RESUMO
Cephalotaxines harbor great medical potential, but their natural source, the endemic conifer Cephalotaxus is highly endangered, creating a conflict between biotechnological valorization and preservation of biodiversity. Here, we construct the whole biosynthetic pathway to the 1-phenethylisoquinoline scaffold, as first committed compound for phenylethylisoquinoline alkaloids (PIAs), combining metabolic modeling, and transcriptome mining of Cephalotaxus hainanensis to infer the biosynthesis for PIA precursor. We identify a novel protein, ChPSS, driving the Pictet-Spengler condensation and show that this enzyme represents the branching point where PIA biosynthesis diverges from the concurrent benzylisoquinoline-alkaloids pathway. We also pinpoint ChDBR as crucial step to form 4-hydroxydihydrocinnamaldehyde diverging from lignin biosynthesis. The elucidation of the early PIA pathway represents an important step toward microbe-based production of these pharmaceutically important alkaloids resolving the conflict between biotechnology and preservation of biodiversity.
Assuntos
Alcaloides , Benzilisoquinolinas , Cephalotaxus , Cephalotaxus/genética , BiotecnologiaRESUMO
Tetracyclic oxindole alkaloids (TOAs), main active ingredients of Uncaria rhynchophylla (UR), has inspired the interest of pharmacologists and chemists because of its great potential in the treatment of the diseases of the nervous system and cardiovascular system and its special spirooxindole scaffold, but the biosynthetic pathway of this compounds is still unknown. In this work, the metabolomics and transcriptomics of hook, leaf and stem of UR were analyzed, and 31 alkaloids and 47,423 unigenes were identified, as well as the relative contents of these alkaloids were evaluated. Based on the above results and literatures, a proposal biosynthetic pathway for TOAs was devised. Furthermore, three unigenes were suggested mediating the biosynthesis of TOAs through the integrated analysis of metabolomics and transcriptomics, and three enzymes, tryptophan decarboxylase, strictosidine synthase and strictosidine-ß-d-glucosidase, were identified as important catalytic enzymes for the synthesis of tryptamine, strictosidine (7) and 4,21-dehydrogeissochizine, respectively, which are considered as the important precursors of TOAs.
Assuntos
Alcaloides , Uncaria , Oxindóis , Alcaloides/metabolismo , Folhas de Planta/metabolismoRESUMO
Organocatalyzed reversible-deactivation radical polymerizations (RDRPs) are attractive for many applications. Here, we developed photoredox-mediated RDRP by activating (hetero)aryl sulfonyl chloride (ArSO2 Cl) initiators with pyridines and designing a novel bis(phenothiazine)arene catalyst. The in situ formed sulfonyl pyridinium intermediates effectively promote controlled chain-growth from ArSO2 Cl, enabling access to various well-defined polymers with high initiation efficiencies and controlled dispersities under mild conditions. This versatile method allows "ON/OFF" temporal control, chain-extension, facile synthesis of different polymer brushes via organocatalyzed grafting reactions from linear chains. Time-resolved fluorescence decay studies and calculations support the reaction mechanism. This work provides a transition-metal-free RDRP to tailor polymers with readily available aromatic initiators, and will promote the design of polymerization leveraged from photoredox catalysis.
RESUMO
A glycosyl transferase, isolated from Angelica decursiva a medical herb rich in coumarin, shows C-glycosyl transferase activity by in vitro activity assay using 5,7-dihydroxycoumarin as substrate, producing a C-glycosylated product at position C'8 along with the main product at C'6 position. Catalytic promiscuity assay shows that AdCGT also displays O- or C-glycosylation activity to other coumarins and flavonoids. When phloretin and 2,4,6-trihydroxyacetophenone were fed as substrates, AdCGT catalyzed the formation of di-C-glycosides. Therefore, AdCGT is a multifunctional glycosyltransferase with a broad substrate acceptability. This work highlights the potential of AdCGT as a catalyst for glycosylation of coumarin and reveals a new regio-selective C-glycosyltransferase, providing a basis for exploring the mechanism of coumarin glycosylation.
Assuntos
Angelica , Angelica/química , Angelica/metabolismo , Biocatálise , Cumarínicos/química , Glicosídeos , Glicosiltransferases/metabolismoRESUMO
S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.
