RESUMEN
ZNF410 is a highly-conserved transcription factor, remarkable in that it recognizes a 15-base pair DNA element but has just a single responsive target gene in mammalian erythroid cells. ZNF410 includes a tandem array of five zinc-fingers (ZFs), surrounded by uncharacterized N- and C-terminal regions. Unexpectedly, full-length ZNF410 has reduced DNA binding affinity, compared to that of the isolated DNA binding ZF array, both in vitro and in cells. AlphaFold predicts a partially-folded N-terminal subdomain that includes a 30-residue long helix, preceded by a hairpin loop rich in acidic (aspartate/glutamate) and serine/threonine residues. This hairpin loop is predicted by AlphaFold to lie against the DNA binding interface of the ZF array. In solution, ZNF410 is a monomer and binds to DNA with 1:1 stoichiometry. Surprisingly, the single best-fit model for the experimental small angle X-ray scattering profile, in the absence of DNA, is the original AlphaFold model with the N-terminal long-helix and the hairpin loop occupying the ZF DNA binding surface. For DNA binding, the hairpin loop presumably must be displaced. After combining biophysical, biochemical, bioinformatic and artificial intelligence-based AlphaFold analyses, we suggest that the hairpin loop mimics the structure and electrostatics of DNA, and provides an additional mechanism, supplementary to sequence specificity, of regulating ZNF410 DNA binding.
Asunto(s)
Factores de Transcripción , Animales , Secuencia de Aminoácidos , Inteligencia Artificial , Mamíferos/genética , Unión Proteica , Dominios Proteicos , Dedos de Zinc/genética , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Interaction between human positive coactivator 4 (PC4), an abundant nuclear protein, and the tumor suppressor protein p53 plays a crucial role in initiating apoptosis. In certain neurodegenerative diseases PC4 assisted-p53-dependent apoptosis may play a central role. Thus, disruption of p53-PC4 interaction may be a good drug target for certain disease pathologies. A p53-derived short peptide (AcPep) that binds the C-terminal domain of PC4 (C-PC4) is known to disrupt PC4-p53 interaction. To fully characterize its binding mode and binding site on PC4, we co-crystallized C-PC4 with the peptide and determined its structure. The crystal, despite exhibiting mass spectrometric signature of the peptide, lacked peptide electron density and showed a novel crystal lattice, when compared to C-PC4 crystals without the peptide. Using peptide-docked models of crystal lattices, corresponding to our structure and the peptide-devoid structure we show the origin of the novel crystal lattice to be dynamically bound peptide at the previously identified putative binding site. The weak binding is proposed to be due to the lack of the N-terminal domain of PC4 (N-PC4), which we experimentally show to be disordered with no effect on PC4 stability. Taking cue from the structure, virtual screening of â¼18.6 million small molecules from the ZINC15 database was performed, followed by toxicity and binding free energy filtering. The novel crystal lattice of C-PC4 in presence of the peptide, the role of the disordered N-PC4 and the high throughput identification of potent small molecules will allow a better understanding and control of p53-PC4 interaction.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enfermedades Neurodegenerativas/patología , Péptidos/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/fisiología , Sitios de Unión , Biología Computacional/métodos , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Evaluación Preclínica de Medicamentos/métodos , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/metabolismo , Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Factores de Transcripción/química , Proteína p53 Supresora de Tumor/químicaRESUMEN
INTRODUCTION: Depression is one of the leading causes of death worldwide. Lower antioxidant concentrations and increased oxidative stress levels contribute to the development of depression. Effective and tolerable medications are urgently needed. Nrf2 and PRDX2 are promising targets in the treatment of oxidative stress and, therefore, promising for the development of novel antidepressants. Ursolic acid (UA), a natural triterpenoid found in various plants is known to exert neuroprotective and antioxidant effects. Skn-1 (which corresponds to human Nrf2) and prdx2 deficient mutants of the nematode Caenorhabditis elegans are suitable models to study the effect of UA on these targets. Additionally, stress assays are used to mimic stress or depressed state. METHODS: We examined the antioxidant activity of UA in Caenorhabditis elegans wildtype and skn-1- and prdx2-deficient strains by H2DCF-DA and juglone assays as well as osmotic and heat stress assays. Additionally, we analyzed the binding of UA to human PRDX2 and Skn-1 proteins by molecular docking and microscale thermophoresis. RESULTS: UA exerted strong antioxidant activities. Additionally, induction of stress resistance towards osmotic and heat stress was observed. qRT-PCR revealed that UA upregulated the gene expression of skn-1 and prdx2. Molecular docking studies supported these findings. CONCLUSION: Our findings implicate that the strong antioxidant activity of UA may exert anti-depressive effects by its interaction with the Skn-1 transcription factor, which is part of a detoxification network, and the antioxidant PRDX2 protein, which protects the organism from the detrimental effects of radical oxygen species.
