RESUMO
Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.
Assuntos
Biomarcadores/metabolismo , Exossomos/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Células COS , Chlorocebus aethiops , Elementos de DNA Transponíveis/genética , Expressão Gênica , Engenharia Genética , Células HEK293 , Humanos , Plasmídeos/metabolismo , Transcrição Gênica , TransgenesRESUMO
Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Glycine max/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência , Glycine max/química , Glycine max/genéticaRESUMO
Over the last several decades, increased agricultural production has been driven by improved agronomic practices and a dramatic increase in the use of nitrogen-containing fertilizers to maximize the yield potential of crops. To reduce input costs and to minimize the potential environmental impacts of nitrogen fertilizer that has been used to optimize yield, an increased understanding of the molecular responses to nitrogen under field conditions is critical for our ability to further improve agricultural sustainability. Using maize (Zea mays) as a model, we have characterized the transcriptional response of plants grown under limiting and sufficient nitrogen conditions and during the recovery of nitrogen-starved plants. We show that a large percentage (approximately 7%) of the maize transcriptome is nitrogen responsive, similar to previous observations in other plant species. Furthermore, we have used statistical approaches to identify a small set of genes whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. Using a composite gene expression scoring system, this single set of biomarker genes can accurately assess nitrogen responses independently of genotype, developmental stage, tissue type, or environment, including in plants grown under controlled environments or in the field. Importantly, the biomarker composite expression response is much more rapid and quantitative than phenotypic observations. Consequently, we have successfully used these biomarkers to monitor nitrogen status in real-time assays of field-grown maize plants under typical production conditions. Our results suggest that biomarkers have the potential to be used as agronomic tools to monitor and optimize nitrogen fertilizer usage to help achieve maximal crop yields.
Assuntos
Biomarcadores , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Nitrogênio/metabolismo , Transcriptoma , Zea mays/genética , Sequência de Bases , Biomarcadores/análise , Produtos Agrícolas , Fertilizantes , Perfilação da Expressão Gênica , Genoma de Planta/genética , Genótipo , Modelos Logísticos , Dados de Sequência Molecular , Nitrogênio/análise , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Análise de Sequência de DNA , Estresse Fisiológico , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismoRESUMO
Corn protein is largely made up of a group of nutritionally limited storage proteins known as zein. The reduction of zein can be achieved by a transcriptional mutation, opaque2 (o2), or a transgene targeting zein through RNA interference (RNAi). Zein reduction results in an increase of more nutritionally balanced non-zein proteins, and therefore enhance the overall quality of corn protein. In this study, the composition of mature kernels and the transcriptional profile of developing kernels of these two types of zein reduced kernels were compared. Both zein reduced kernels contained higher levels of lysine and tryptophan and free amino acids were 10-20-folds more abundant than the wild-type counterpart. We also found that free lysine contributed partially to the increased lysine in o2 kernels while protein-bound lysine was mainly responsible for the increased lysine in transgenic zein reduction (TZR) kernels. Although they had relatively similar gene expression patterns in developing endosperm, o2 kernels had greater transcriptional changes than TZR kernels in general. A number of transcripts that were specifically down-regulated in o2 were identified. Many promoter sequences of these transcripts contain putative O2 binding motifs, suggesting that their expression is directly regulated by O2.
Assuntos
Proteínas de Ligação a DNA/genética , Endosperma/genética , Mutação/genética , Proteínas de Plantas/genética , Interferência de RNA , Fatores de Transcrição/genética , Transcrição Gênica , Zea mays/genética , Zeína/genética , Aminoácidos/análise , Northern Blotting , Endosperma/ultraestrutura , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Regulação para Cima/genética , Zea mays/ultraestrutura , Zeína/metabolismoRESUMO
Exosomes are secreted organelles that have the same topology as the cell and bud outward (outward is defined as away from the cytoplasm) from endosome membranes or endosome-like domains of plasma membrane. Here we describe an exosomal protein-sorting pathway in Jurkat T cells that selects cargo proteins on the basis of both higher-order oligomerization (the oligomerization of oligomers) and plasma membrane association, acts on proteins seemingly without regard to their function, sequence, topology, or mechanism of membrane association, and appears to operate independently of class E vacuolar protein-sorting (VPS) function. We also show that higher-order oligomerization is sufficient to target plasma membrane proteins to HIV virus-like particles, that diverse Gag proteins possess exosomal-sorting information, and that higher-order oligomerization is a primary determinant of HIV Gag budding/exosomal sorting. In addition, we provide evidence that both the HIV late domain and class E VPS function promote HIV budding by unexpectedly complex, seemingly indirect mechanisms. These results support the hypothesis that HIV and other retroviruses are generated by a normal, nonviral pathway of exosome biogenesis.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Produtos do Gene gag/metabolismo , HIV/fisiologia , Proteínas de Membrana/metabolismo , Humanos , Células Jurkat , Células K562 , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Integral peroxisomal membrane proteins (PMPs) are synthesized in the cytoplasm and imported posttranslationally. Here, we demonstrate that PEX19 binds and stabilizes newly synthesized PMPs in the cytosol, binds to multiple PMP targeting signals (mPTSs), interacts with the hydrophobic domains of PMP targeting signals, and is essential for PMP targeting and import. These results show that PEX19 functions as both a chaperone and an import receptor for newly synthesized PMPs. We also demonstrate the existence of two PMP import mechanisms and two classes of mPTSs: class 1 mPTSs, which are bound by PEX19 and imported in a PEX19-dependent manner, and class 2 mPTSs, which are not bound by PEX19 and mediate protein import independently of PEX19.
