RESUMO
Uridine 5'-diphospho-glulcuronosyltransferase 2B17 (UGT2B17) is important in the metabolism of steroids and orally administered drugs due to its high interindividual variability. However, the structural basis governing the substrate selectivity or inhibition of UGT2B17 remains poorly understood. This study investigated 76 FDA-approved drugs and 20 steroids known to undergo glucuronidation for their metabolism by UGT2B17. Specifically, we assessed the substrate selectivity for UGT2B17 over other UGT enzymes using recombinant human UGT2B17 (rUGT2B17), human intestinal microsomes, and human liver microsomes. The quantitative contribution of intestinal UGT2B17 in the glucuronidation of these compounds was characterized using intestinal microsomes isolated from UGT2B17 expressors and nonexpressors. In addition, a structure-based pharmacophore model for UGT2B17 substrates was built and validated using the studied pool of substrates and nonsubstrates. The results show that UGT2B17 could metabolize 23 out of 96 compounds from various chemical classes, including alcohols and carboxylic acids, particularly in the intestine. Interestingly, amines were less susceptible to UGT2B17 metabolism, though they could inhibit the enzyme. Three main pharmacophoric features of UGT2B17 substrates include (1) the presence of an accessible -OH or -COOH group near His35 residue, (2) a hydrophobic functional group at â¼4.5-5 Å from feature 1, and (3) an aromatic ring â¼5-7 Å from feature 2. Most of the studied compounds inhibited UGT2B17 activity irrespective of their substrate potential, indicating the possibility of multiple mechanisms. These data suggest that UGT2B17 is promiscuous in substrate selectivity and inhibition and has a high potential to produce significant variability in the absorption and disposition of orally administered drugs.
Assuntos
Glucuronosiltransferase , Esteroides , Humanos , Glucuronosiltransferase/metabolismo , Uridina , Antígenos de Histocompatibilidade Menor/metabolismoRESUMO
Aphids are sap-sucking insects responsible for crop losses and a severe threat to crop production. Proteins in the aphid saliva are integral in establishing an interaction between aphids and plants and are responsible for host plant adaptation. The cotton aphid, Aphis gossypii (Hemiptera: Aphididae) is a major pest of Gossypium hirsutum. Despite extensive studies of the salivary proteins of various aphid species, the components of A. gossypii salivary glands are unknown. In this study, we identified 123,008 transcripts from the salivary gland of A. gossypii. Among those, 2933 proteins have signal peptides with no transmembrane domain known to be secreted from the cell upon feeding. The transcriptome includes proteins with more comprehensive functions such as digestion, detoxification, regulating host defenses, regulation of salivary glands, and a large set of uncharacterized proteins. Comparative analysis of salivary proteins of different aphids and other insects with A. gossypii revealed that 183 and 88 orthologous clusters were common in the Aphididae and non-Aphididae groups, respectively. The structure prediction for highly expressed salivary proteins indicated that most possess an intrinsically disordered region. These results provide valuable reference data for exploring novel functions of salivary proteins in A. gossypii with their host interactions. The identified proteins may help develop a sustainable way to manage aphid pests.
Assuntos
Afídeos , Proteínas de Insetos , Glândulas Salivares , Transcriptoma , Animais , Afídeos/genética , Afídeos/metabolismo , Glândulas Salivares/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas de Insetos/química , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismo , Gossypium/genética , Gossypium/metabolismo , Perfilação da Expressão GênicaRESUMO
Glory lily (Gloriosa superba), an ornamental climbing plant, contains the bioactive compound colchicine, attracting attention from the pharmaceutical industry. However, soil-borne pathogens have emerged as a serious threat to the cultivation of glory lily, leading to substantial economic losses in the southern parts of India. Among these, the three major pathogens are Macrophomina phaseolina, Fusarium oxysporum, and Agroathelia rolfsii, causing dry root rot (also referred to as charcoal rot), wilt, and stem rot, respectively. Here, we characterised these pathogens using morphological characteristics and phylogenetic analysis of DNA sequences related to the internal transcribed spacer (ITS) of ribosomal DNA, calmodulin (CAL) and translation elongation factor (TEF)-1α. Further, in the pathogenicity tests, the inoculation of M. phaseolina alone resulted in lesions measuring 7.54±0.01 mm on tubers and 90% seedling mortality. This severity was comparable to the simultaneous inoculation of all three pathogens, indicating the prominence of dry root rot among soil-borne diseases. This study marks the first detailed investigation of soil-borne pathogens combined infection in G. superba, contributing to the understanding of fungal disease complexity in medicinal plants.
