RESUMO
MAIN CONCLUSION: Two glycosyltransferase genes belonging to UGT88 family were identified to have 6'-deoxychalcone 4'-glucosyltransferase activity in dahlia. 6'-Deoxychalcones (isoliquiritigenin and butein) are important pigments for yellow and orange to red flower color. 6'-Deoxychalcones are glucosylated at the 4'-position in vivo, but the genes encoding 6'-deoxychalcone 4'-glucosyltransferase have not yet been identified. In our previous study, it was indicated that snapdragon (Antirrhinum majus) chalcone 4'-O-glucosyltransferase (Am4'CGT) has isoliquiritigenin 4'-glucosylation activity. Therefore, to identify genes encoding 6'-deoxychalcone 4'-glucosyltransferase in dahlia (Dahlia variabilis), genes expressed in ray florets that shared high homology with Am4'CGT were explored. As a result, c34671_g1_i1 and c35662_g1_i1 were selected as candidate genes for 6'-deoxychalcone 4'-glucosyltransferases in dahlia. We conducted transient co-overexpression of three genes (c34671_g1_i1 or c35662_g1_i1, dahlia aldo-keto reductase1 (DvAKR1) or soybean (Glycine max) chalcone reductase5 (GmCHR5), and chili pepper (Capsicum annuum) MYB transcription factor (CaMYBA)) in Nicotiana benthamiana by agroinfiltration. Transient overexpression of c34671_g1_i1, DvAKR1, and CaMYBA resulted in increase in the accumulation of isoliquiritigenin 4'-glucosides, isoliquiritigenin 4'-O-glucoside, and isoliquiritigenin 4'-O-[6-O-(malonyl)-glucoside]. However, transient overexpression of c35662_g1_i1, DvAKR1, and CaMYBA did not increase accumulation of isoliquiritigenin 4'-glucosides. Using GmCHR5 instead of DvAKR1 showed similar results suggesting that c34671_g1_i1 has isoliquiritigenin 4'-glucosyltransferase activity. In addition, we conducted co-overexpression of four genes (c34671_g1_i1, c35662_g1_i1 or Am4'CGT, DvAKR1 or GmCHR5, CaMYBA, and chalcone 3-hydroxylase from dahlia). Accumulation of butein 4'-O-glucoside and butein 4'-O-[6-O-(malonyl)-glucoside] was detected for c35662_g1_i1, suggesting that c35662_g1_i1 has butein 4'-glucosyltransferase activity. Recombinant enzyme analysis also supported butein 4'-glucosyltransferases activity of c35662_g1_i1. Therefore, our results suggested that both c34671_g1_i1 and c35662_g1_i1 are 6'-deoxychalcone 4'-glucosyltransferases but with different substrate preference.
Assuntos
Capsicum , Chalcona , Chalconas , Dahlia , Glucosiltransferases/genética , Glucosídeos , Glycine maxRESUMO
MAIN CONCLUSION: A novel gene belonging to the aldo-keto reductase 13 family is involved in isoliquiritigenin biosynthesis in dahlia. The yellow pigments of dahlia flowers are derived from 6'-deoxychalcones, which are synthesized via a two-step process, involving the conversion of 3-malonyl-CoA and 4-coumaloyl-CoA into isoliquiritigenin in the first step, and the subsequent generation of butein from isoliquiritigenin. The first step reaction is catalyzed by chalcone synthase (CHS) and aldo-keto reductase (AKR). AKR has been implicated in the isoflavone biosynthesis in legumes, however, isolation of butein biosynthesis related AKR members are yet to be reported. A comparative RNA-seq analysis between two dahlia cultivars, 'Shukuhai' and its butein-deficient lateral mutant 'Rinka', was used in this study to identify a novel AKR gene involved in 6'-deoxychalcone biosynthesis. DvAKR1 encoded a AKR 13 sub-family protein with significant differential expression levels, and was phylogenetically distinct from the chalcone reductases, which belongs to the AKR 4A sub-family in legumes. DNA sequence variation and expression profiles of DvAKR1 gene were correlated with 6'-deoxychalcone accumulation in the tested dahlia cultivars. A single over-expression analysis of DvAKR1 was not sufficient to initiate the accumulation of isoliquiritigenin in tobacco, in contrast, its co-overexpression with a chalcone 4'-O-glucosyltransferase (Am4'CGT) from Antirrhinum majus and a MYB transcription factor, CaMYBA from Capsicum annuum successfully induced isoliquiritigenin accumulation. In addition, DvAKR1 homologous gene expression was detected in Coreopsideae species accumulating 6'-deoxychalcone, but not in Asteraceae species lacking 6'-deoxychalcone production. These results not only demonstrate the involvement of DvAKR1 in the biosynthesis of 6'-deoxychalcone in dahlia, but also show that 6'-deoxychalcone occurrence in Coreopsideae species developed evolutionarily independent from legume species.
