RESUMO
Closely spaced promoters are ubiquitous in prokaryotic and eukaryotic genomes. How their structure and dynamics relate remains unclear, particularly for tandem formations. To study their transcriptional interference, we engineered two pairs and one trio of synthetic promoters in nonoverlapping, tandem formation, in single-copy plasmids transformed into Escherichia coli cells. From in vivo measurements, we found that these promoters in tandem formation can have attenuated transcription rates. The attenuation strength can be widely fine-tuned by the promoters' positioning, natural regulatory mechanisms, and other factors, including the antibiotic rifampicin, which is known to hamper RNAP promoter escape. From this, and supported by in silico models, we concluded that the attenuation in these constructs emerges from premature terminations generated by collisions between RNAPs elongating from upstream promoters and RNAPs occupying downstream promoters. Moreover, we found that these collisions can cause one or both RNAPs to falloff. Finally, the broad spectrum of possible, externally regulated, attenuation strengths observed in our synthetic tandem promoters suggests that they could become useful as externally controllable regulators of future synthetic circuits.
Assuntos
Escherichia coli , Regiões Promotoras Genéticas , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Plasmídeos/genética , Regulação Bacteriana da Expressão GênicaRESUMO
The topology of the transcription factor network (TFN) of Escherichia coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for green fluorescent protein (GFP) fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.IMPORTANCECells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries.
Assuntos
Escherichia coli , Regulação Bacteriana da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas/genéticaRESUMO
Cold shock adaptability is a key survival skill of gut bacteria of warm-blooded animals. Escherichia coli cold shock responses are controlled by a complex multi-gene, timely-ordered transcriptional program. We investigated its underlying mechanisms. Having identified short-term, cold shock repressed genes, we show that their responsiveness is unrelated to their transcription factors or global regulators, while their single-cell protein numbers' variability increases after cold shock. We hypothesized that some cold shock repressed genes could be triggered by high propensity for transcription locking due to changes in DNA supercoiling (likely due to DNA relaxation caused by an overall reduction in negative supercoiling). Concomitantly, we found that nearly half of cold shock repressed genes are also highly responsive to gyrase inhibition (albeit most genes responsive to gyrase inhibition are not cold shock responsive). Further, their response strengths to cold shock and gyrase inhibition correlate. Meanwhile, under cold shock, nucleoid density increases, and gyrases and nucleoid become more colocalized. Moreover, the cellular energy decreases, which may hinder positive supercoils resolution. Overall, we conclude that sensitivity to diminished negative supercoiling is a core feature of E. coli's short-term, cold shock transcriptional program, and could be used to regulate the temperature sensitivity of synthetic circuits.
Assuntos
DNA Super-Helicoidal , Escherichia coli , Resposta ao Choque Frio/genética , DNA/metabolismo , DNA Girase/genética , DNA Girase/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
BACKGROUND: During the second wave of the COVID-19 pandemic, an unusual increase in cases of mucormycosis was observed in India, owing to immunological dysregulation caused by the SARS-CoV-2 and the use of broad-spectrum antibiotics, particularly in patients with poorly controlled diabetes with ketoacidosis to have contributed to the rise, and it has been declared an epidemic in several states of India. Because of the black colouring of dead and dying tissue caused by the fungus, it was dubbed "black fungus" by several Indian media outlets. In this study, attempts were taken to unmask novel therapeutic options to treat mucormycosis disease. Rhizopus species is the primary fungi responsible for 70% of mucormycosis cases. RESULTS: We chose three important proteins from the Rhizopus delemar such as CotH3, Lanosterol 14 alpha-demethylase and Mucoricin which plays a crucial role in the virulence of Mucorales. Initially, we explored the physiochemical, structural and functional insights of proteins and later using AutoDock Vina, we applied computational protein-ligand binding modelling to perform a virtual screening around 300 selected compounds against these three proteins, including FDA-approved drugs, FDA-unapproved drugs, investigational-only drugs and natural bioactive compounds. ADME parameters, toxicity risk and biological activity of those compounds were approximated via in silico methods. Our computational studies identified six ligands as potential inhibitors against Rhizopus delemar, including 12,28-Oxamanzamine A, vialinin B and deoxytopsentin for CotH3; pramiconazole and saperconazole for Lanosterol 14 alpha-demethylase; and Hesperidin for Mucoricin. Interestingly, 12,28-Oxamanzamine A showed a maximum binding affinity with all three proteins (CotH3: - 10.2 kcal/mol Lanosterol 14 alpha-demethylase: - 10.9 kcal/mol Mucoricin: - 8.6 kcal/mol). CONCLUSIONS: In summary, our investigation identified 12,28-Oxamanzamine A, vialinin B, deoxytopsentin, pramiconazole, saperconazole and hesperidin as potent bioactive compounds for treating mucormycosis that may be considered for further optimisation techniques and in vitro and in vivo studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s42269-022-00704-4.