RESUMO
In mammalian cells, all-trans farnesol, a 15-carbon isoprenol, is a product of the mevalonate pathway. It is the natural substrate of alcohol dehydrogenase and a substrate for CYP2E1, two enzymes implicated in ethanol metabolism. Studies have shown that farnesol is present in the human brain and inhibits voltage-gated Ca2+ channels at much lower concentrations than ethanol. Here we show that farnesol modulates the activity of γ-aminobutyric acid type A receptors (GABAARs), some of which also mediate the sedative activity of ethanol. Electrophysiology experiments performed in HEK cells expressing human α1ß3γ2 or α6ß3γ2 GABAARs revealed that farnesol increased chloride currents through positive allosteric modulation of these receptors and showed dependence on both the alcoholic functional group of farnesol and the length of the alkyl chain for activity. In silico studies using long-timescale unbiased all-atom molecular dynamics (MD) simulations of the human α1ß3γ2 GABAA receptors revealed that farnesol modulates the channel by directly binding to the transmembrane neurosteroid-binding site, after partitioning into the surrounding membrane and reaching the receptor by lateral diffusion. Channel activation by farnesol was further characterized by several structural and dynamic variables, such as global twisting of the receptor's extracellular domain, tilting of the transmembrane M2 helices, radius, cross-sectional area, hydration status, and electrostatic potential of the channel pore. Our results expand the pharmacological activities of farnesol to yet another class of ion channels implicated in neurotransmission, thus providing a novel path for understanding and treatment of diseases involving GABAA receptor dysfunction.
Assuntos
Neuroesteroides , Receptores de GABA-A , Humanos , Sítios de Ligação , Farneseno Álcool/farmacologia , Ácido gama-Aminobutírico/farmacologia , Domínios Proteicos , Receptores de GABA-A/metabolismoRESUMO
The drugs salmeterol, formoterol, and salbutamol constitute the frontline treatment of asthma and other chronic pulmonary diseases. These drugs activate the ß2-adrenergic receptors (ß2-AR), a class A G protein-coupled receptor (GPCR), and differ significantly in their clinical onset and duration of actions. According to the microkinetic model, the long duration of action of salmeterol and formoterol compared with salbutamol were attributed, at least in part, to their high lipophilicity and increased local concentrations in the membrane near the receptor. However, the structural and molecular bases of how the lipophilic drugs reach the binding site of the receptor from the surrounding membrane remain unknown. Using a variety of classic and enhanced molecular dynamics simulation techniques, we investigated the membrane partitioning characteristics, binding, and unbinding mechanisms of the ligands. The obtained results offer remarkable insight into the functional role of membrane lipids in the ligand association process. Strikingly, salmeterol entered the binding site from the bilayer through transmembrane helices 1 and 7. The entry was preceded by membrane-facilitated rearrangement and presentation of its phenyl-alkoxy-alkyl tail as a passkey to an access route gated by F193, a residue known to be critical for salmeterol's affinity. Formoterol's access is through the aqueous path shared by other ß2-AR agents. We observed a novel secondary path for salbutamol that is distinct from its primary route. Our study offers a mechanistic description for the membrane-facilitated access and binding of ligands to a membrane protein and establishes a groundwork for recognizing membrane lipids as an integral component in the molecular recognition process. SIGNIFICANCE STATEMENT: The cell membrane's functional role behind the duration of action of long-acting ß2-adrenergic receptor (ß2-AR) agonists such as salmeterol has been a subject of debate for a long time. This study investigated the binding and unbinding mechanisms of the three commonly used ß2-AR agonists, salmeterol, formoterol, and salbutamol, using advanced simulation techniques. The obtained results offer unprecedented insights into the active role of membrane lipids in facilitating access and binding of the ligands, affecting the molecular recognition process and thus their pharmacology.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Membrana Celular/metabolismo , Simulação de Acoplamento Molecular/métodos , Albuterol/química , Albuterol/metabolismo , Sítios de Ligação/fisiologia , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Fumarato de Formoterol/química , Fumarato de Formoterol/metabolismo , Humanos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Xinafoato de Salmeterol/química , Xinafoato de Salmeterol/metabolismoRESUMO