Assuntos
Aspergillus flavus/enzimologia , Proteínas Fúngicas/química , Metiltransferases/química , S-Adenosil-Homocisteína/química , S-Adenosilmetionina/química , Sequência de Aminoácidos , Aspergillus flavus/química , Aspergillus flavus/classificação , Domínio Catalítico , Cristalografia por Raios X , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Ligação de Hidrogênio , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Peucedani Radix is the dry root of Peucedanum praeruptorum of the umbelliferous family, but the dry root of Angelica decursiva was also the source of Peucedani Radix in the past. As one of the most popular traditional Chinese medicinal herbs, the certified source of Peucedani Radix is still disputed. To better understand the relationship between A. decursiva and P. praeruptorum, we sequenced their chloroplast (cp) genomes. The gene structure, codon usage bias, repeat, simple sequence repeat (SSR), as well as their borders of inverted repeat (IR) regions of the two cp genomes are analyzed to identify potential genetic markers. Great variation is exhibited in the repeat sequences of IR, large single copy regions and the SSRs of the two cp genomes, which can be used as molecular markers to distinguish them. The phylogenetic analysis also indicates that they belong to two different genera in Apiaceae family: A. decursiva is an Angelica plant and P. praeruptorum is a Peucedanum plant. Our observations suggest that the two species are somewhere different in gene features, which contributes to support A. decursiva as an independent species from P. praeruptorum. The results also provide new evidence that A. decursiva should not be regarded as the certified source of Peucedani Radix in taxonomy. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01097-w.
RESUMO
Serotonin N-acetyltransferase (SNAT) is the key rate-limiting enzyme in melatonin biosynthesis. It mediates melatonin biosynthesis in plants by using serotonin and 5-methoxytryptamine (5-MT), but little is known of its underlying mechanisms. Herein, we present a detailed reaction mechanism of a SNAT from Oryza sativa through combined structural and molecular dynamics (MD) analysis. We report the crystal structures of plant SNAT in the apo and binary/ternary complex forms with acetyl-CoA (AcCoA), serotonin, and 5-MT. OsSNAT exhibits a unique enzymatically active dimeric fold not found in the known structures of arylalkylamine N-acetyltransferase (AANAT) family. The key residues W188, D189, D226, N220, and Y233 located around the active pocket are important in catalysis, confirmed by site-directed mutagenesis. Combined with MD simulations, we hypothesize a novel plausible catalytic mechanism in which D226 and Y233 function as catalytic base and acid during the acetyl-transfer reaction.
Assuntos
Arilalquilamina N-Acetiltransferase/química , Proteínas de Plantas/química , 5-Metoxitriptamina/química , 5-Metoxitriptamina/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Arilalquilamina N-Acetiltransferase/genética , Arilalquilamina N-Acetiltransferase/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Oryza/enzimologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Serotonina/química , Serotonina/metabolismoRESUMO
KEY MESSAGE: Psoralen synthase and angelicin synthase responsible for the formation of psoralen and angelicin in Peucedanum praeruptorum Dunn were identified and functionally characterized, respectively. Furanocoumarins were reported to possess several activities such as anticancer, anti-inflammatory and neuroprotective, and function as phytotoxin and allelochemical in plants. Furanocoumarins are the main bioactive ingredient in P. praeruptorum which is a commonly used traditional Chinese medicine. Phenylalanine ammonia lyase (PAL), 4-coumarate: CoA ligase (4CL), p-coumaroyl CoA 2'-hyfroxylase (C2'H) were cloned previously to elucidate the biosynthetic mechanism of coumarin lactone ring. However, the genes involved in complex coumarins in P. praeruptorum have not been explored. Herein, putative psoralen synthase CYP71AJ49 and angelicin synthase CYP71AJ51 were cloned from P. praeruptorum. In vivo and in vitro yeast assays were conducted to confirm their activities. Furthermore, the results of High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS) verified that CYP71AJ49 catalyzed the conversion of marmesin to psoralen, and CYP71AJ51 catalyzed columbianetin to angelicin. Subsequently, the expression profile showed that CYP71AJ49 and CYP71AJ51 were easily affected by environmental conditions, especially UV and temperature. The genes tissue-specific expression and compounds tissue-specific distribution pattern indicated the existence of substance transport in P. praeruptorum. Phylogenetic analysis was conducted with 27 CYP71AJs, CYP71AJ49 and CYP71AJ51 were classified in I-4 and I-2, respectively. These results provide further insight to understand the biosynthetic mechanism of complex coumarins.
