaining "Uncleaved" and "Completely Cleaved" Reelin proteins. The N-t cleavage splits Reelin into two distinct fragments and virtually abolishes its signaling activity. These findings provide an important biochemical basis for the function of Reelin proteolysis in brain development and function.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Peptídeo Hidrolases/metabolismo , Serina Endopeptidases/metabolismo , Clorometilcetonas de Aminoácidos , Animais , Furina/antagonistas & inibidores , Furina/metabolismo , Heparina/química , Humanos , Camundongos , Camundongos Endogâmicos ICR , Oligopeptídeos/farmacologia , Peptídeo Hidrolases/química
13.
Proc Natl Acad Sci U S A ; 104(24): 9988-93, 2007 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-17548821

RESUMO

Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of "reelin repeats" and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We found that both receptor-binding and subsequent Dab1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats (R5-6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0-A crystal structure from the R5-6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5-6 revealed that two Lys residues (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.


Assuntos
Moléculas de Adesão Celular Neuronais/química , Proteínas da Matriz Extracelular/química , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Receptores de Lipoproteínas/metabolismo , Serina Endopeptidases/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Células Cultivadas , Córtex Cerebral/citologia , Células Clonais , Sequência Conservada , Cricetinae , Cricetulus , Cristalografia por Raios X , Análise Mutacional de DNA , Dissulfetos/química , Eletroporação , Ácido Glutâmico/química , Histidina/química , Humanos , Proteínas Relacionadas a Receptor de LDL , Ligantes , Camundongos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Zinco/metabolismo
14.
J Biol Chem ; 282(28): 20544-52, 2007 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-17504759

RESUMO

Reelin is a very large secreted glycoprotein essential for correct development of the mammalian brain. It is also implicated in higher functions and diseases of human brain. However, whether or not secretion of Reelin is regulated and how Reelin transmits signals remain largely unknown. Reelin protein is composed of an N-terminal F-spondin-like domain, Reelin repeats, and a short and highly basic C-terminal region (CTR). The primary sequence of CTR is almost completely conserved among vertebrates except fishes, indicating its importance. A prevailing idea regarding the function of CTR is that it is required for the secretion of Reelin, although this remains unproven. Here we aimed to clarify the function of Reelin CTR. Neither deleting most of CTR nor replacing CTR with unrelated amino acids affected secretion efficiency, indicating that CTR is not absolutely required for the secretion of Reelin. We also found that Reelin mutants without CTR were less potent in activating the downstream signaling in cortical neurons. Although these mutants were able to bind to the Reelin receptor ectodomain as efficiently as wild-type Reelin, quite interestingly, their ability to bind to the isolated cell membrane bearing Reelin receptors or receptor-expressing cells (including cortical neurons) was much weaker than that of wild-type Reelin. Therefore, it is concluded that the CTR of Reelin is not essential for its secretion but is required for efficient activation of downstream signaling events, presumably via binding to an unidentified "co-receptor" molecule(s) on the cell membrane.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/metabolismo , Córtex Cerebelar/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Substituição de Aminoácidos , Animais , Encefalopatias/genética , Encefalopatias/metabolismo , Encefalopatias/patologia , Células COS , Moléculas de Adesão Celular Neuronais/genética , Membrana Celular/patologia , Cercopithecus aethiops , Córtex Cerebelar/patologia , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Serina Endopeptidases/genética , Transdução de Sinais/genética
15.
Plant Cell Rep ; 25(7): 676-88, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16496151

RESUMO

Polysaccharide-linked hydroxycinnamoyl esters (PHEs) over-accumulate in the internodes of a rice (Oryza sativa L.) mutant, Fukei 71 (F71). This accumulation is accompanied by over-expression of phenylalanine ammonialyase (PAL). In this study, we show that only one member of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) family expresses in close correlation with PAL. Furthermore, substrate availability to DAHPS is promoted by down-regulating the expression of plastidic pyruvate kinase (PKp), a competitor of DAHPS. Since the over-production of PHEs is caused by D50 gene disruption, these results suggest that specific enzymes in the phenylpropanoid and shikimate pathways are coordinately up-regulated. In addition, the results indicate that carbon-flow into the shikimate pathway is modified for the synthesis of PHEs, and is probably controlled by D50.


