RESUMEN
Polyphyllin VII is a biologically active herbal monomer extracted from the traditional Chinese herbal medicine Chonglou. Many studies have demonstrated the anticancer activity of polyphyllin VII against various types of cancers, such as colon, liver, and lung cancer, but its effect on breast cancer has not been elucidated. In this study, we demonstrate that polyphyllin VII inhibited proliferation, increased production of intracellular reactive oxygen species, and decreased mitochondrial membrane potential in breast cancer cells. Notably, polyphyllin VII also induced apoptosis via the mitochondrial pathway. Transcriptome sequencing was used to analyze the targets of PPVII in regulating breast cancer cells. Mechanistic studies showed that polyphyllin VII downregulated Son of Sevenless1 (SOS1) and inhibited the MAPK/ERK pathway. Furthermore, PPVII exerted strong antitumor effects in vivo in nude mice injected with breast cancer cells. Our results suggest that PPVII may promote apoptosis through regulating the SOS1/MAPK/ERK pathway, making it a possible candidate target for the treatment of breast cancer.
Asunto(s)
Apoptosis , Neoplasias de la Mama , Regulación hacia Abajo , Sistema de Señalización de MAP Quinasas , Proteína SOS1 , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Animales , Humanos , Femenino , Regulación hacia Abajo/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína SOS1/metabolismo , Proteína SOS1/genética , Ratones Desnudos , Saponinas/farmacología , Saponinas/uso terapéutico , Proliferación Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ratones , Línea Celular Tumoral , Medicamentos Herbarios Chinos/farmacología , Fitoterapia , Antineoplásicos Fitogénicos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones Endogámicos BALB CRESUMEN
Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.
Asunto(s)
Bombyx , Proteínas de Unión al ADN , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Bombyx/genética , Bombyx/metabolismo , Islas de CpG , Conformación Proteica en Lámina beta , Metilación de ADN , GenómicaRESUMEN
Monolithic Al2O3-SiO2 composite aerogels were synthesized by using inexpensive aluminum chloride hexahydrate (AlCl3·6H2O) and tetraethyl orthosilicate (TEOS). By adjusting the molar ratio of Al and Si, the best ratio of high-temperature resistance was found. The resultant aerogels (Al:Si = 9:1) exhibit high thermal performance, which can be identified by the low linear shrinkage of 5% and high specific surface area (SSA) of 283 m2/g at 1200 °C. Alumina in these aerogels mainly exists in the boehmite phase and gradually transforms into the θ-Al2O3 phase in the process of heating to 1200 °C. No α-Al2O3 is detected in the heating process. These Al2O3-SiO2 composite aerogels are derived from a simple, low-priced and safe method. With their high thermal performance, these aerogels will have a wide application in high-temperature field.
RESUMEN
DNA methylation and transcription factors play roles in gene expression and animal development. In insects, DNA methylation modifies gene bodies, but how DNA methylation and transcription factors regulate gene expression is unclear. In this study, we investigated the mechanism that regulates the expression of Bombyx mori Zinc finger protein 615 (ZnF 615), which is a downstream gene of DNA methyltransferase 1 (Dnmt1), and its effects on the regulation of embryonic development. By progressively truncating the ZnF 615 promoter, it was found that the -223 and -190 nt region, which contains homeobox (Hox) protein cis-regulatory elements (CREs), had the greatest impact on the transcription of ZnF 615. RNA interference (RNAi)-mediated knockdown and overexpression of Hox family genes showed that Hox A1-like can enhance the messenger RNA level of ZnF 615. Further studies showed that Hox A1-like regulates ZnF 615 expression by directly binding to the -223 and -190 nt region of its promoter. Simultaneous RNAi-mediated knockdown or overexpression of Hox A1-like and Dnmt1 significantly inhibited or enhanced the regulatory effect of either gene alone on ZnF 615 expression, suggesting that both DNA methylation of gene bodies and binding of transcription factors to promoters are essential for gene expression. RNAi-mediated knockdown of Hox A1-like and Dnmt1 showed that the embryonic development was retarded and the hatching rate was decreased. Taken together, these data suggest that Hox A1-like and DNA methylation enhance the expression of ZnF 615, thereby affecting the development of B. mori embryos.