Assuntos
Apiaceae/enzimologia , Apiaceae/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Furocumarinas/metabolismo , Proteínas de Plantas/metabolismo , Apiaceae/genética , China , Cromatografia Líquida de Alta Pressão/métodos , Coenzima A Ligases/genética , Cumarínicos/metabolismo , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Furocumarinas/química , Furocumarinas/genética , Regulação da Expressão Gênica de Plantas , Cinética , Medicina Tradicional Chinesa , Fenilalanina Amônia-Liase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray/métodos , TranscriptomaRESUMO
Fluoropolymers have found broad applications for many decades. Considerable efforts have focused on expanding access toward main-chain fluorinated polymers. In contrast to previous polymerizations of gaseous fluoroethylenes conducted at elevated temperatures and with high-pressure metallic vessels, we here report the development of a photoorganocatalyzed reversible-deactivation radical alternating copolymerization of chlorotrifluoroethylene (CTFE) and vinyl ethers (VEs) at room temperature and ambient pressure by exposing to LED light irradiation. This method enables the synthesis of various fluorinated alternating copolymers with low D and high chain-end fidelity, allowing an iterative switch of the copolymerization between "ON" and "OFF" states, the preparation of fluorinated block alternating copolymers, as well as postsynthetic modifications.
RESUMO
In hepatocellular carcinoma (HCC), dysregulated expression of DDX5 (DEAD box protein 5) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. Here we present evidence to show that, by interacting with autophagic receptor p62, DDX5 promotes autophagy and suppresses tumorigenesis. DDX5 inversely correlated with p62/sequestosome 1 (SQSTM1) expression in hepatitis B virus (HBV)-associated and non-HBV-associated HCCs. Patients with low DDX5 expression showed poor prognosis after tumor resection. We found that DDX5 overexpression induced, while DDX5 knockdown attenuated, autophagic flux in HepG2 and Huh7 cells. DDX5 promoted p62 degradation and markedly reduced the half-life of p62. Moreover, DDX5 overexpression dramatically reduced, while DDX5 knockdown promoted, cancer cell growth and tumorigenesis in vitro and in vivo. We found that DDX5 bound to p62 and interfered with p62/TRAF6 (tumor necrosis factor receptor-associated factor 6) interaction. Further findings revealed that the N-terminal domain of DDX5, involved in the interaction with p62, was sufficient to induce autophagy independent of its RNA binding and helicase activity. DDX5 overexpression decreased p62/TRAF6-mediated lysine 63-linked ubiquitination of mammalian target of rapamycin (mTOR) and subsequently inhibited the mTOR signaling pathway. Knockdown of TRAF6 blocked DDX5-induced autophagy. Furthermore, we showed that miR-17-5p downregulated DDX5 and impaired autophagy. Inhibition of miR-17-5p promoted autophagic flux and suppressed tumor growth in HCC xenograft models. Conclusion: Our findings define a noncanonical pathway that links miR-17-5p, DDX5, p62/TRAF6, autophagy, and HCC. These findings open an avenue for the treatment of HCC.
Assuntos
Autofagia/fisiologia , Carcinogênese , RNA Helicases DEAD-box/fisiologia , Proteína Sequestossoma-1/fisiologia , Humanos , Neoplasias Hepáticas , Células Tumorais CultivadasRESUMO
Telocytes are novel interstitial cells with a specific structure:the body has an elliptical shape or a triangle shape,with slender and thin protrusions that connect with other cells to form a complex 3D network.This article summarizes the structural characteristics and identification Methods of Telocytes and demonstrates their potential functions as a new target for disease prevention and treatment.
Assuntos
TelócitosRESUMO
Topology influences the properties and applications of polymers. Consequently, considerable efforts have been made to control topological structures. In this work, we have developed a photoorganocatalyzed divergent synthetic approach based on reversible-deactivation radical polymerization (RDRP) that enables the preparation of both linear and branched fluoropolymers of low dispersity (Ð), a tunable degree of branching and high chain-end fidelity by exposure to LED light irradiation under metal-free conditions. This method promotes the generation of complicated structures (e.g., necklace-like and mop-like fluoropolymers) via chain-extension photo-RDRP, and provides a novel and versatile platform to access fluoropolymer electrolytes with high Li-ion transference number and good ionic conductivity, which should create improved opportunities for advanced material engineering.