Assuntos
Chalconas , Dahlia , Aldeído Redutase/metabolismo , Aldo-Ceto Redutases/genética , Aldo-Ceto Redutases/metabolismo , Chalconas/metabolismo , Coenzima A/metabolismo , Dahlia/genéticaRESUMO
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PtdOH) and regulates the balance between two lipid second messengers: diacylglycerol and PtdOH. Several lines of evidence suggest that the η isozyme of DGK is involved in the pathogenesis of bipolar disorder. However, the detailed molecular mechanisms regulating the pathophysiological functions remain unclear. One reason is that it is difficult to detect the cellular activity of DGKη. To overcome this difficulty, we utilized protein myristoylation and a cellular PtdOH sensor, the N-terminal region of α-synuclein (α-Syn-N). Although DGKη expressed in COS-7 cells was broadly distributed in the cytoplasm, myristoylated (Myr)-AcGFP-DGKη and Myr-AcGFP-DGKη-KD (inactive (kinase-dead) mutant) were substantially localized in the plasma membrane. Moreover, DsRed monomer-α-Syn-N significantly colocalized with Myr-AcGFP-DGKη but not Myr-AcGFP-DGKη-KD at the plasma membrane. When COS-7 cells were osmotically shocked, all DGKη constructs were exclusively translocated to osmotic shock-responsive granules (OSRG). DsRed monomer-α-Syn-N markedly colocalized with only Myr-AcGFP-DGKη at OSRG and exhibited a higher signal/background ratio (3.4) than Myr-AcGFP-DGKη at the plasma membrane in unstimulated COS-7 cells (2.5), indicating that α-Syn-N more effectively detects Myr-AcGFP-DGKη activity in OSRG. Therefore, these results demonstrated that the combination of myristoylation and the PtdOH sensor effectively detects DGKη activity in cells and that this method is convenient to examine the molecular functions of DGKη. Moreover, this method will be useful for the development of drugs targeting DGKη. Furthermore, the combination of myristoylation (intensive accumulation in membranes) and α-Syn-N can be applicable to assays for various cytosolic PtdOH-generating enzymes.
Assuntos
Diacilglicerol Quinase/metabolismo , Ácidos Fosfatídicos/análise , Animais , Técnicas Biossensoriais/métodos , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Diacilglicerol Quinase/genética , Células HEK293 , Humanos , Isoenzimas , Pressão Osmótica , Ácidos Fosfatídicos/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
Phosphatidic acid (PA) is the simplest phospholipid and is involved in the regulation of various cellular events. Recently, we developed a new PA sensor, the N-terminal region of α-synuclein (α-Syn-N). However, whether α-Syn-N can sense physiologically produced, endogenous PA remains unclear. We first established an inactive PA sensor (α-Syn-N-KQ) as a negative control by replacing all eleven lysine residues with glutamine residues. Using confocal microscopy, we next verified that α-Syn-N, but not α-Syn-N-KQ, detected PA in macrophagic phagosomes in which PA is known to be enriched, further indicating that α-Syn-N can be used as a reliable PA sensor in cells. Finally, because PA generated during neuronal differentiation is critical for neurite outgrowth, we investigated the subcellular distribution of PA using α-Syn-N. We found that α-Syn-N, but not α-Syn-N-KQ, accumulated at the peripheral regions (close to the plasma membrane) of neuronal growth cones. Experiments using a phospholipase D (PLD) inhibitor strongly suggested that PA in the peripheral regions of the growth cone was primarily produced by PLD. Our findings provide a reliable sensor of endogenous PA and novel insights into the distribution of PA during neuronal differentiation.