Assuntos
3-Desoxi-7-Fosfo-Heptulonato Sintase/metabolismo , Parede Celular/metabolismo , Ácidos Cumáricos/metabolismo , Ésteres/metabolismo , Oryza/citologia , Oryza/enzimologia , Polissacarídeos/metabolismo , 3-Desoxi-7-Fosfo-Heptulonato Sintase/química , 3-Desoxi-7-Fosfo-Heptulonato Sintase/genética , Sequência de Aminoácidos , Parede Celular/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Oryza/genética , Filogenia
16.
Plant Cell Rep ; 24(8): 487-93, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15838683

RESUMO

Both polysaccharide-linked hydroxycinnamoyl esters (PHEs) and lignin are biosynthesized via the phenylpropanoid pathway. In the abnormal internode parenchyma of the rice (Oryza sativa L.) mutant Fukei 71, which has a defective recessive gene (d50), the biosynthesis of lignin and PHEs differs. . The polysaccharide-linked ferulate and p-coumarate have been shown to accumulate to high levels in the irregularly shaped and collapsed internode parenchyma cells of Fukei 71 without an accompanying overaccumulation of lignin as a result of the defective d50 gene. In the present study we demonstrated that in this abnormal parenchyma tissue of Fukei 71 the expression of phenylalanine ammonia lyase (PAL) and glutamine synthetase (GS) were ectopically induced with the ectopic accumulation of PHEs, suggesting that the d50 gene may play a role as a controlling element in the biosynthesis of PHEs during cell-wall formation in the grasses.


Assuntos
Oryza/enzimologia , Fenilalanina Amônia-Liase/metabolismo , Polissacarídeos/metabolismo , Sequência de Bases , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Primers do DNA , Ésteres , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Imuno-Histoquímica , Fenilalanina Amônia-Liase/genética , Transcrição Genética
17.
Biochim Biophys Acta ; 1600(1-2): 61-7, 2002 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-12445460

RESUMO

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand (PEF) protein family. ALG-2 forms a homodimer, a heterodimer with another PEF protein, peflin, and a complex with its interacting protein, named Alix or AIP1. We previously identified annexin XI as a novel ALG-2-binding partner. Both the N-terminal regulatory domain of annexin XI (Anx11N) and the ALG-2-binding domain of Alix/AIP1 are rich in Pro, Gly, Ala, Tyr and Gln. This PGAYQ-biased amino acid composition is also found in the N-terminal extension of annexin VII (Anx7N). Using recombinant ALG-2 proteins and the glutathione S-transferase (GST) fusion proteins of Anx7N and Anx11N, the direct Ca(2+)-dependent interaction was analyzed by a biotin-tagged ALG-2 overlay assay and by a real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. Both GST-Anx7N and GST-Anx11N showed similar binding kinetics against ALG-2 as well as ALG-2-DeltaN23, which lacked the hydrophobic N-terminal region. Two binding sites were predicted in both Anx7N and Anx11N, and the dissociation constants (K(d)) were estimated to be approximately 40-60 nM for the high-affinity site and 500-700 nM for the low-affinity site.


Assuntos
Anexinas/química , Anexinas/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/farmacologia , Anexina A7/química , Anexina A7/genética , Anexina A7/metabolismo , Anexinas/genética , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Motivos EF Hand , Humanos , Cinética , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície
18.
Biochem Biophys Res Commun ; 291(5): 1166-72, 2002 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11883939

RESUMO

The apoptosis-linked protein ALG-2 is a Ca(2+)-binding protein that belongs to the penta-EF-hand protein family. ALG-2 forms a homodimer, a heterodimer with another penta-EF-hand protein, peflin, and a complex with its interacting protein, named AIP1 or Alix. By yeast two-hybrid screening using human ALG-2 as bait, we isolated a cDNA of a novel ALG-2-interacting protein, which turned out to be annexin XI. Deletion analysis revealed that ALG-2 interacted with the N-terminal domain of annexin XI (AnxN), which has an amino acid sequence similar to that of the C-terminal region of AIP1/Alix. Using recombinant biotin-tagged ALG-2 and the glutathione S-transferase (GST) fusion protein of AnxN, the direct interaction was analyzed by an ALG-2 overlay assay and by real-time interaction analysis with a surface plasmon resonance (SPR) biosensor. The dissociation constant (K(d)) was estimated to be approximately 70 nM. The Ca(2+)-dependent fluorescence change of ALG-2 in the presence of the hydrophobicity fluorescent probe 2-p-toluidinylnaphthalene-6-sulfonate (TNS) was inhibited by mixing with GST-AnxN, suggesting that the Pro/Gly/Tyr/Ala-rich hydrophobic region in AnxN masked the Ca(2+)-dependently exposed hydrophobic surface of ALG-2.


Assuntos
Anexinas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Sequência de Aminoácidos , Anexinas/química , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Técnicas Biossensoriais , Corantes Fluorescentes/metabolismo , Humanos , Dados de Sequência Molecular , Naftalenossulfonatos/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Técnicas do Sistema de Duplo-Híbrido
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