RESUMO
Ultra-high-molecular-weight (UHMW) polymers display outstanding properties and hold potential for wide applications. However, their precise synthesis remains challenging. Herein, we developed a novel reversible-deactivation radical polymerization based on the strong and selective fluorine-fluorine interaction, allowing chain-transfer agents to spontaneously differentiate into two groups that take charge of the chain growth and reversible deactivation of the growing chains, respectively. This method enables dramatically improved livingness of propagation, providing UHMW polymers with a surprisingly narrow molecular weight distribution (D≈1.1) from a variety of fluorinated (meth)acrylates and acrylamide at quantitative conversions under visible-light irradiation. Inâ situ chain-end extensions from UHMW polymers facilitated the synthesis of well-defined block copolymers, revealing the excellent chain-end fidelity achieved by this method.
RESUMO
Substance P (SP) is a candidate mediator along the brain-skin axis and can mimic the effects of stress to regulate melanogenesis. Previously, we and others have found that the regulation of SP for pigmentary function was mediated by neurokinin 1 receptor (NK1R). Emerging evidence has accumulated that psychologic stress can induce dysfunction in the cutaneous serotonin 5-hydroxytryptamine (5-HT)-5-HT1A/1B receptor system, thereby resulting in skin hypopigmentation. Moreover, NK1R and 5-HTR (except 5-HT3) belong to GPCR. The present study aimed at assessing the possible existence of NK1R-5-HTR interactions and related melanogenic functions. Western blot and PCR detection revealed that SP reduced expression of 5-HT1A receptor via the NK1 receptor. Biochemical analyses showed that NK1R and 5-HT1AR could colocalize and interact in a cell and in the skin. When the N terminus of the NK1R protein was removed NK1R surface targeting was prevented, the interaction between NK1R-5-HT1AR decreased, and the depigmentation caused by SP and WAY100635 could be rescued. Importantly, pharmaceutical coadministration of NK1R agonist (SP) and 5-HT1A antagonist (WAY100635) enhanced the NK1-5-HT1A receptor coimmunoprecipitation along with the depigmentary response. SP and WAY100635 cooperation elicited activation of a signaling cascade (the extracellular, regulated protein kinase p-JNK signaling pathway) and inhibition of p70S6K1 phosphorylation and greatly reduced melanin production in vitro and in vivo in mice and zebrafish. Moreover, the SP-induced depigmentation response did not be occur in 5-htr1aa+/- zebrafish embryos. Taken together, the results of our systemic study increases our knowledge of the roles of NK1R and 5-HT1AR in melanogenesis and provides possible, novel therapeutic strategies for treatment of skin hypo/hyperpigmentation.-Wu, H., Zhao, Y., Huang, Q., Cai, M., Pan, Q., Fu, M., An, X., Xia, Z., Liu, M., Jin, Y., He, L., Shang, J. NK1R/5-HT1AR interaction is related to the regulation of melanogenesis.
Assuntos
Melaninas/biossíntese , Receptor 5-HT1A de Serotonina/metabolismo , Receptores da Neurocinina-1/metabolismo , Pigmentação da Pele , Pele/metabolismo , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Melaninas/genética , Camundongos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Receptor 5-HT1A de Serotonina/genética , Receptores da Neurocinina-1/genética , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Pele/patologia , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Substância P/metabolismo , Substância P/farmacologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
The objective of this study is to identify individual-level factors and health venue utilization patterns associated with uptake of pre-exposure prophylaxis (PrEP) and to evaluate whether PrEP uptake behavior is further diffused among young men who have sex with men (YMSM) through health venue referral networks. A sample of 543 HIV-seronegative YMSM aged 16-29 were recruited in 2014-2016 in Chicago, IL, and Houston, TX. Stochastic social network models were estimated to model PrEP uptake. PrEP uptake was associated with more utilization of health venues in Houston and higher levels of sexual risk behavior in Chicago. In Houston, both Hispanic and Black YMSM compared to White YMSM were less likely to take PrEP. No evidence was found to support the spread of PrEP uptake via referral networks, which highlights the need for more effective PrEP referral network systems to scale up PrEP implementation among at-risk YMSM.