Assuntos
Citosol/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Peroxissomos/metabolismo , Linhagem Celular , Citosol/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/metabolismo , Peroxissomos/ultraestrutura , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/fisiologia , Interferência de RNA , Transdução de Sinais/fisiologiaRESUMO
PEX19 is a chaperone and import receptor for newly synthesized, class I peroxisomal membrane proteins (PMPs). PEX19 binds these PMPs in the cytoplasm and delivers them to the peroxisome for subsequent insertion into the peroxisome membrane, indicating that there may be a PEX19 docking factor in the peroxisome membrane. Here we show that PEX3 is required for PEX19 to dock at peroxisomes, interacts specifically with the docking domain of PEX19, and is required for recruitment of the PEX19 docking domain to peroxisomes. PEX3 is also sufficient to dock PEX19 at heterologous organelles and binds PEX19 via a conserved motif that is essential for this docking activity and for PEX3 function in general. Not surprisingly, transient inhibition of PEX3 abrogates class I PMP import but has no effect on class II PMP import or peroxisomal matrix protein import. Taken together, these results suggest that PEX3 plays a selective, essential, and direct role in PMP import as a docking factor for PEX19.
Assuntos
Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Motivos de Aminoácidos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Lipoproteínas/genética , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Peroxinas , Peroxissomos/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
It is currently estimated that 11 million Canadians are living with diabetes or prediabetes. Although hyperglycemia is associated with serious complications, it is well established that improved glycemic control reduces the risk of microvascular complications and can also reduce cardiovascular (CV) complications over the long term. The UKPDS and ADVANCE landmark trials have resulted in diabetes guidelines recommending an A1C target of ≤ 7.0% for most patients or a target of ≤ 6.5% to further reduce the risk of nephropathy and retinopathy in those with type 2 diabetes (T2D), if it can be achieved safely. However, half of the people with T2D in Canada are not achieving these glycemic targets, despite advances in diabetes pharmacological management. There are many contributing factors to account for this poor outcome; however, one of the major factors is the delay in treatment advancement, particularly a resistance to insulin initiation and intensification. To simplify the process of initiating and titrating insulin in T2D patients, a group of Canadian experts reviewed the evidence and best clinical practices with the goal of providing guidance and practical recommendations to the diabetes healthcare community at large. This expert panel included general practitioners (GPs), nurses, nurse practitioners, endocrinologists, dieticians, pharmacists, and a psychologist. This article summarizes the panel recommendations.
RESUMO
3(2H)-Furanones can be prepared by a catalytic asymmetric protocol from enynones, which, if electron-rich, require only one reagent and involve two reactions in a single operation--a domino process.
Assuntos
Furanos/química , Alcinos/química , Catálise , Furanos/síntese química , Modelos Moleculares , Estrutura Molecular , EstereoisomerismoRESUMO
Zellweger syndrome is a lethal neurological disorder characterized by severe defects in peroxisomal protein import. The resulting defects in peroxisome metabolism and the accumulation of peroxisomal substrates are thought to cause the other Zellweger syndrome phenotypes, including neuronal migration defects, hypotonia, a developmental delay, and neonatal lethality. These phenotypes are also manifested in mouse models of Zellweger syndrome generated by disruption of the PEX5 or PEX2 gene. Here we show that mice lacking peroxisomal membrane protein PEX11 beta display several pathologic features shared by these mouse models of Zellweger syndrome, including neuronal migration defects, enhanced neuronal apoptosis, a developmental delay, hypotonia, and neonatal lethality. However, PEX11 beta deficiency differs significantly from Zellweger syndrome and Zellweger syndrome mice in that it is not characterized by a detectable defect in peroxisomal protein import and displays only mild defects in peroxisomal fatty acid beta-oxidation and peroxisomal ether lipid biosynthesis. These results demonstrate that the neurological pathologic features of Zellweger syndrome can occur without peroxisomal enzyme mislocalization and challenge current models of Zellweger syndrome pathogenesis.