Asunto(s)
Caenorhabditis elegans/efectos de los fármacos , Depresión/genética , Estrés Fisiológico/efectos de los fármacos , Triterpenos/farmacología , Animales , Antidepresivos/farmacología , Antioxidantes/farmacología , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Simulación del Acoplamiento Molecular , Mutación , Estrés Oxidativo/efectos de los fármacos , Peroxirredoxinas/genética , Sustancias Protectoras/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Triterpenos/química , Ácido UrsólicoRESUMEN
Lonicera macranthoides Hand-Mazz is an important medicinal plant widely distributed in southern China that has long been used in Chinese traditional medicines. Chlorogenic acid (CGA, 3-caffeoylquinic acid) is the major biologically active ingredient in L. macranthoides. Although key CGA biosynthetic genes have been well documented, their transcriptional regulation remains largely unknown. In this study, we observed that a R2R3 MYB transcription factor LmMYB15 showed a significant correlation with CGA content, indicating its potential role in CGA biosynthesis. A yeast two-hybrid assay suggested that LmMYB15 functions as a transcriptional activator. Overexpression of LmMYB15 in tobacco led to increased accumulation of CGA compared to those in wild-type leaves. To elucidate its functional mechanism, genome-wide DAP-seq was employed and identified the conserved binding motifs of LmMYB15, that is [(C/T) (C/T) (C/T) ACCTA(C/A) (C/T) (A/T)], as well as its direct downstream target genes, including 4CL, MYB3, MYB4, KNAT6/7, IAA26, and ETR2. Subsequently, yeast one-hybrid and dual-luciferase reporter assays verified that LmMYB15 could bind and activate the promoters of 4CL, MYB3 and MYB4, thereby facilitating CGA biosynthesis and phenylpropanoid metabolism. Our findings provide a new track for breeding strategies aiming to enhance CGA content in L. macranthoides that can significantly contribute to better mechanical properties.
Asunto(s)
Ácido Clorogénico/metabolismo , Lonicera/genética , Proteínas de Plantas/genética , Metabolismo Secundario , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Lonicera/metabolismo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Bromodomain and PHD finger containing transcription factor (BPTF) is a multidomain protein that regulates the transcription of chromatin and is related to many cancers. Herein, we report the screening-based discovery of Cpd1, a compound with micromolar affinity to the BPTF bromodomain. Through structure-guided optimization, we synthesized a variety of new inhibitors. Among these compounds, Cpd8 and Cpd10 were highly potent and selective inhibitors, with KD values of 428 nM and 655 nM in ITC assays, respectively. The high activity was explained by the cocrystal structure of Cpd8 in complex with the BPTF bromodomain protein. Cpd8 and Cpd10 were able to stabilize the BPTF bromodomain protein in cells in a cellular thermal shift assay (CETSA). Cpd8 downregulated c-MYC expression in A549 cells. All experiments prove that these two compounds are potential BPTF inhibitors.