Assuntos
Infecções por HIV/prevenção & controle , Homossexualidade Masculina/estatística & dados numéricos , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Profilaxia Pré-Exposição , Adolescente , Adulto , Conhecimentos, Atitudes e Prática em Saúde , Inquéritos Epidemiológicos , Humanos , Masculino , Profilaxia Pré-Exposição/estatística & dados numéricos , Encaminhamento e Consulta , Estados Unidos , Adulto JovemRESUMO
2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) are two typical polybrominated diphenyl ethers (PBDEs), and studies have proven that these PBDs can disrupt the behaviors and physical function of aquatic organisms. However, little is known about the compositional impacts of BDE-47/BDE-99 compound pollution on the feeding behavior of Daphnia magna. In this study, a response surface methodology (RSM) was introduced into the combined toxicity assessment of BDE-47 and BDE-99 on the feeding depression of D. magna. Low concentrations of BDE-47 (9.2⯵g/L) and BDE-99 (5.4⯵g/L) had no effect on the feeding behavior of D. magna; nevertheless, the feeding depression was strengthened, and a concentration-dependent effect was observed with increasing concentrations of BDE-47 and BDE-99. The results of RSM indicated that the mixture of BDE-47 and BDE-99 can enhance their toxicity on the feeding behavior of D. magna. Moreover, real-time PCR (qPCR) analysis showed that the down-regulation of α-amylase (AMS) appeared in most of the exposed D. magna. However, there were significant different in the gene expression of trypsin, superoxide dismutase (SOD) and catalase (CAT) between the exposure and control groups. The change in the enzyme activity of AMS, trypsin, SOD and CAT implied that BDE-47 and BDE-99 cause damage to the digestive and antioxidative systems of D. magna. Correlation analysis indicated that a significant positive correlation existed between the gene expression and enzyme activity of SOD and CAT. Our results contribute to the understanding of toxicity caused by BDE-47/BDE-99 compound pollution in D. magna and help to improve traditional toxicity assessment methods for aquatic environments.
Assuntos
Antioxidantes/metabolismo , Daphnia/efeitos dos fármacos , Digestão/efeitos dos fármacos , Comportamento Alimentar/efeitos dos fármacos , Éteres Difenil Halogenados/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Catalase/genética , Daphnia/enzimologia , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Superóxido Dismutase/genéticaRESUMO
Methoxylated coumarins represent a large proportion of officinal value coumarins while only one enzyme specific to bergaptol O-methylation (BMT) has been identified to date. The multiple types of methoxylated coumarins indicate that at least one unknown enzyme participates in the O-methylation of other hydroxylated coumarins and remains to be identified. Combined transcriptome and metabonomics analysis revealed that an enzyme similar to caffeic acid O-methyltransferase (COMT-S, S is short for similar) was involved in catalyzing all the hydroxylated coumarins in Peucedanum praeruptorum. However, the precise molecular mechanism of its substrate heterozygosis remains unsolved. Pursuing this question, we determined the crystal structure of COMT-S to clarify its substrate preference. The result revealed that Asn132, Asp271, and Asn325 govern the substrate heterozygosis of COMT-S. A single mutation, such as N132A, determines the catalytic selectivity of hydroxyl groups in esculetin and also causes production differences in bergapten. Evolution-based analysis indicated that BMT was only recently derived as a paralogue of caffeic acid O-methyltransferase (COMT) via gene duplication, occurring before the Apiaceae family divergence between 37 and 100 mya. The present study identified the previously unknown O-methylation steps in coumarin biosynthesis. The crystallographic and mutational studies provided a deeper understanding of the substrate preference, which can be used for producing specific O-methylation coumarins. Moreover, the evolutionary relationship between BMT and COMT-S was clarified to facilitate understanding of evolutionary events in the Apiaceae family.
Assuntos
Apiaceae/metabolismo , Vias Biossintéticas , Cumarínicos/metabolismo , Sequência de Aminoácidos , Apiaceae/química , Apiaceae/genética , Cumarínicos/química , Mineração de Dados , Evolução Molecular , Furocumarinas/química , Furocumarinas/metabolismo , Duplicação Gênica , Heterozigoto , Metilação , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , S-Adenosil-Homocisteína/química , S-Adenosil-Homocisteína/metabolismo , Análise de Sequência de RNA , Especificidade por Substrato , Transcriptoma/genética , Umbeliferonas/química , Umbeliferonas/metabolismoRESUMO
Fluorinated polymers are important materials that are widely used in many areas. Herein, we report the development of a metal-free photocontrolled radical polymerization of semifluorinated (meth)acrylates with a new visible-light-absorbing organocatalyst. This method enabled the production of a variety of semifluorinated polymers with narrow molar-weight distributions from semifluorinated trithiocarbonates or perfluoroalkyl iodides. The high performance of "ON/OFF" control and chain-extension experiments further demonstrate the utility and reliability of this method. Furthermore, to streamline the preparation of semifluorinated polymers, a scalable continuous-flow approach has been developed. Given the broad interest in fluorinated materials and photopolymerization, we expect that this method will facilitate the development of advanced materials with unique properties.