RESUMO
Tracking the localization and dynamics of the intracellular bioactive lipid phosphatidic acid (PA) is important for understanding diverse biological phenomena. Although several PA sensors have been developed, better ones are still needed for comprehensive PA detection in cells. We recently found that α-synuclein (α-Syn) selectively and strongly bound to PA in vitro. Here, we revealed that the N-terminal region of α-Syn (α-Syn-N) specifically bound to PA, with a dissociation constant of 6.6 µm. α-Syn-N colocalized with PA-producing enzymes, diacylglycerol kinase (DGK) ß at the plasma membrane (PM), myristoylated DGKζ at the Golgi apparatus, phorbol ester-stimulated DGKγ at the PM, and phospholipase D2 at the PM and Golgi but not with the phosphatidylinositol-4,5-bisphosphate-producing enzyme in COS-7 cells. However, α-Syn-N failed to colocalize with them in the presence of their inhibitors and/or their inactive mutants. These results indicate that α-Syn-N specifically binds to cellular PA and can be applied as an excellent PA sensor.
Assuntos
Diacilglicerol Quinase/genética , Lipídeos/genética , alfa-Sinucleína/genética , Animais , Células COS , Chlorocebus aethiops , Complexo de Golgi/genética , Humanos , Lipídeos/química , Ácidos Fosfatídicos/química , Ácidos Fosfatídicos/genética , Fosfatidilinositóis , Fosfolipase D/química , Fosfolipase D/genética , Ligação Proteica , Transdução de Sinais , alfa-Sinucleína/químicaRESUMO
Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP-DGKζ cDNA with a consensus N-myristoylation sequence at the 5' end (Myr-AcGFP-DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (LC-MS/MS), we showed that Myr-AcGFP-DGKζ, but not AcGFP-DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr-AcGFP-DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity-dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid-metabolizing enzymes, including other DGK isozymes.
Assuntos
Diacilglicerol Quinase/análise , Ácidos Mirísticos/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromatografia Líquida , Diacilglicerol Quinase/metabolismo , Humanos , Espectrometria de Massas em TandemRESUMO
Thalidomide was originally used as a sedative and found to be a teratogen, but now thalidomide and its derivatives are widely used to treat haematologic malignancies. Accumulated evidence suggests that thalidomide suppresses nerve cell death in neurologic model mice. However, detailed molecular mechanisms are unknown. Here we examined the molecular mechanism of thalidomide's neuroprotective effects, focusing on its target protein, cereblon (CRBN), and its binding protein, AMP-activated protein kinase (AMPK), which plays an important role in maintaining intracellular energy homeostasis in the brain. We used a cerebral ischemia rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Thalidomide treatment significantly decreased the infarct volume and neurological deficits of MCAO/R rats. AMPK was the key signalling protein in this mechanism. Furthermore, we considered that the AMPK-CRBN interaction was altered when neuroprotective action by thalidomide occurred in cells under ischemic conditions. Binding was strong between AMPK and CRBN in normal SH-SY5Y cells, but was weakened by the addition of H2O2. However, when thalidomide was administered at the same time as H2O2, the binding of AMPK and CRBN was partly restored. These results suggest that thalidomide inhibits the activity of AMPK via CRBN under oxidative stress and suppresses nerve cell death.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteases Dependentes de ATP/genética , Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Talidomida/farmacologia , Complexos Ubiquitina-Proteína Ligase/genética , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Proteases Dependentes de ATP/antagonistas & inibidores , Proteases Dependentes de ATP/metabolismo , Animais , Isquemia Encefálica/enzimologia , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Reposicionamento de Medicamentos , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Imunossupressores/farmacologia , Infarto da Artéria Cerebral Média/enzimologia , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/patologia , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Complexos Ubiquitina-Proteína Ligase/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Cereblon (CRBN) is encoded by a candidate gene for autosomal recessive nonsyndromic intellectual disability (ID). The nonsense mutation, R419X, causes deletion of 24 amino acids at the C-terminus of CRBN, leading to mild ID. Although abnormal CRBN function may be associated with ID disease onset, its cellular mechanism is still unclear. Here, we examine the role of CRBN in aggresome formation and cytoprotection. In the presence of a proteasome inhibitor, exogenous CRBN formed perinuclear inclusions and co-localized with aggresome markers. Endogenous CRBN also formed perinuclear inclusions under the same condition. Treatment with a microtubule destabilizer or an inhibitor of the E3 ubiquitin ligase activity of CRBN blocked formation of CRBN inclusions. Biochemical analysis showed CRBN containing inclusions were high-molecular weight, ubiquitin-positive. CRBN overexpression in cultured cells suppressed cell death induced by proteasome inhibitor. Furthermore, knockdown of endogenous CRBN in cultured cells increased cell death induced by proteasome inhibitor, compared with control cells. Our results show CRBN is recruited to aggresome and has functional roles in cytoprotection against ubiquitin-proteasome system impaired condition.