RESUMO
Myo-inositol is one of the vital nutritional requirements for the Leishmania parasites' survival and virulence in the mammalian host. . Myo-inositol-1-phosphate synthase (MIPS) is responsible for the synthesis of myo-inositol in Leishmania, which plays a vital role in Leishmania's virulence to mammalian hosts. Earlier studies suggest MIP synthase as a potential drug target against which valproate was used as a drug. So, MIP synthase can be used as a target for anti-leishmanial drugs, and its inhibition may help in preventing leishmaniasis. The present study aims to identify valproate's potent analogs as drugs against MIP synthase of L. donovani (Ld-MIPS) with minimum side effects and toxicity to host.In this study, the three-dimensional structure of Ld-MIPS was built, followed by active site prediction. Ligand-based virtual screening was done using hybrid similarity recognition methods. The best 123 valproate analogs were filtered based on their quantitative structure activity relationship (QSAR) properties and were docked against Ld-MIPS using FlexX, PyRx and iGEMDOCK software. The topmost five ligands were selected for molecular dynamics simulation and pharmacokinetic analysis based on the docking score. Simulation studies up to 30 ns revealed that all five lead molecules bound with Ld-MIPS throughout MD simulation and there was no variation in their backbone. All the chosen inhibitors exhibited good pharmacokinetics/ADMET predictions with an excellent absorption profile, metabolism, oral bioavailability, solubility, excretion, and minimal toxicity, suggesting that these inhibitors may further be developed as anti-leishmaniasis drugs to prevent the spread of leishmaniasis.Communicated by Ramaswamy H. Sarma.
Assuntos
Leishmania donovani , Leishmaniose , Animais , Inositol/farmacologia , Ligantes , Mamíferos , Simulação de Dinâmica Molecular , Mio-Inositol-1-Fosfato Sintase , Ácido Valproico/farmacologiaRESUMO
Flavonoids are polyphenolic compounds spotted in various fruits, vegetables, barks, tea plants, and stems and many more natural commodities. They have a multitude of applications through their anti-inflammatory, anti-oxidative, anti-carcinogenic properties, along with the ability to assist in the stimulation of bone formation. Bone, a rigid connective body tissue made up of cells embedded in a mineralised matrix is maintained by an assemblage of pathways assisting osteoblastogenesis and osteoclastogenesis. These have a significant impact on a plethora of bone diseases. The homeostasis between osteoblast and osteoclast formation decides the integrity and structure of the bone. The flavonoids discussed here are quercetin, kaempferol, icariin, myricetin, naringin, daidzein, luteolin, genistein, hesperidin, apigenin and several other flavonoids. The effects these flavonoids have on the mitogen activated protein kinase (MAPK), nuclear factor kappa ß (NF-kß), Wnt/ß-catenin and bone morphogenetic protein 2/SMAD (BMP2/SMAD) signalling pathways, and apoptotic pathways lead to impacts on bone remodelling. In addition, these polyphenols regulate angiogenesis, decrease the levels of inflammatory cytokines and play a crucial role in scavenging reactive oxygen species (ROS). Considering these important effects of flavonoids, they may be regarded as a promising agent in treating bone-related ailments in the future.