Deficiency of succinate semialdehyde dehydrogenase (SSADH; aldehyde dehydrogenase 5a1 (ALDH5A1), OMIM 271980, 610045), the second enzyme of GABA degradation, represents a rare autosomal-recessively inherited disorder which manifests metabolically as gamma-hydroxybutyric aciduria. The neurological phenotype includes intellectual disability, autism spectrum, epilepsy and sleep and behavior disturbances. Approximately 70 variants have been reported in the ALDH5A1 gene, half of them being missense variants. In this study, 34 missense variants, of which 22 novel, were evaluated by in silico analyses using PolyPhen2 and SIFT prediction tools. Subsequently, the effect of these variants on SSADH activity was studied by transient overexpression in HEK293 cells. These studies showed severe enzymatic activity impairment for 27 out of 34 alleles, normal activity for one allele and a broad range of residual activities (25 to 74%) for six alleles. To better evaluate the alleles that showed residual activity above 25%, we generated an SSADH-deficient HEK293-Flp-In cell line using CRISPR-Cas9, in which these alleles were stably expressed. This model proved essential in the classification as deficient for one out of the seven studied alleles. For 8 out of 34 addressed alleles, there were discrepant results among the used prediction tools, and/or in correlating the results of the prediction tools with the functional data. In case of diagnostic urgency of missense alleles, we propose the use of the transient transfection model for confirmation of their effect on the SSADH catalytic function, since this model resulted in fast and robust functional characterization for the majority of the tested variants. In selected cases, stable transfections can be considered and may prove valuable.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Deficiências do Desenvolvimento/patologia , Mutação de Sentido Incorreto , Succinato-Semialdeído Desidrogenase/deficiência , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Simulação por Computador , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Células HEK293 , Humanos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismoRESUMO
Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.
Assuntos
Acroleína/análogos & derivados , Cinnamomum zeylanicum/química , Citocromo P-450 CYP2A6/antagonistas & inibidores , Interações Ervas-Drogas , Acroleína/química , Acroleína/farmacologia , Área Sob a Curva , Citocromo P-450 CYP2A6/isolamento & purificação , Citocromo P-450 CYP2A6/metabolismo , Citocromo P-450 CYP2A6/ultraestrutura , Avaliação Pré-Clínica de Medicamentos , Humanos , Letrozol/farmacocinética , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Nicotina/farmacocinética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Resolvins D1 and D2 (RvDs) are structural isomers and metabolites of docosahexaenoic acid, an omega-3 fatty acid, enzymatically produced in our body in response to acute inflammation or microbial invasion. Resolvins have been shown to play an essential role in the resolution of inflammation, tissue repair, and return to homeostasis and thus are actively pursued as potential therapeutics in treating inflammatory disorders and infectious diseases. However, effective in vivo delivery of RvDs continues to be a challenging task. Recent studies demonstrated that RvD1 or RvD2 loaded in cell membrane-derived nanovesicles significantly increased therapeutic efficacy in treating murine peritonitis and ischemic stroke, respectively. The mechanistic details of how the subtle structural difference between RvD1 and RvD2 alters their molecular interactions with the membrane lipids of the nanovesicles and thus affects the loading efficiency remain unknown. Here, we report the encapsulation profiles of the neutral and ionized species of both RvD1 and RvD2 determined with the cell membrane-derived nanovesicles at pH values 5.4 and 7.4, respectively. Also, we performed microsecond time-scale all-atom molecular dynamics (MD) simulations in explicit water to elucidate the molecular interactions of both neutral and ionized species of RvD1 and RvD2 with the lipid bilayer using a model membrane system, containing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. We found that the differences in the position and chirality of hydroxyl groups in RvD1 and RvD2 affected their location, orientation, and conformations within the bilayer. Surprisingly, the deprotonation of their carboxyl group caused their orientation and conformation to change from a fully extended one that is oriented in parallel to the membrane plane to a J-shaped bent conformation that is oriented perpendicular to the bilayer plane. Our studies offer valuable insight into the molecular interactions of RvD1/D2 with the lipid bilayer in atomistic details and provide a mechanistic explanation for the observed differences in the encapsulation profiles of RvD1 and RvD2, which may facilitate the rational design of nanovesicle-based therapeutics for treating inflammatory diseases.