Asunto(s)
Bombyx , Animales , Metilación de ADN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Desarrollo Embrionario/genética , Expresión Génica , Dedos de Zinc , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismoRESUMEN
Tetrazine-based bio-orthogonally activated fluorogenic probes have drawn great attention due to their excellent performance in bioimaging; however, most of them suffer from aggregation-caused quenching (ACQ) problems. Herein, we developed a set of novel tetrazine-modified tetraphenylenes (TPEs) as bio-orthogonally activated aggregation-induced emission (AIE) fluorogenic probes. Both the fluorescence and AIE features are quenched by tetrazine, which is mediated by the through-bond energy-transfer (TBET) mechanism, and are activated upon converting tetrazine to pyridazine via the inverse electron-demand Diels-Alder (iEDDA) reaction. The activated cycloadducts displayed a notable fluorescence enhancement, a large Stokes shift, a high fluorescence quantum yield, and evident AIE-active features. Manipulating the length and position of the π-linker enables fine-tuning of the photophysical properties of the probes, while an overlong planar π-linker leads to AIE-to-ACQ transformation. We also designed bi-tetrazyl-substituted probes, which exhibited a higher turn-on ratio than the mono-tetrazyl analogs owing to the 'double-quenched' function. When they reacted with double-clickable linkers, fluorescent macrocycles were obtained because of the restriction of the free rotation of the phenyl rings of TPE. Using an organelle-pretargeting strategy, we succeeded in applying these probes for mitochondria-specific bio-orthogonal imaging in live cells under no-wash conditions, which is expected to provide a powerful tool for biomedical applications.
Asunto(s)
Colorantes Fluorescentes , Compuestos Heterocíclicos , Colorantes Fluorescentes/química , Reacción de Cicloadición , Espectrometría de Fluorescencia , Compuestos Heterocíclicos/química , ElectronesRESUMEN
Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression. However, the mechanisms by which DNA methylation regulates these processes in insects remain unclear. Here, we studied the impacts of DNA methylation on early embryonic development in Bombyx mori. Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5' region of protein metabolism-related genes. The transcription factor gene zinc finger protein 615 ( ZnF615) was methylated by DNA methyltransferase 1 (Dnmt1) to be up-regulated and bind to protein metabolism-related genes. Dnmt1 RNA interference (RNAi) revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615. The same sites in the ZnF615 gene were methylated in ovaries and embryos. Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout. Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo. Thus, Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.
Asunto(s)
Bombyx , Animales , Bombyx/genética , Bombyx/metabolismo , Metilación de ADN , Desarrollo Embrionario/genética , Femenino , Factores de Transcripción/genética , Dedos de ZincRESUMEN
RNA interference (RNAi) is a technique used for posttranscriptional gene silencing, but lepidopteran insects are not sensitive to RNAi. Here, we present a protocol for knocking down the expression level of target genes by RNAi in Bombyx mori embryos. We describe the preparation of double-stranded RNAs (dsRNAs) of target genes, followed by microinjection of embryos at different developmental stages, with single or mixed dsRNA. Finally, we use RT-qPCR to verify RNAi efficiency. For complete details on the use and execution of this protocol, please refer to Xu et al. (2021).
Asunto(s)
Bombyx , Animales , Bombyx/genética , Insectos/genética , Interferencia de ARN , ARN Bicatenario/genéticaRESUMEN
We report a passively mode-locked Pr:LiYF4 (Pr:YLF) visible laser using a palladium diselenide (PdSe2) as a saturable absorber (SA) for the first time, to the best of our knowledge. The nonlinear optical properties of two-dimensional (2D) PdSe2 nanosheets in the visible band were studied by the open-aperture Z-scan technique. The results indicate the significant saturable absorption properties of PdSe2 nanosheets in the visible region. Furthermore, the continuous wave mode-locked (CWML) visible laser based on PdSe2 SA was successfully realized. Ultrashort pulses as short as 35 ps were obtained at 639.5â nm with a repetition rate of 80.3â MHz and a maximum output power of 116â mW. The corresponding pulse energy was 1.44 nJ and peak power was 41.3 W. These results indicate that 2D PdSe2 SA is a promising high stability passively mode-locked device for ultrafast solid-state visible lasers.