Assuntos
Citoproteção/fisiologia , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Códon sem Sentido , Citoproteção/genética , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Humanos , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/metabolismo , Corpos de Inclusão Intranuclear/patologia , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Nocodazol/farmacologia , Células PC12 , Peptídeo Hidrolases/deficiência , Peptídeo Hidrolases/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , RNA Interferente Pequeno/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Talidomida/análogos & derivados , Talidomida/farmacologia , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A series of hydroxy-amide functionalized azolium salts have been designed and synthesized for Cu-catalyzed asymmetric conjugate addition reaction. The (CH(2))(2)-bridged hydroxy-amide functionalized azolium ligand precursors 2, in addition to the previously reported CH(2)-bridged azolium salts 1, have been prepared from readily available enantiopure ß-amino alcohols. The combination of a Cu species with 1 or 2 efficiently promoted the 1,4-addition reaction of cyclic enones with dialkylzincs. For example, the reaction of 2-cyclohepten-1-one (17) with Bu(2)Zn in the presence of catalytic amounts of Cu(OTf)(2) and 1 gave (S)-3-butylcycloheptanone (20) in 99% yield and 96% ee. On the other hand, when the reaction was carried out under the influence of Cu(OTf)(2) combined with 2, (R)-20 in preference to (S)-20 was obtained in 98% yield and 80% ee. In this manner, the enantioselecvity was switched by controlling the structure of chiral ligand. Additionally, the reversal of enantioselectivity was also achieved by changing the Cu precatalyst from Cu(OTf)(2) to Cu(acac)(2) with the same ligand. The combination of Cu(acac)(2) with CH(2)-bridged azolium salt 1 in the reaction of 17 with Bu(2)Zn led to formation of (R)-20 as a major product in 55% yield and 80% ee. This result was in contrast to the Cu(OTf)(2)/1 catalytic system, where the 1,4-adduct with opposite configuration was obtained. Moreover, use of the Cu(acac)(2)/2 catalytic system produced (S)-20, while (R)-20 was formed by the Cu(OTf)(2)/2 catalytic system. Thus, it was found that either varying the linker of the chiral ligands or changing the counterion of Cu species between a OTf and acac ligand initially on the metal led to dual enantioselective control in the 1,4-addition reaction.
RESUMO
Desmosine, a crosslinking amino acid of elastin, is an attractive biomarker for diagnosis of chronic obstructive pulmonary disease (COPD). In this study, the first total synthesis of (+)-desmosine was achieved in 11% overall yield in 13 steps utilizing stepwise and regioselective Sonogashira cross-coupling reactions.
Assuntos
Reagentes de Ligações Cruzadas/síntese química , Desmosina/síntese química , Elastina/química , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Biomarcadores/química , Reagentes de Ligações Cruzadas/química , Desmosina/química , Estrutura Molecular , EstereoisomerismoRESUMO
The effect of consuming bonito extract (BE) on cerebral blood flow was evaluated in stroke-prone spontaneously hypertensive rats (SHRSP), a cerebrovascular disease model. BE dissolved in drinking water was given to 5-week-old male SHRSP for 7 weeks. Tap water was given to the control group. At the age of 12 weeks, blood flow and vascular diameter were measured in the middle cerebral artery. Both cerebral blood flow and cerebral vessel width were greater in the BE group than in the control group. Also, stroke occurred in 7 (with death in 2) of the 8 animals in the control group but in none of the 6 animals in the BE group. To clarify its mechanism, the expressions of nitrogen oxide synthase (NOS) and the superoxide dismutase activity (SOD) in the brain were evaluated. NOS mRNA expression and SOD activity in the cerebrum were higher in the BE group. These results suggest that the consumption of BE suppresses the decrease of cerebral blood flow and reduces the risk of stroke to maintain vasorelaxation through the production of nitrogen oxide and suppression of active oxygen generation.