Asunto(s)
Proteínas del Tejido Nervioso/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Células A549 , Antígenos Nucleares/química , Antígenos Nucleares/genética , Calorimetría , Cristalografía por Rayos X , Diseño de Fármacos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Fluorometría , Regulación de la Expresión Génica/efectos de los fármacos , Genes myc , Células HEK293 , Humanos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Dominios Proteicos , Estabilidad Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: The latest coronavirus SARS-CoV-2, discovered in China and rapidly spread Worldwide. COVID-19 affected millions of people and killed hundreds of thousands worldwide. There are many ongoing studies investigating drug(s) suitable for preventing and/or treating this pandemic; however, there are no specific drugs or vaccines available to treat or prevent SARS-CoV-2 as of today. METHODS: Fifty-eight fragrance materials, which are classified as allergen fragrance molecules, were selected and used in this study. Docking simulations were carried out using four functional proteins; the Covid19 Main Protase (MPro), Receptor binding domain (RBD) of spike protein, Nucleocapsid, and host Bromodomain protein (BRD2), as target macromolecules. Three different software, AutoDock, AutoDock Vina (Vina), and Molegro Virtual Docker (MVD), running a total of four different docking protocol with optimized energy functions were used. Results were compared with the five molecules reported in the literature as potential drugs against COVID-19. Virtual screening was carried out using Vina, molecules satisfying our cut-off (- 6.5 kcal/mol) binding affinity was confirmed by MVD. Selected molecules were analyzed using the flexible docking protocol of Vina and AutoDock default settings. RESULTS: Ten out of 58 allergen fragrance molecules were selected for further docking studies. MPro and BRD2 are potential targets for the tested allergen fragrance molecules, while RBD and Nucleocapsid showed weak binding energies. According to AutoDock results, three molecules, Benzyl Cinnamate, Dihydroambrettolide, and Galaxolide, had good binding affinities to BRD2. While Dihydroambrettolide and Galaxolide showed the potential to bind to MPro, Sclareol and Vertofix had the best calculated binding affinities to this target. When the flexible docking results analyzed, all the molecules tested had better calculated binding affinities as expected. Benzyl Benzoate and Benzyl Salicylate showed good binding affinities to BRD2. In the case of MPro, Sclareol had the lowest binding affinity among all the tested allergen fragrance molecules. CONCLUSION: Allergen fragrance molecules are readily available, cost-efficient, and shown to be safe for human use. Results showed that several of these molecules had comparable binding affinities as the potential drug molecules reported in the literature to target proteins. Thus, these allergen molecules at correct doses could have significant health benefits.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Tratamiento Farmacológico de COVID-19 , COVID-19/inmunología , Odorantes , Perfumes/química , SARS-CoV-2/química , SARS-CoV-2/inmunología , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/metabolismo , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alérgenos/administración & dosificación , Alérgenos/uso terapéutico , Benzopiranos/química , Benzopiranos/metabolismo , Compuestos de Bencilo/química , Compuestos de Bencilo/metabolismo , Cinamatos/química , Cinamatos/metabolismo , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/metabolismo , Proteínas de la Nucleocápside de Coronavirus/química , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Diterpenos/química , Diterpenos/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Perfumes/administración & dosificación , Perfumes/uso terapéutico , Fosfoproteínas/química , Fosfoproteínas/metabolismo , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
MRG15 is a transcription factor containing the methyl-lysine reader chromodomain. Despite its involvement in different physiological and pathological states, to date the role of this protein has not been fully elucidated due to the lack of a specific and potent chemical probe.In this work, we report the development of a microscale thermophoresis (MST)-based assay for the study of MRG15-ligand binding interactions. After the development, the assay was validated using a small focused library and UNC1215 as the reference compound, to yield the identification of 10 MRG15 ligands with affinities ranging from 37.8 nM to 59.1 µM.Hence, our method is robust, convenient, and fast and could be applied to other methylation reader domain-containing proteins for the identification of new chemical probes.
Asunto(s)
Desarrollo de Medicamentos/métodos , Evaluación Preclínica de Medicamentos/métodos , Factores de Transcripción/química , Ligandos , Unión Proteica , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Plants produce thousands of chemically diverse secondary metabolites, many of which have valuable pharmaceutical properties. There is much interest in the synthesis of these pharmaceuticallyvaluable compounds, including the key enzymes and the transcription factors involved. The function and regulatory mechanism of transcription factors in biotic and abiotic stresses have been studied in depth. However, their regulatory roles in the biosynthesis of bioactive compounds, especially in medicinal plants, have only begun. Here, we review what is currently known about how transcription factors contribute to the synthesis of bioactive compounds (alkaloids, terpenoids, flavonoids, and phenolic acids) in medicinal plants. Recent progress has been made in the cloning and characterization of transcription factors in medicinal plants on the genome scale. So far, several large transcription factors have been identified in MYB, WRKY, bHLH, ZIP, AP2/ERF transcription factors. These transcription factors have been predicted to regulate bioactive compound production. These transcription factors positively or negatively regulate the expression of multiple genes encoding key enzymes, and thereby control the metabolic flow through the biosynthetic pathway. Although the research addressing this niche topic is in its infancy, significant progress has been made, and advances in high-throughput sequencing technology are expected to accelerate the discovery of key regulatory transcription factors in medicinal plants. This review is likely to be useful for those interested in the synthesis of pharmaceutically- valuable plant compounds, especially those aiming to breed or engineer plants that produce greater yields of these compounds.