Assuntos
Ácidos Docosa-Hexaenoicos/química , Simulação de Dinâmica Molecular , Colesterol/química , Bicamadas Lipídicas/química , Nanopartículas/química , Nanotecnologia/métodos , Fosfatidilcolinas/químicaRESUMO
Maize is the predominant food source for the world population, but lack of lysine and tryptophan in maize endosperm cannot fulfill the nutritional requirements of humans. Hence, the improvement of lysine and tryptophan content is the ultimate goal of maize biofortification programs. In the present study, the marker-assisted backcross (MABC) breeding strategy was used to enhance the lysine and tryptophan content of the elite maize inbred line UMI1230 by introgressing opaque 2 (o2) gene from the VQL1. During the transfer of the gene into UMI1230, SSR marker umc1066 tightly associated with o2 used for foreground selection. Background recovery was estimated using 168 SSR markers. Phenotype screening for morphological traits was adopted to choose plants parallel to UMI1230. As a result, four BC2F3 improved lines (DBT5-1-14/25-5/25-8/25-8/25, DBT5-1-14/25-5/25-8/25-7/25, DBT5-1-14/25-5/25-8/25-10/25 and DBT5-1-14/25-5/25-8/25-12/25) with o2 were developed. The improved line's background genome recovery varied between 90.60 and 94.80%. Also, the improved lines had better agronomic performance along with increased lysine (0.311-0.331%) and tryptophan (0.040-0.048%) contents. In summary, the MABC breeding strategy has successfully improved the levels of lysine and tryptophan in UMI1230 without affecting agronomic performance. The improved line's hold great potential as donors in biofortification programs in maize.
RESUMO
The ligand-binding sites of many G protein-coupled receptors (GPCRs) are situated around and deeply embedded within the central pocket formed by their seven transmembrane-spanning α-helical domains. Generally, these binding sites are assumed accessible to endogenous ligands from the aqueous phase. Recent advances in the structural biology of GPCRs, along with biophysical and computational studies, suggest that amphiphilic and lipophilic molecules may gain access to these receptors by first partitioning into the membrane and then reaching the binding site via lateral diffusion through the lipid bilayer. In addition, several crystal structures of class A and class B GPCRs bound to their ligands offer unprecedented details on the existence of lipid-facing allosteric binding sites outside the transmembrane helices that can only be reached via lipid pathways. The highly organized structure of the lipid bilayer may direct lipophilic or amphiphilic drugs to a specific depth within the bilayer, changing local concentration of the drug near the binding site and affecting its binding kinetics. Additionally, the constraints of the lipid bilayer, including its composition and biophysical properties, may play a critical role in "pre-organizing" ligand molecules in an optimal orientation and conformation to facilitate receptor binding. Despite its clear involvement in molecular recognition processes, the critical role of the membrane in binding ligands to lipid-exposed transmembrane binding sites remains poorly understood and warrants comprehensive investigation. Understanding the mechanistic basis of the structure-membrane interaction relationship of drugs will not only provide useful insights about receptor binding kinetics but will also enhance our ability to take advantage of the apparent membrane contributions when designing drugs that target transmembrane proteins with improved efficacy and safety. In this minireview, we summarize recent structural and computational studies on membrane contributions to binding processes, elucidating both lipid pathways of ligand access and binding mechanisms for several orthosteric and allosteric ligands of class A and class B GPCRs.