RESUMEN
A series of naphthalimide-tetrazines were developed as bioorthogonal fluorogenic probes, which could produce significant fluorescence enhancement, notable aggregation-induced emission (AIE) characters and multicolor emissions after bioorthogonal reaction with strained dienophiles. Manipulating the π-bridge in the fluorophore skeleton allows fine-tuning of the emission wavelength and influences the AIE-active properties. With these probes, we succeeded in no-wash fluorogenic protein labeling and mitochondria-selective bioorthogonal imaging in live cells.
Asunto(s)
Colorantes Fluorescentes/química , Naftalimidas/química , Tetrazoles/química , Células HeLa , Humanos , Microscopía Fluorescente/métodos , Mitocondrias/química , Mitocondrias/metabolismoRESUMEN
A smart fluorescence "turn-on" probe which contained a dansyl amide fluorophore and an N-oxide group was designed based on the bioorthogonal decaging reaction between N-oxide and the boron reagent. The reaction proceeds in a rapid kinetics (k2 =57.1±2.5â m-1 s-1 ), and the resulting reduction product showcases prominent fluorescence enhancement (up to 72-fold). Time dependent density functional theoretical (TD-DFT) calculation revealed that the process of photoinduced electron transfer (PET) from the N-oxide moiety to the dansyl amide fluorophore accounts for the quenching mechanism of N-oxide. This probe also showed high selectivity over various nucleophilic amino acids and good biocompatibility in physiological conditions. The successful application of the probe in HaloTag protein labeling and HepG2 live-cell imaging proves it a valuable tool for visualization of biomolecules.
RESUMEN
The third intracellular loop (ICL3) in the cytosolic face of glucagon receptor (GCGR) experiences significant conformational transition during receptor activation. It thus offers an attractive site for the introduction of organic fluorophores in our efforts to construct fluorescence-based GPCR biosensors. Herein, we report our confocal microscopic study of intracellular fluorescent labeling of ICL3 using a bioorthogonal chemistry strategy. Our approach involves the site-specific introduction of a strained alkene amino acid into the ICL3 through genetic code expansion, followed by a highly specific inverse electron-demand Diels-Alder reaction with the fluorescent tetrazine probes. Among the three strained alkene amino acids examined, both SphK and 2'-aTCOK offered successful fluorescent labeling of GCGR ICL3 with the appropriate tetrazine probes. At the same time, 4'-TCOK gave high background fluorescence due to its intracellular retention. The fluorescent tetrazine probes were designed following a computational model for background-free intracellular fluorescent labeling; however, their performance varied significantly in live-cell imaging as the strong non-specific signals interfered with the specific ones. Among all GCGR ICL3 mutants bearing a strained alkene, the H339SphK/2'-aTCOK mutants provided the best reaction partners for the BODIPY-Tz1/4 reagents in the bioorthogonal labeling reactions. The results from this study highlight the challenges in identifying bioorthogonal reactant pairs suitable for intracellular labeling of low-abundance receptors in live-cell imaging studies.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Compuestos Heterocíclicos con 1 Anillo/química , Receptores de Glucagón/química , Colorantes Fluorescentes/síntesis química , Células HEK293 , Humanos , Estructura MolecularRESUMEN
We reported a series of nitroso-modified naphthylene-based fluorophores as novel bioorthogonal fluorescence turn-on probes. The cycloadducts from nitroso-diene Diels-Alder reaction could be either photochemically or spontaneously transformed into highly fluorescent rearrangement products with remarkable photophysical properties including significant fluorescence enhancement, large Stokes shift, high fluorescence quantum yield, superior photostability, and distinct solvatochromic effect. This strategy is suitable for selective labeling of diene-modified proteins and visualizing specific organelles in live mammalian cells under no-wash conditions.