Asunto(s)
Plantas Medicinales/química , Plantas Medicinales/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Alcaloides/química , Alcaloides/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Medicinales/metabolismo , Estrés Fisiológico/fisiología , Terpenos/química , Terpenos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
KEY MESSAGE: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.
Asunto(s)
Antocianinas/biosíntesis , Fagopyrum/metabolismo , Proteínas de Plantas/metabolismo , Proantocianidinas/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Arabidopsis , Fagopyrum/genética , Flavonoides/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Estrés Fisiológico , Nicotiana/genética , Factores de Transcripción/químicaRESUMEN
Retinoic acid-related orphan receptor beta (RORß) is a transcription factor (TF) and marker of layer 4 (L4) neurons, which are distinctive both in transcriptional identity and the ability to form aggregates such as barrels in rodent somatosensory cortex. However, the relationship between transcriptional identity and L4 cytoarchitecture is largely unknown. We find RORß is required in the cortex for L4 aggregation into barrels and thalamocortical afferent (TCA) segregation. Interestingly, barrel organization also degrades with age in wildtype mice. Loss of RORß delays excitatory input and disrupts gene expression and chromatin accessibility, with down-regulation of L4 and up-regulation of L5 genes, suggesting a disruption in cellular specification. Expression and binding site accessibility change for many other TFs, including closure of neurodevelopmental TF binding sites and increased expression and binding capacity of activity-regulated TFs. Lastly, a putative target of RORß, Thsd7a, is down-regulated without RORß, and Thsd7a knock-out alone disrupts TCA organization in adult barrels.
Asunto(s)
Neuronas , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Corteza Somatosensorial , Animales , Antígenos de Superficie/química , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Femenino , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Neuronas/química , Neuronas/citología , Neuronas/metabolismo , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Corteza Somatosensorial/química , Corteza Somatosensorial/citología , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/fisiología , Tálamo/química , Tálamo/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma/genéticaRESUMEN
l-Theanine, a non-proteinaceous amino acid abundantly present in tea (Camellia sinensis), contributes to the umami flavor of tea and has beneficial effects on human health. While key l-theanine biosynthetic genes have been well documented, their transcriptional regulation remains poorly understood. In this study, we determined the l-theanine contents in tea leaves of two cultivars at three developmental stages and investigated the expression patterns of the l-theanine biosynthetic genes CsGS1 and CsGS2. Additionally, we identified an R2R3-MYB transcription factor, CsMYB73, belonging to subgroup 22 of the R2R3-MYB family. CsMYB73 expression negatively correlated with l-theanine accumulation during leaf maturation. We found that CsMYB73, as a nuclear protein, binds to the promoter regions of CsGS1 and CsGS2 via MYB recognition sequences and represses the transcription of CsGS1 and CsGS2 in tobacco leaves. Collectively, our results demonstrate that CsMYB73 is a transcriptional repressor involved in l-theanine biosynthesis in tea plants. Our findings might contribute to future tea plant breeding strategies.