Assuntos
Sítio Alostérico/fisiologia , Ligantes , Bicamadas Lipídicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Sítios de Ligação/fisiologia , Humanos , Bicamadas Lipídicas/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/químicaRESUMO
Aberrant access to genetic information disrupts cellular homeostasis and can lead to cancer development. One molecular mechanism that regulates access to genetic information includes recognition of histone modifications, which is carried out by protein modules that interact with chromatin and serve as landing pads for enzymatic activities that regulate gene expression. The ING3 tumor suppressor protein contains a plant homeodomain (PHD) that reads the epigenetic code via recognition of histone H3 tri-methylated at lysine 4 (H3K4me3), and this domain is lost or mutated in various human cancers. However, the molecular mechanisms targeting ING3 to histones and the role of this interaction in the cell remain elusive. Thus, we employed biochemical and structural biology approaches to investigate the interaction of the ING3 PHD finger (ING3PHD) with the active transcription mark H3K4me3. Our results demonstrate that association of the ING3PHD with H3K4me3 is in the sub-micromolar range (KD ranging between 0.63 and 0.93 µm) and is about 200-fold stronger than with the unmodified histone H3. NMR and computational studies revealed an aromatic cage composed of Tyr-362, Ser-369, and Trp-385 that accommodate the tri-methylated side chain of H3K4. Mutational analysis confirmed the critical importance of Tyr-362 and Trp-385 in mediating the ING3PHD-H3K4me3 interaction. Finally, the biological relevance of ING3PHD-H3K4me3 binding was demonstrated by the failure of ING3PHD mutant proteins to enhance ING3-mediated DNA damage-dependent cell death. Together, our results reveal the molecular mechanism of H3K4me3 selection by the ING3PHD and suggest that this interaction is important for mediating ING3 tumor suppressive activities.
Assuntos
Histonas/química , Proteínas de Homeodomínio/química , Proteínas Supressoras de Tumor/química , Substituição de Aminoácidos , Morte Celular , Dano ao DNA , Epigênese Genética , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metilação , Mutação de Sentido Incorreto , Ressonância Magnética Nuclear Biomolecular , Domínios RING Finger , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Background: Optimization of the antibiotics for patients with infections due to MDR Pseudomonas aeruginosa (MDR-PA) often requires consideration of alternate dose and infusion times that can be influenced by renal function. Objectives: We sought to identify ceftolozane/tazobactam dosing schemes that optimized the probability of target attainment (PTA) against infections due to MDR-PA with ceftolozane/tazobactam MICs between 4 and 32 mg/L across different categories of renal function. Methods: A prior validated ceftolozane/tazobactam population pharmacokinetic model was used for Monte Carlo simulation of 128 alternate permutations of dose, infusion time and renal function in 5000 cases/permutation. Four ceftolozane/tazobactam doses (250/125 mg to 2/1 g) every 8 h with infusion durations of 1-7 h and as continuous infusions were simulated. The model simulated ceftolozane/tazobactam clearance as a function of creatinine clearance (CLCR) within four categories of estimated renal function: 15-29, 30-50, 51-120 and 121-180 mL/min. The PTA was benchmarked on 40% free ceftolozane/tazobactam concentration time above the MIC. Results: The 512 alternate scenarios identified the current ceftolozane/tazobactam dose of 1/0.5 g to be optimal for MICs ≤32 mg/L (CLCR 15-50 mL/min), ≤16 mg/L (CLCR 51-120 mL/min) and ≤8 mg/L (CLCR 121-180 mL/min). Extended infusion of 4-5 h had a higher PTA than shorter and continuous infusions in simulations of augmented renal clearance across infections with MICs of 4-32 mg/L. Conclusions: Extended infusion ceftolozane/tazobactam regimens should be investigated as a potential dosing solution to improve the PTA against infections due to MDR-PA with higher ceftolozane/tazobactam MICs.