Assuntos
Proteínas de Membrana/genética , Neurônios/patologia , Peroxissomos/metabolismo , Animais , Movimento Celular , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Retardo do Crescimento Fetal/genética , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mitocôndrias/ultraestrutura , Hipotonia Muscular/genética , Transporte Proteico , Síndrome de Zellweger/metabolismo , Síndrome de Zellweger/patologiaRESUMO
The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. As part of our effort to understand the molecular and physiological functions of PEX11 proteins, we disrupted the mouse PEX11alpha gene. Overexpression of PEX11alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, we find that PEX11alpha(-/-) mice have normal peroxisome abundance and that cells lacking both PEX11alpha and PEX11beta, a second mammalian PEX11 gene, have no greater defect in peroxisome abundance than do cells lacking only PEX11beta. Finally, we report the identification of a third mammalian PEX11 gene, PEX11gamma, and show that it too encodes a peroxisomal protein.
Assuntos
Proteínas de Membrana/genética , Proliferadores de Peroxissomos/farmacologia , Peroxissomos/efeitos dos fármacos , Peroxissomos/metabolismo , Fenilbutiratos/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Dieta , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Marcação de Genes , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/classificação , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Oxirredução , Proliferadores de Peroxissomos/administração & dosagem , Peroxissomos/ultraestrutura , Fenótipo , Filogenia , Plasmalogênios/metabolismo , Alinhamento de Sequência , Distribuição TecidualRESUMO
In plants and microbes, sucrose phosphate synthase (SPS) is an important enzyme in sucrose biosynthesis. Several different isozymes of SPS exist in plants. Genomic and EST sequence data from Arabidopsis, rice and maize has been analyzed. This analysis has revealed that the Arabidopsis genome contains four unique SPS genes. The rice databases (Monsanto proprietary, and public databases) contain five unique full-length SPS genes. Using the Monsanto maize EST and genomic sequence databases, we have identified five full length and two partial SPS sequences, bringing the total number of presently known maize SPS genes to at least seven. Phylogenetic analysis of all known SPS sequences revealed several putative evolutionary branches of SPS. We have classified SPS genes into three major groups in higher plants, all with distinct features from the known microbial SPS genes. Furthermore, this analysis suggests evolutionary divergence of monocotyledonous (monocot) and dicotyledonous (dicot) SPS sequences. The evidence suggests that several gene duplication events occurred at various points during evolution, both before and after the monocot/dicot split. It appears that at least one of the major forms of SPS genes may have evolved after the divergence of monocots and dicots. In addition, several more recent gene duplication events may have occurred after maize/rice speciation, giving rise to additional SPS genes in maize. Some of the variants lack one or more of the presently known regulatory sites, implying that this evolutionary divergence may have given rise to enzymes with functional differences. We present evidence from transcript distribution studies using cDNA libraries as well as transcriptional profiling experiments and propose that specific SPS genes have diverse patterns of expression that are sometimes responsive to environmental signals. Our data suggests that higher plant SPS isozymes differ with respect to their patterns of expression and regulation and that our proposed phylogenetic classification reflects specific functional categories for higher plant SPS isozymes.
Assuntos
Arabidopsis/enzimologia , Glucosiltransferases/metabolismo , Oryza/enzimologia , Filogenia , Proteínas de Plantas/metabolismo , Zea mays/enzimologia , Sequência de Aminoácidos , Arabidopsis/genética , Ritmo Circadiano , Temperatura Baixa , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Fertilização , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Glucosiltransferases/classificação , Glucosiltransferases/genética , Isoenzimas/classificação , Isoenzimas/metabolismo , Luz , Dados de Sequência Molecular , Oryza/genética , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Alinhamento de Sequência , Análise de Sequência de Proteína , Zea mays/genéticaRESUMO
The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.