RESUMO
KEY MESSAGE: A p-coumaroyl CoA 2'-hydroxylase responsible for the formation of coumarin lactone ring was identified from Peucedanum praeruptorum Dunn and functionally characterized in vitro. Coumarins are important plant secondary metabolites with a variety of biological activities. Ortho-hydroxylation of cinnamates leads to the formation of coumarin lactone ring and is generally thought to be a key step in coumarin biosynthesis. However, ortho-hydroxylases, especially p-coumaroyl CoA 2'-hydroxylase (C2'H) responsible for the biosynthesis of the most common coumarin skeleton, have received insufficient attention. Here, a putative ortho-hydroxylase PpC2'H was isolated from P. praeruptorum Dunn, a traditional Chinese medicinal herb rich in coumarins. Expression profile indicated that PpC2'H exhibited the highest transcript level in roots and could be up-regulated by MeJA elicitation. Subcellular localization of PpC2'H was demonstrated to be cytosol in planta. In order to functionally characterize PpC2'H, the purified recombinant protein was incubated with various potential substrates. HPLC-ESI-MS analysis indicated that PpC2'H catalyzed the conversion of p-coumaroyl CoA into hydroxylated intermediate, which then underwent spontaneous lactonization to generate umbelliferone. Our data also showed that light would promote the spontaneous process. In addition, based on homology modeling and site-directed mutagenesis, amino acid residues Phe-130, Lys-141, Asn-207, His-224, Asp-226, His-282 and Phe-298 were verified essential for enzymatic activity. These findings provide insight into structure-function relationship of this pivotal ortho-hydroxylase and also contribute to elucidating the biosynthetic mechanism of coumarin skeleton.
Assuntos
Apiaceae/enzimologia , Vias Biossintéticas , Cumarínicos/metabolismo , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas/genética , Cromatografia Líquida de Alta Pressão , Cumarínicos/química , DNA Complementar/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Cinética , Luz , Oxigenases de Função Mista/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade de Órgãos/genética , Especificidade de Órgãos/efeitos da radiação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Homologia Estrutural de Proteína , Frações Subcelulares/enzimologia , Transcriptoma/genética , Transcriptoma/efeitos da radiaçãoRESUMO
Polymer conjugation is an attractive approach for delivering insoluble and highly toxic drugs to tumors. However, most reports in the literature only disclose the optimal composition without emphasizing rational design or composition optimization to achieve maximized biological effects. In this study, we aimed to demonstrate that composition of a polymer conjugate would determine its physiochemical characteristics, tumor penetration, and, ultimately, the in vivo efficacy. We also aimed to examine whether the tumor spheroid model could generate comparable results with the in vivo tumor model in terms of tumor penetration and efficacy of the various polymer conjugates. We have designed a polymer conjugate delivery system for a chemotherapeutic drug podophyllotoxin (PPT) by covalently conjugating PPT and polyethylene glycol (PEG) with acetylated carboxymethyl cellulose to yield conjugates containing various amounts of PPT and PEG. Depending on the composition, these conjugates self-assembled into nanoparticles (NPs) with different physicochemical properties. Conjugates with an increased PPT content formed particles with an increased diameter. In the present study, we selected three conjugates representing compositions containing high, medium, and low drug content, and compared their particle formation, drug release kinetics, their ability to penetrate tumor spheroid and in vivo s.c. tumor, and finally their antitumor efficacy in spheroid culture and an in vivo s.c. tumor model. We found that the low drug content conjugate formed smaller NPs (20 nm) compared to the high drug content conjugates (30-120 nm), and displayed faster drug release kinetics (5%/day vs 1-3%/day), improved tumor penetration, and enhanced antitumor efficacy in both the spheroid model and s.c. tumor model. In particular, the low drug content conjugate preferentially accumulated in the hypovascular region within the tumor, inducing complete regression of s.c. tumors and the metastasis to the lungs. Our data indicate composition optimization is needed to select the optimal conjugate, and tumor spheroid culture is a robust screening tool to help select the optimal formulation.