Assuntos
Antibacterianos/administração & dosagem , Cefalosporinas/administração & dosagem , Ácido Penicilânico/análogos & derivados , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/farmacocinética , Antibacterianos/uso terapêutico , Cefalosporinas/farmacocinética , Cefalosporinas/uso terapêutico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Combinação de Medicamentos , Feminino , Humanos , Infusões Intravenosas , Testes de Função Renal , Masculino , Testes de Sensibilidade Microbiana , Método de Monte Carlo , Ácido Penicilânico/administração & dosagem , Ácido Penicilânico/farmacocinética , Ácido Penicilânico/uso terapêutico , Infecções por Pseudomonas/microbiologia , TazobactamRESUMO
BACKGROUND: The production of metabolites via in vitro culture is promoted by the availability of fully defined metabolic pathways. Withanolides, the major bioactive phytochemicals of Withania somnifera, have been well studied for their pharmacological activities. However, only a few attempts have been made to identify key candidate genes involved in withanolide biosynthesis. Understanding the steps involved in withanolide biosynthesis is essential for metabolic engineering of this plant to increase withanolide production. RESULTS: Transcriptome sequencing was performed on in vitro adventitious root and leaf tissues using the Illumina platform. We obtained a total of 177,156 assembled transcripts with an average unigene length of 1,033 bp. About 13% of the transcripts were unique to in vitro adventitious roots but no unique transcripts were observed in in vitro-grown leaves. A putative withanolide biosynthetic pathway was deduced by mapping the assembled transcripts to the KEGG database, and the expression of candidate withanolide biosynthesis genes -were validated by qRT PCR. The accumulation pattern of withaferin A and withanolide A varied according to the type of tissue and the culture period. Further, we demonstrated that in vitro leaf extracts exhibit anticancer activity against human gastric adenocarcinoma cell lines at sub G1 phase. CONCLUSIONS: We report here a validated large-scale transcriptome data set and the potential biological activity of in vitro cultures of W. somnifera. This study provides important information to enhance tissue-specific expression and accumulation of secondary metabolites, paving the way for industrialization of in vitro cultures of W. somnifera.
Assuntos
Transcriptoma , Withania/metabolismo , Vitanolídeos/metabolismo , Antioxidantes/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Etiquetas de Sequências Expressas , Humanos , Repetições de Microssatélites/genética , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Análise de Sequência de RNA , Withania/genética , Vitanolídeos/isolamento & purificação , Vitanolídeos/toxicidadeRESUMO
Solvation of drugs in the core (C) and headgroup (H) strata of phospholipid bilayers affects their physiological transport rates and accumulation. These characteristics, especially a complete drug distribution profile across the bilayer strata, are tedious to obtain experimentally, to the point that even simplified preferred locations are only available for a few dozen compounds. Recently, we showed that the partition coefficient (P) values in the system of hydrated diacetyl phosphatidylcholine (DAcPC) and n-hexadecane (C16), as surrogates of the H- and C-strata of the bilayer composed of the most abundant mammalian phospholipid, PC, agree well with the preferred bilayer location of compounds. High P values are typical for lipophiles accumulating in the core, and low P values are characteristic of cephalophiles preferring the headgroups. This simple pattern does not hold for most compounds, which usually have more even distribution and may also accumulate at the H/C interface. To model complete distribution, the correlates of solvation energies are needed for each drug state in the bilayer: (1) for the H-stratum it is the DAcPC/W P value, calculated as the ratio of the C16/W and C16/DAcPC (W for water) P values; (2) for the C-stratum, the C16/W P value; (3) for the H/C interface, the P values for all plausible molecular poses are characterized using the fragment DAcPC/W and C16/W solvation parameters for the parts of the molecule embedded in the H- and C-strata, respectively. The correlates, each scaled by two Collander coefficients, were used in a nonlinear, mass-balance based model of intrabilayer distribution, which was applied to the easily measurable overall P values of compounds in the DMPC (M = myristoyl) bilayers and monolayers as the dependent variables. The calibrated model for 107 neutral compounds explains 94% of experimental variance, achieves similar cross-validation levels, and agrees well with the nontrivial, experimentally determined bilayer locations for 27 compounds. The resulting structure-based prediction system for intrabilayer distribution will facilitate more realistic modeling of passive transport and drug interactions with those integral membrane proteins, which have the binding sites located in the bilayer, such as some enzymes, influx and efflux transporters, and receptors. If only overall bilayer accumulation is of interest, the 1-octanol/W P values suffice to model the studied set.