Assuntos
Acridinas/síntese química , Acridinas/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Telomerase/antagonistas & inibidores , Cristalografia por Raios X , DNA/efeitos dos fármacos , DNA/metabolismo , DNA de Neoplasias/biossíntese , DNA de Neoplasias/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodaminas/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Deleção de Sequência/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/genética , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Peso Corporal/genética , Núcleo Celular/metabolismo , Sequência Consenso , Expressão Gênica , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Protoplastos/metabolismo , Reprodução , Fatores de Transcrição/química , Transcrição Gênica , Zea mays/citologia , Zea mays/fisiologiaRESUMO
Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glycine max/genética , Sementes/crescimento & desenvolvimento , Sementes/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Relógios Biológicos/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Análise de Sequência com Séries de Oligonucleotídeos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodução/genética , Supressão GenéticaRESUMO
Syntheses of aryloxyalkanoic acid hydroxyamides are described, all of which are potent inhibitors of histone deacetylase, some being more potent in vitro than trichostatin A (IC(50)=3 nM). Variation of the substituents on the benzene ring as well as fusion of a second ring have marked effects on potency, in vitro IC(50) values down to 1 nM being obtained.
Assuntos
Amidas/química , Antineoplásicos/química , Inibidores Enzimáticos/química , Inibidores de Histona Desacetilases , Amidas/síntese química , Amidas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Histona Desacetilases/química , Humanos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Matsumoto and colleagues recently identified PEX26 as the gene responsible for complementation group 8 of the peroxisome biogenesis disorders and showed that it encodes an integral peroxisomal membrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminus that interacts with the PEX1/PEX6 heterodimer through direct binding to the latter. They proposed that PEX26 functions as the peroxisomal docking factor for the PEX1/PEX6 heterodimer. Here, we identify new PEX26 disease alleles, localize the PEX6-binding domain to the N-terminal half of the protein (aa 29-174), and show that, at the cellular level, PEX26 deficiency impairs peroxisomal import of both PTS1- and PTS2-targeted matrix proteins. Also, we find that PEX26 undergoes alternative splicing to produce several splice forms--including one, PEX26- delta ex5, that maintains frame and encodes an isoform lacking the transmembrane domain of full-length PEX26 (PEX26-FL). Despite its cytosolic location, PEX26- delta ex5 rescues peroxisome biogenesis in PEX26-deficient cells as efficiently as does PEX26-FL. To test our observation that a peroxisomal location is not required for PEX26 function, we made a chimeric protein (PEX26-Mito) with PEX26 as its N-terminus and the targeting segment of a mitochondrial outer membrane protein (OMP25) at its C-terminus. We found PEX26-Mito localized to the mitochondria and directed all detectable PEX6 and a fraction of PEX1 to this extraperoxisomal location; yet PEX26-Mito retains the full ability to rescue peroxisome biogenesis in PEX26-deficient cells. On the basis of these observations, we suggest that a peroxisomal localization of PEX26 and PEX6 is not required for their function and that the interaction of PEX6 with PEX1 is dynamic. This model predicts that, once activated in an extraperoxisomal location, PEX1 moves to the peroxisome and completes the function of the PEX1/6 heterodimer.
Assuntos
Processamento Alternativo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Genéticos , Peroxissomos/metabolismo , Alelos , Sequência de Aminoácidos/genética , Linhagem Celular , Mapeamento Cromossômico , Citosol/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons , Fibroblastos/metabolismo , Frequência do Gene , Humanos , Proteínas de Membrana/química , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Receptor 2 de Sinal de Orientação para Peroxissomos , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/química , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Pele/citologiaRESUMO
The synthesis and evaluation of a group of 2,6-, 2,7- and 3,6-bis-aminoalkylamido acridones are reported, which show a similar level of activity against telomerase in vitro compared to their acridine counterparts. Computer modelling and calculations of relative binding energies suggest an equivalent binding mode to human intramolecular G-quadruplex DNA, but with significantly reduced affinity, as a result of the limited delocalisation of the acridone chromophore compared to the acridine system. Thermal melting studies on acridone and acridine quadruplex complexes using a FRET approach support these predictions. Long-term cell proliferation studies at sub-cytotoxic doses with two representative acridones using the SKOV3 cell line, show that neither compound produces growth arrest, in contrast with the effects produced by the tri-substituted acridine compound BRACO-19. It is concluded that telomerase inhibitory activity is a necessary though by itself insufficient property in order for cellular growth arrest to occur at sub-toxic concentrations, and that tight quadruplex binding is also required.