Asunto(s)
Amida Sintasas/genética , Camellia sinensis/genética , Glutamatos/biosíntesis , Proteínas de Plantas/genética , Factores de Transcripción/genética , Amida Sintasas/metabolismo , Secuencia de Aminoácidos , Camellia sinensis/enzimología , Filogenia , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Pseudomonas aeruginosa has become a global concern due to its extreme resistance to most of the last resort antibiotics. Present study focuses on the screening of potential molecular targets involved in regulation of biofilm formation in P. aeruginosa and identification of potential natural lead molecules against these targets by molecular modelling, docking and simulation studies. Response regulator (GacA) and transcriptional activator (RhlR) involved in biofilm formation in P. aeruginosa were identified as molecular targets by metabolic pathway analysis and the three dimensional structures of these proteins were predicted by homology modelling and validated. By thorough literature survey, 78 lead molecules were screened and their pharmacokinetic profiles were determined and best two of them selected. The binding potential of selected lead molecules against GacA and RhlR were predicted by molecular docking and their binding energy was compared with the interaction of meropenem and its usual target penicillin binding protein-3. The stabilities of best docked complex were studied by molecular dynamic (MD) simulation. This study showed that Celastrol present in Celastrus paniculatus and Rotiorinol present in Chaetomium cupreum showed better binding affinities with GacA (binding energy -7.2 kcal/mol) and RhlR (binding energy -8.0 kcal/mol) respectively in comparison with the binding of Meropenem and its target (binding energy -6.2 kcal/mol). MD simulation studies showed that GacA-Celastrol and RhlR-Rotiorinol complexes demonstrated conformational stability throughout the simulation. This study highlights the application of GacA and RhlR as prospective targets and Celastrol and Rotiorinol are the potential lead molecules towards biofilm producing drug resistant P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/química , Biopelículas/efectos de los fármacos , Triterpenos Pentacíclicos/farmacología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos , Redes y Vías Metabólicas/efectos de los fármacos , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Factores de Transcripción/químicaRESUMEN
Calcineurin B-like protein-interacting protein kinases (CIPKs), as key regulators, play an important role in plant growth and development and the response to various stresses. In the present study, we identified 80 and 78 CIPK genes in the Gossypium hirsutum and G. barbadense, respectively. The phylogenetic and gene structure analysis divided the cotton CIPK genes into five groups which were classified into an exon-rich clade and an exon-poor clade. A synteny analysis showed that segmental duplication contributed to the expansion of Gossypium CIPK gene family, and purifying selection played a major role in the evolution of the gene family in cotton. Analyses of expression profiles showed that GhCIPK genes had temporal and spatial specificity and could be induced by various abiotic stresses. Fourteen GhCIPK genes were found to contain 17 non-synonymous single nucleotide polymorphisms (SNPs) and co-localized with oil or protein content quantitative trait loci (QTLs). Additionally, five SNPs from four GhCIPKs were found to be significantly associated with oil content in one of the three field tests. Although most GhCIPK genes were not associated with natural variations in cotton oil content, the overexpression of the GhCIPK6 gene reduced the oil content and increased C18:1 and C18:1+C18:1d6 in transgenic cotton as compared to wild-type plants. In addition, we predicted the potential molecular regulatory mechanisms of the GhCIPK genes. In brief, these results enhance our understanding of the roles of CIPK genes in oil synthesis and stress responses.
Asunto(s)
Genoma de Planta , Gossypium/genética , Aceites de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinasas/genética , Cromosomas de las Plantas , Ácidos Grasos/metabolismo , Duplicación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Gossypium/química , Gossypium/metabolismo , MicroARNs/metabolismo , Familia de Multigenes , Filogenia , Aceites de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/química , Plantas Modificadas Genéticamente/metabolismo , Polimorfismo de Nucleótido Simple , Proteínas Serina-Treonina Quinasas/clasificación , Proteínas Serina-Treonina Quinasas/metabolismo , Sitios de Carácter Cuantitativo , Elementos Reguladores de la Transcripción/genética , Sales (Química)/farmacología , Semillas/química , Semillas/metabolismo , Estrés Fisiológico , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
SQUAMOSA promoter-binding protein (SBP)-box gene family is one kind of plant-specific transcription factor that plays important roles in the process of resisting abiotic stress. The SBP-box gene family has been studied in many species, but their functions are not yet clear in Camellia sinensis var. sinensis (CSS) (tea) plants. In our study, 25 SBP-box genes in the CSS were identified in the reference genome and classified into six groups based on a phylogenetic tree. The expression pattern of CsSBP genes under temperature stresses showed that CsSBPs were involved in the process of resisting temperature stresses. CsSBP8 had a positive effect on the anthocyanin accumulation during high temperature exposures, but CsSBP12 has a high correlation with anthocyanin accumulation during both high and low temperature. This study provides a foundation for the further study of CsSBP genes involved in the anthocyanin biosynthesis pathway during the temperature stress in tea.