Assuntos
Bicamadas Lipídicas/química , Fosfolipídeos/química , Alcanos/química , Interações Hidrofóbicas e Hidrofílicas , Fosfatidilcolinas/químicaRESUMO
The optimal interaction of drugs with plasma membranes and membranes of subcellular organelles is a prerequisite for desirable pharmacology. Importantly, for drugs targeting the transmembrane lipid-facing sites of integral membrane proteins, the relative affinity of a drug to the bilayer lipids compared to the surrounding aqueous phase affects the partitioning, access, and binding of the drug to the target site. Molecular dynamics (MD) simulations, including enhanced sampling techniques such as steered MD, umbrella sampling (US), and metadynamics, offer valuable insights into the interactions of drugs with the membrane lipids and water in atomistic detail. However, these methods are computationally prohibitive for the high-throughput screening of drug candidates. This study shows that applying denoising diffusion probabilistic models (DDPMs), a generative AI method, to US simulation data reduces the computational cost significantly. Specifically, the models used only partial (one-third) data from the US simulations and reproduced the complete potential of mean force (PMF) profiles for three FDA-approved drugs (ß2-adrenergic agonists) and â¼20 biologically relevant chemicals with known experimentally characterized bilayer locations. Intriguingly, the model can predict the solvation-free energies for partitioning and crossing the bilayer, preferred bilayer locations (low-energy well), and orientations of the ligands with high accuracy. The results indicate that DDPMs can be used to characterize the complete membrane partitioning profile of drug molecules using fewer umbrella sampling simulations at select positions along the bilayer normal (z-axis), irrespective of their amphiphilic-lipophilic-cephalophilic characteristics.
Assuntos
Inteligência Artificial , Simulação de Dinâmica Molecular , Difusão , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Estatísticos , Membrana Celular/química , Membrana Celular/metabolismoRESUMO
Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme ß-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9â¯H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant's defense mechanisms against the root rot pathogen.
Assuntos
Ascomicetos , Bacillus , Bacillus/metabolismo , Bacillus subtilis/metabolismo , Desenvolvimento Vegetal , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologiaRESUMO
Silver (Ag) is a non-essential heavy metal with substantial environmental toxicity but an excellent promotor for plant organogenesis. It is used as an elicitor for secondary metabolite production and for in planta synthesis of metal nanoparticles (MNPs). In the present study, the Ag accumulation and reduction capability of in vitro shoots of Withania somnifera and the toxicity and elicitation effect of Ag on in vitro shoots were explored. In vitro shoot cultures of W. somnifera were treated with different concentrations of silver nitrate for a specific treatment period. Growth index, withaferin A, elemental and electron microscopy analyses were done on silver-treated in vitro shoots of W. somnifera. 1 mM silver nitrate treatment for 12 days period was found to give increased growth index (1.425 ± 0.05c) and withaferin A (2.568 ± 0.08e mg g-1) content. The concentration of bioaccumulated Ag in 1 mM silver nitrate treated in vitro shoot was found to be 50.8 ppm. The presence of nano-Ag was also found in the leaves of 1 mM silver nitrate-treated in vitro shoots. In summary, this is the first report portraying the bioaccumulation and in planta reduction capability of the in vitro shoot system of W. somnifera, which makes it a potential medicinal plant of commercial value for silver contaminated soils.