Asunto(s)
Colorantes Fluorescentes/química , Compuestos Nitrosos/química , Reacción de Cicloadición , Fluorescencia , Ionóforos/química , Estructura Molecular , Imagen ÓpticaRESUMEN
SYL927 and SYL930, two aminopropanediol analogues, are novel Sphingosine-1-phosphate receptor 1 (S1P1) modulators with higher selectivity and pharmacological activity compared with FTY720. Although the immunosuppressive activity of SYLs has been well demonstrated, information regarding the metabolic fates of the two chemicals is limited except for the CYP-catalyzed hydroxylation of SYL930. In this study, the biotransformation schemes of the two promising chemicals were investigated and compared using liver microsomes, S9 fractions and recombinant enzymes, and relevant molecular mechanism was primarily demonstrated by ligand-enzyme docking analysis (CDOCKER). As a result, the hydroxylation at alkyl chain on oxazole ring by the action of CYPs was found for both SYLs in vivo. The SULT-catalyzed sulfonation of the hydroxide was observed for SYL927 while the ADH/ALDH-catalyzed oxidation was only discovered for SYL930. The docking analysis suggested that specific non-covalent forces and/or bonding conformations of the hydroxides with biomacromolecules might be involved in the disparate metabolism of SYLs. Exploring the metabolic characteristics will help clarify the substance base for efficacy and safety of the two drugs. The uncovered structure-metabolism relationship in this study may provide an implication for the design and optimization for other S1P modulators.
Asunto(s)
Clorhidrato de Fingolimod , Receptores de Lisoesfingolípidos , Hidroxilación , Inmunosupresores/metabolismo , Microsomas Hepáticos/metabolismo , Receptores de Lisoesfingolípidos/metabolismoRESUMEN
Bioorthogonal chemistry has offered an invaluable reactivity-based tool to chemical biology owing to its exquisite specificity in tagging a diverse set of biomolecules in their native environment. Despite tremendous progress in the field over the past decade, designing a suitable bioorthogonal chemical probe to investigate a given biological system remains a challenge. In this Perspective, we put forward a series of fitness factors that can be used to assess the performance of bioorthogonal chemical probes. The consideration of these criteria should encourage continuous innovation in bioorthogonal probe development as well as enhance the quality of biological data.
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Sondas Moleculares/química , Permeabilidad de la Membrana Celular , Lípidos/química , Sondas Moleculares/toxicidad , Peso Molecular , Solubilidad , Agua/químicaRESUMEN
4-Oxime-1,8-naphthalimide was reported as a novel bioorthogonal turn-on probe based on 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxide and alkenes/alkynes. The resulting isoxazole products displayed dramatically strong fluorescence enhancement upon photoirradiation through isoxazole-oxazole photoisomerization. This new methodology was successfully applied for in situ fluorogenic protein labeling.
Asunto(s)
Colorantes Fluorescentes/química , Naftalimidas/química , Oximas/química , Proteínas/química , Reacción de Cicloadición , Coloración y EtiquetadoRESUMEN
A panel of three lipid-modified, functionalized biphenyl cross-linkers (fBph) were synthesized and subsequently employed in the preparation of the stapled oxyntomodulin (OXM) analogs. In a luciferase-based reporter assay, these stapled OXM analogs showed varying degree of potency in activating GLP-1R and GCGR, presumably due to the disparate effect of the lipid chains on the local environment close to the ligand-receptor binding interface. In particular, the fBph-1 cross-linked peptide with the lipid chain attached to position-3 of the biphenyl cross-linker exhibited the highest dual agonist activity.