Asunto(s)
Camellia sinensis/genética , Proteínas de Plantas/genética , Estrés Fisiológico/genética , Factores de Transcripción/genética , Antocianinas/metabolismo , Camellia sinensis/metabolismo , Genoma de Planta , MicroARNs/metabolismo , Familia de Multigenes , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificación , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas , Alineación de Secuencia , Análisis de Secuencia de Proteína , Temperatura , Factores de Transcripción/química , Factores de Transcripción/clasificación , Factores de Transcripción/metabolismoRESUMEN
Bivalent chemical degraders provide a catalytic route to selectively degrade disease-associated proteins. By linking target-specific ligands with E3 ubiquitin ligase recruiting ligands, these compounds facilitate targeted protein ubiquitination and degradation by the proteasome. Due to the complexity of this multistep mechanism, the development of effective degrader molecules remains a difficult, lengthy, and unpredictable process. Since degraders are large heterobifunctional molecules, the efficacy of these compounds may be limited by poor cell permeability, and an efficient and reliable method to quantify the cell permeability of these compounds is lacking. Herein, we demonstrate that by the addition of a chloroalkane tag on the BRD4 specific degrader, MZ1, cell permeability can be quantified via the chloroalkane penetration assay. By extending this analysis to individual components of the degrader molecule, we have obtained structure-permeability relationships that will be informative for future degrader development, particularly as degraders move into the clinic as potential therapeutics.
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Dipéptidos/química , Dipéptidos/metabolismo , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Hidrocarburos/química , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Técnicas Biosensibles , Proteínas de Ciclo Celular/química , Línea Celular , Permeabilidad de la Membrana Celular , Evaluación Preclínica de Medicamentos , Humanos , Ligandos , Estructura Molecular , Relación Estructura-Actividad , Factores de Transcripción/química , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors that play a crucial role in growth and development, as well as metabolic processes. However, knowledge of the function of LBD proteins in Camellia sinensis is limited, and no systematic investigations of the LBD family have been reported. In this study, we identified 54 LBD genes in Camellia sinensis. The expression patterns of CsLBDs in different tissues and their transcription responses to exogenous hormones and abiotic stress were determined by RNA-seq, which showed that CsLBDs may have diverse functions. Analysis of the structural gene promoters revealed that the promoters of CsC4H, CsDFR and CsUGT84A, the structural genes involved in flavonoid biosynthesis, contained LBD recognition binding sites. The integrative analysis of CsLBD expression levels and metabolite accumulation also suggested that CsLBDs are involved in the regulation of flavonoid synthesis. Among them, CsLOB_3, CsLBD36_2 and CsLBD41_2, localized in the nucleus, were selected for functional characterization. Yeast two-hybrid assays revealed that CsLBD36_2 and CsLBD41_2 have self-activation activities, and CsLOB_3 and CsLBD36_2 can directly bind to the cis-element and significantly increase the activity of the CsC4H, CsDFR and CsUGT84A promoter. Our results present a comprehensive characterization of the 54 CsLBDs in Camellia sinensis and provide new insight into the important role that CsLBDs play in abiotic and flavonoid biosynthesis.
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Camellia sinensis/metabolismo , Flavonoides/biosíntesis , Ligasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Camellia sinensis/citología , Camellia sinensis/genética , Camellia sinensis/fisiología , Regulación de la Expresión Génica de las Plantas , Espacio Intracelular/metabolismo , Anotación de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiones Promotoras Genéticas/genética , Transporte de Proteínas , Alineación de Secuencia , Factores de Transcripción/genética , Activación TranscripcionalRESUMEN
C-repeat binding factors (CBFs) are key signaling genes that can be rapidly induced by cold and bind to the C-repeat/dehydration-responsive motif (CRT/DRE) in the promoter region of the downstream cold-responsive (COR) genes, which play a vital role in the plant response to low temperature. However, the CBF family in tea plants has not yet been elucidated, and the possible target genes regulated by this family under low temperature are still unclear. In this study, we identified five CsCBF family genes in the tea plant genome and analyzed their phylogenetic tree, conserved domains and motifs, and cis-elements. These results indicate that CsCBF3 may be unique in the CsCBF family. This is further supported by our findings from the low-temperature treatment: all the CsCBF genes except CsCBF3 were significantly induced after treatment at 4 °C. The expression profiles of eight tea plant tissues showed that CsCBFs were mainly expressed in winter mature leaves, roots and fruits. Furthermore, 685 potential target genes were identified by transcriptome data and CRT/DRE element information. These target genes play a functional role under the low temperatures of winter through multiple pathways, including carbohydrate metabolism, lipid metabolism, cell wall modification, circadian rhythm, calcium signaling, transcriptional cascade, and hormone signaling pathways. Our findings will further the understanding of the stress regulatory network of CsCBFs in tea plants.