Assuntos
Nanopartículas Metálicas , Brotos de Planta , Prata , Withania , Vitanolídeos , Withania/metabolismo , Withania/crescimento & desenvolvimento , Withania/efeitos dos fármacos , Vitanolídeos/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Prata/farmacologia , Nitrato de Prata/farmacologiaRESUMO
Aphid salivary proteins mediate the interaction between aphids and their host plants. Moreover, these proteins facilitate digestion, detoxification of secondary metabolites, as well as activation and suppression of plant defenses. The cowpea aphid, Aphis craccivora, is an important sucking pest of leguminous crops worldwide. Although aphid saliva plays an important role in aphid plant interactions, knowledge of the cowpea aphid salivary proteins is limited. In this study, we performed transcriptomic and LC-MS/MS analyses to identify the proteins present in the salivary glands and saliva of A. craccivora. A total of 1,08,275 assembled transcripts were identified in the salivary glands of aphids. Of all these assembled transcripts, 53,714 (49.11%) and 53,577 (49.48%) transcripts showed high similarity to known proteins in the Nr and UniProt databases, respectively. A total of 2159 proteins were predicted as secretory proteins from the salivary gland transcriptome dataset, which contain digestive enzymes, detoxification enzymes, previously known effectors and elicitors, and potential proteins whose functions have yet to be determined. The proteomic analysis of aphid saliva resulted in the identification of 171 proteins. Tissue-specific expression of selected genes using RT-PCR showed that three genes were expressed only in the salivary glands. Overall, our results provide a comprehensive repertoire of cowpea aphid salivary proteins from the salivary gland and saliva, which will be a good resource for future effector functional studies and might also be useful for sustainable aphid management.
Assuntos
Afídeos , Vigna , Animais , Transcriptoma , Afídeos/genética , Afídeos/metabolismo , Vigna/genética , Espectrometria de Massa com Cromatografia Líquida , Cromatografia Líquida , Proteômica/métodos , Espectrometria de Massas em Tandem , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/metabolismoRESUMO
Type-I interferons (IFN) are key antiviral factors that induce several cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity. Apart from ISGylation, unconjugated ISG15 is also released from cells during immuno-stimulation and virus infection. The role of extracellular ISG15 during virus infection was unknown. We now show that extracellular ISG15 acts as a soluble antiviral factor to restrict virus infection in human epithelial cells via an IFN-independent mechanism. Additionally, we demonstrated that extracellular ISG15 induces expression of intracellular ISG15 and ISGylation in the absence of IFN. Mechanistic studies revealed that extracellular ISG15 interacts with cell surface integrin (α5ß1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified ISG15 protein as a new soluble factor that confers IFN-independent antiviral activity by inducing ISGylation. Additionally, we have unfolded an IFN-independent non-canonical ISGylation by extracellular ISG15 that operates via the integrin-FAK pathway.
RESUMO
Stromules are stroma-filled tubules that extend from the surface of plastids and allow the transfer of proteins as large as 550 kDa between interconnected plastids. The aim of the present study was to determine if plastid DNA or plastid ribosomes are able to enter stromules, potentially permitting the transfer of genetic information between plastids. Plastid DNA and ribosomes were marked with green fluorescent protein (GFP) fusions to LacI, the lac repressor, which binds to lacO-related sequences in plastid DNA, and to plastid ribosomal proteins Rpl1 and Rps2, respectively. Fluorescence from GFP-LacI co-localised with plastid DNA in nucleoids in all tissues of transgenic tobacco (Nicotiana tabacum L.) examined and there was no indication of its presence in stromules, not even in hypocotyl epidermal cells, which contain abundant stromules. Fluorescence from Rpl1-GFP and Rps2-GFP was also observed in a punctate pattern in chloroplasts of tobacco and Arabidopsis [Arabidopsis thaliana (L.) Heynh.], and fluorescent stromules were not detected. Rpl1-GFP was shown to assemble into ribosomes and was co-localised with plastid DNA. In contrast, in hypocotyl epidermal cells of dark-grown Arabidopsis seedlings, fluorescence from Rpl1-GFP was more evenly distributed in plastids and was observed in stromules on a total of only four plastids (<0.02% of the plastids observed). These observations indicate that plastid DNA and plastid ribosomes do not routinely move into stromules in tobacco and Arabidopsis, and suggest that transfer of genetic information by this route is likely to be a very rare event, if it occurs at all.