Assuntos
Remodelação Óssea/efeitos dos fármacos , Flavonoides/administração & dosagem , Flavonoides/classificação , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/classificação , Anti-Inflamatórios/metabolismo , Doenças Ósseas/tratamento farmacológico , Doenças Ósseas/metabolismo , Remodelação Óssea/fisiologia , Flavonoides/metabolismo , Humanos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Leishmania donovani, causes leishmaniasis, a global health trouble with around 89 different countries and its population under its risk. Replication initiation events have been instrumental in regulating the DNA duplication and as the small subunit of L. donovani nuclear DNA primase (Ld-PriS) inherits the catalytic site, it plays a vital role in DNA replication. In this study we have aimed Ld-PriS for the first time as a prospective target for the application of drug against Leishmania parasite. 3-D structures of Ld-PriS were built and ligand-based virtual screening was performed using hybrid similarity recognition techniques. Ligands from the ZINC database were used for the screening purposes based on known DNA primase inhibitor Sphingosine as a query. Top 150 ligands were taken into consideration for molecular docking against the query protein (Ld-PriS) using PyRx and iGEMDOCK softwares. Top five compounds with the best docking score were selected for pharmacokinetic investigation and molecular dynamic simulation. These top five screened inhibitors showed very poor binding affinity toward the catalytic subunit of human primase indicating their safety toward the host normal replication mechanism. The top five compounds showed good pharmacokinetic profiles and ADMET predictions revealed good absorption, solubility, permeability, uniform distribution, proper metabolism, minimal toxicity and good bioavailability. Simulation studies upto 50 ns revealed the three leads ZINC000009219046, ZINC000025998119 and ZINC000004677901 bind with Ld-PriS throughout the simulation and there were no huge variations in their backbone suggesting that these three may play as potential lead compounds for developing new drug against leishmaniasis.Communicated by Ramaswamy H. Sarma.
Assuntos
Leishmania donovani , Leishmaniose , Preparações Farmacêuticas , Domínio Catalítico , DNA , DNA Primase , Descoberta de Drogas , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estudos ProspectivosRESUMO
WHO has declared the outbreak of COVID-19 as a public health emergency of international concern. The ever-growing new cases have called for an urgent emergency for specific anti-COVID-19 drugs. Three structural proteins (Membrane, Envelope and Nucleocapsid protein) play an essential role in the assembly and formation of the infectious virion particles. Thus, the present study was designed to identify potential drug candidates from the unique collection of 548 anti-viral compounds (natural and synthetic anti-viral), which target SARS-CoV-2 structural proteins. High-end molecular docking analysis was performed to characterize the binding affinity of the selected drugs-the ligand, with the SARS-CoV-2 structural proteins, while high-level Simulation studies analyzed the stability of drug-protein interactions. The present study identified rutin, a bioflavonoid and the antibiotic, doxycycline, as the most potent inhibitor of SARS-CoV-2 envelope protein. Caffeic acid and ferulic acid were found to inhibit SARS-CoV-2 membrane protein while the anti-viral agent's simeprevir and grazoprevir showed a high binding affinity for nucleocapsid protein. All these compounds not only showed excellent pharmacokinetic properties, absorption, metabolism, minimal toxicity and bioavailability but were also remain stabilized at the active site of proteins during the MD simulation. Thus, the identified lead compounds may act as potential molecules for the development of effective drugs against SARS-CoV-2 by inhibiting the envelope formation, virion assembly and viral pathogenesis.
Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Proteínas do Nucleocapsídeo/química , Proteínas do Envelope Viral/química , Proteínas da Matriz Viral/química , Vírion/efeitos dos fármacos , Amidas , Sequência de Aminoácidos , Antivirais/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , COVID-19 , Ácidos Cafeicos/química , Ácidos Cafeicos/farmacologia , Carbamatos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Ciclopropanos , Doxiciclina/química , Doxiciclina/farmacologia , Expressão Gênica , Humanos , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Nucleocapsídeo/antagonistas & inibidores , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Quinoxalinas/química , Quinoxalinas/farmacologia , Rutina/química , Rutina/farmacologia , SARS-CoV-2 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Simeprevir/química , Simeprevir/farmacologia , Sulfonamidas , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Vírion/genéticaRESUMO
Over the past decade, the available crystal structures have almost doubled in Protein Data Bank (PDB) providing the research community with a series of similar crystal structures to choose from for future docking studies. With the steady growth in the number of high-resolution three-dimensional protein structures, ligand docking-based virtual screening of chemical libraries to a receptor plays a critical role in the drug discovery process by identifying new drug candidates. Thus, identifying potential candidates among all the available structures in a database for docking studies is of utmost importance. Our work examined whether one could use the resolution of a number of known structures, without considering other parameters, to choose a good experimental structure for various docking studies to find more useful drug leads. We expected that a good experimental structure for docking studies to be the one that gave favorable docking with the largest number of ligands among the experimental structures to be selected. We chose three protein test systems for our study, all belonging to the family of MAPK: (1) JNK1, (2) JNK2, and (3) JNK3. On analysis of the results, the best resolution structures showed significant variations from the expected values in their result, whereas the poor resolution structures proved to be better candidates for docking studies.