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Camellia sinensis/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencias de Aminoácidos , Sitios de Unión , Frío , Secuencia Conservada , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/fisiología , Estrés Fisiológico , Factores de Transcripción/química , Factores de Transcripción/fisiologíaRESUMEN
Lonicera japonica Thunb. is a widely used medicinal plant and is rich in a variety of active ingredients. Flavonoids are one of the important components in L. japonica and their content is an important indicator for evaluating the quality of this herb. To study the regulation of flavonoid biosynthesis in L. japonica, an R2R3-MYB transcription factor gene LjaMYB12 was isolated and characterized. Bioinformatics analysis indicated that LjaMYB12 belonged to the subgroup 7, with a typical R2R3 DNA-binding domain and conserved subgroup 7 motifs. The transcriptional level of LjaMYB12 was proportional to the total flavonoid content during the development of L. japonica flowers. Subcellular localization analysis revealed that LjaMYB12 localized to the nucleus. Transactivation activity assay indicated that LjaMYB12 was a transcriptional activator. Then, ectopic expression of LjaMYB12 in Arabidopsis could increase PAL activity and flavonoid content and promote transcription of a range of flavonoid biosynthetic genes. Interestingly, the fold changes of downstream genes in the flavonoid biosynthetic pathway were significantly higher than that of the upstream genes, which suggested that LjaMYB12 may have different regulatory patterns for the upstream and downstream pathways of flavonoid biosynthesis. The results provided here will effectively facilitate the study of subgroup 7 MYBs and transcriptional regulation of flavonoid biosynthesis in L. japonica.
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Arabidopsis/genética , Arabidopsis/metabolismo , Flavonoides/metabolismo , Genes de Plantas , Lonicera/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Núcleo Celular/metabolismo , Flores/genética , Regulación de la Expresión Génica de las Plantas , Fenilanina Amoníaco-Liasa/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Activación TranscripcionalRESUMEN
We introduce SAR-by-Space, a concept to drastically accelerate structure-activity relationship (SAR) elucidation by synthesizing neighboring compounds that originate from vast chemical spaces. The space navigation is accomplished within minutes on affordable standard computer hardware using a tree-based molecule descriptor and dynamic programming. Maximizing the synthetic accessibility of the results from the computer is achieved by applying a careful selection of building blocks in combination with suitably chosen reactions; a decade of in-house quality control shows that this is a crucial part in the process. The REAL Space is the largest chemical space of commercially available compounds, counting 11 billion molecules as of today. It was used to mine actives against bromodomain 4 (BRD4). Before synthesis, compounds were docked into the binding site using a scoring function, which incorporates intrinsic desolvation terms, thus avoiding time-consuming simulations. Five micromolecular hits have been identified and verified within less than six weeks, including the measurement of IC50 values. We conclude that this procedure is a substantial time-saver, accelerating both ligand- and structure-based approaches in hit generation and lead optimization stages.
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Biología Computacional/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Sitios de Unión , Bases de Datos de Compuestos Químicos , Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
The MYB proteins represent a large family of transcription factors and play important roles in development, senescence, and stress responses in plants. In the current study, 233 MYB transcription factor-encoding genes were identified and analyzed in the potato genome, including 119 R1-MYB, 112 R2R3-MYB, and two R1R2R3-MYB members. R2R3-MYB is the most abundant MYB subclass and potato R2R3-MYB members together with their Arabidopsis homologs were divided into 35 well-supported subgroups as the result of phylogenetic analyses. Analyses on gene structure and protein motif revealed that members from the same subgroup shared similar exon/intron and motif organization, further supporting the results of phylogenetic analyses. Evolution of the potato MYB family was studied via syntenic analysis. Forty-one pairs of StMYB genes were predicted to have arisen from tandem or segmental duplication events, which played important roles in the expansion of the StMYB family. Expression profiling revealed that the StMYB genes were expressed in various tissues and several StMYB genes were identified to be induced by different stress conditions. Notably, StMYB030 was found to act as the homolog of AtMYB44 and was significantly up-regulated by salt and drought stress treatments. Furthermore, overexpression of StMYB030 in Arabidopsis enhanced salt stress tolerance of transgenic plants. The results from this study provided information for further functional analysis and for crop improvements through genetic manipulation of these StMYB genes.