RESUMEN
Photo-cross-linkers are invaluable tools for identifying drug targets and off-targets as well as probing the binding sites in medicinal chemistry and chemical biology. In this review, we summarize recent development of the ligand-based and genetically encoded photo-cross-linkers and their use in biological system. In particular, we highlight our discovery of 2-aryl-5-carboxytetrazole (ACT) as a novel class of photo-cross-linkers and their successful applications in drug target identification as well as identifying transient protein-protein interaction complexes in mammalian cells.
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Investigación Biomédica , Procesos Fotoquímicos , Proteínas/química , Sistemas de Liberación de Medicamentos , Humanos , Etiquetas de Fotoafinidad , Unión ProteicaRESUMEN
Three γ-heteroatom-substituted N-methylpyrroletetrazole-lysines (mPyTXKs) were synthesized and subsequently incorporated into proteins site-specifically via genetic code expansion. The γ-seleno-substituted derivative, mPyTSeK, showed excellent incorporation efficiency in Escherichia coli and allowed site-selective photo-cross-linking of the GST dimer. Furthermore, the mPyTSeK-cross-linked GST dimer can be cleaved under mild oxidative conditions. The incorporation of mPyTXKs into proteins in mammalian cells was also demonstrated. Lastly, the recombinantly expressed mPyTSeK-encoded Grb2 was shown to covalently capture its interaction partner, EGFR, in mammalian cell lysate, which was subsequently released after treatment with H2O2.
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Reactivos de Enlaces Cruzados/efectos de la radiación , Glutatión Transferasa/genética , Lisina/análogos & derivados , Lisina/genética , Ingeniería de Proteínas , Tetrazoles/efectos de la radiación , Animales , Reactivos de Enlaces Cruzados/síntesis química , Reactivos de Enlaces Cruzados/toxicidad , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/efectos de la radiación , Escherichia coli , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/efectos de la radiación , Glutatión Transferasa/química , Glutatión Transferasa/efectos de la radiación , Células HEK293 , Humanos , Peróxido de Hidrógeno/química , Lisina/efectos de la radiación , Lisina/toxicidad , Schistosoma japonicum , Tetrazoles/síntesis química , Tetrazoles/toxicidad , Rayos UltravioletaRESUMEN
The genetically encoded photo-cross-linkers promise to offer a temporally controlled tool to map transient and dynamic protein-protein interaction complexes in living cells. Here we report the synthesis of a panel of 2-aryl-5-carboxytetrazole-lysine analogs (ACTKs) and their site-specific incorporation into proteins via amber codon suppression in Escherichia coli and mammalian cells. Among five ACTKs investigated, N-methylpyrroletetrazole-lysine (mPyTK) was found to give robust and site-selective photo-cross-linking reactivity in E. coli when placed at an appropriate site at the protein interaction interface. A comparison study indicated that mPyTK exhibits higher photo-cross-linking efficiency than a diazirine-based photo-cross-linker, AbK, when placed at the same location of the interaction interface in vitro. When mPyTK was introduced into the adapter protein Grb2, it enabled the photocapture of EGFR in a stimulus-dependent manner. The design of mPyTK along with the identification of its cognate aminoacyl-tRNA synthetase makes it possible to map transient protein-protein interactions and their interfaces in living cells.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Reactivos de Enlaces Cruzados/química , Proteínas de Escherichia coli/química , Proteína Adaptadora GRB2/química , Código Genético/genética , Tetrazoles/química , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Proteínas de Escherichia coli/genética , Proteína Adaptadora GRB2/genética , Humanos , Lisina/química , Modelos Moleculares , Estructura Molecular , Procesos FotoquímicosRESUMEN
SYL927 and SYL930 are selective S1P1 agonists under preclinical development. However, during their pharmacokinetic studies we detected two metabolites in rat blood that were tentatively identified as monohydroxylated metabolites of SYL927 and SYL930 based on LC-MS/MS data. In this study, we designed and synthesized possible monohydroxylated products 6a-e and used them as references to confirm the structures of the two metabolites detected by LC-MS/MS. We also evaluated the in vitro and in vivo biological activities of these two metabolites.