RESUMO
Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.
Assuntos
Enoil-CoA Hidratase/ultraestrutura , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/ultraestrutura , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Simulação por Computador , Cristalização , Dimerização , Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Ativação Enzimática , Ácido Graxo Sintase Tipo II/metabolismo , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Mycobacterium tuberculosis/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Transdução de Sinais/fisiologia , Relação Estrutura-AtividadeRESUMO
Expression of METTL3, a SAM dependent methyltransferase, which deposits m6A on mRNA is linked to poor prognosis in Acute Myeloid Leukaemia and other type of cancers. Down regulation of this epitranscriptomic regulator has been found to inhibit cancer progression. Silencing the methyltransferase activity of METTL3 is a lucrative strategy to design anticancer drugs. In this study 3600 commercially available molecules were screened against METTL3 using brute force screening approach. However, none of these compounds take advantage of the unique Y-shaped binding cavity of the protein, raising the need for de novo drug designing strategies. As such, 125 branched, Y-shaped molecules were designed by "stitching" together the chemical fragments of the best inhibitors that interact strongly with the METTL3 binding pocket. This results in molecules that have the three-dimensional structure and functional groups which enable it to fit in the METTL3 cavity like fingers in a glove, having unprecedented selectivity and binding affinities. The designed compounds were further refined based on Lipinski's rule, docking score and synthetic accessibility. The molecules faring well in these criteria were simulated for 100 ns to check the stability of the protein inhibitor complex followed by binding free energy calculation.Communicated by Ramaswamy H. Sarma.
RESUMO
Epitranscriptomic modification is a dynamic modification of RNAs. Epitranscriptomic writer proteins are methyltransferases, such as METTL3 and METTL16. The up regulation of METTL3 have been found to be linked to different cancers and targeting METTL3 is an effective way to reduce tumour progression. Drug development against METTL3 is an active field of research. METTL16, SAM dependent methyltransferase, is another writer protein, that has been found to be upregulated in hepatocellular carcinoma and gastric cancer. In this pioneering study METTL16 has been targeted for virtual drug screening for the very first time using brute force strategy to identify a drug molecule that could be repurposed for the treatment of the disease caused. An unbiased library of the commercially available drug molecules has been used for screening using a multipoint validation process developed for this work, which includes molecular docking, ADMET analysis, protein-ligand interaction analysis, Molecular Dynamics Simulation, binding energy calculation via Molecular Mechanics Poisson-Boltzmann Surface Area method. Upon the in-silico screening of over 650 drugs the authors have found NIL and VXL passed the validation process. The data strongly indicates the potency of these two drugs in the treatment of disease where METTL16 needs to be inhibited.
Assuntos
Simulação de Dinâmica Molecular , RNA , Simulação de Acoplamento Molecular , Sítios de Ligação , Avaliação Pré-Clínica de MedicamentosRESUMO
INTRODUCTION: M6A modification in transcriptome is critical in regulating different cellular processes, including cancer. In human beings, METTL3 is the major m6A writer that works in association with METTL14, an accessory protein. Extensive study revealed that cancer progression for acute myeloid leukemia, gastric cancer, colorectal cancer, hepatocellular carcinoma, and lung cancer is directly contributed by irregular expression of METTL3. OBJECTIVE: Targeting METTL3 has opened a new window in the development of novel inhibitors/drugs. METHODS: In this study, commercially available natural compounds were randomly screened to avoid the bias of screening small molecules on the basis of structural similarity. From 810 compounds that were screened, 80 commercially available compounds were showing better score when compared with the existing substrate/substrate-analogue and the inhibitor bound crystal structures in terms of docking score and binding energy calculation. RESULTS AND CONCLUSION: Among this pool of compounds, the best seven small molecules have been selected and further validated by different computational tools like binding energy calculation, molecular dynamics simulation, ADME analysis, and toxicity prediction. The novel hits found in this study can function as lead compounds which can be developed into inhibitors as well as drugs, specific against METTL3.
Assuntos
Leucemia Mieloide Aguda , Simulação de Dinâmica Molecular , Humanos , Avaliação Pré-Clínica de Medicamentos , Simulação de Acoplamento Molecular , Leucemia Mieloide Aguda/tratamento farmacológico , MetiltransferasesRESUMO
Long non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra- or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1-containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs.
Assuntos
Regulação Fúngica da Expressão Gênica , Meiose/genética , Mitose/genética , RNA Fúngico/genética , RNA Longo não Codificante/genética , Schizosaccharomyces/genética , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Timely and accurate expression of the genetic information relies on the integration of environmental cues and the activation of regulatory networks involving transcriptional and post-transcriptional mechanisms. In fission yeast, meiosis-specific transcripts are selectively targeted for degradation during mitosis by the EMC complex, composed of Erh1, the ortholog of human ERH, and the YTH family RNA-binding protein Mmi1. Here, we present the crystal structure of Erh1 and show that it assembles as a homodimer. Mutations of amino acid residues to disrupt Erh1 homodimer formation result in loss-of-function phenotypes, similar to erh1∆ cells: expression of meiotic genes is derepressed in mitotic cells and meiosis progression is severely compromised. Interestingly, formation of Erh1 homodimer is dispensable for interaction with Mmi1, suggesting that only fully assembled EMC complexes consisting of two Mmi1 molecules bridged by an Erh1 dimer are functionally competent. We also show that Erh1 does not contribute to Mmi1-dependent down-regulation of the meiosis regulator Mei2, supporting the notion that Mmi1 performs additional functions beyond EMC. Overall, our results provide a structural basis for the assembly of the EMC complex and highlight its biological relevance in gametogenic gene silencing and meiosis progression.
Assuntos
Proteínas de Transporte/química , Inativação Gênica/fisiologia , Meiose/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Cristalografia por Raios X , Complexos Multiproteicos/metabolismo , Conformação Proteica , Multimerização Proteica , Schizosaccharomyces/genéticaRESUMO
The control of gene expression is a multi-layered process occurring at the level of DNA, RNA, and proteins. With the emergence of highly sensitive techniques, new aspects of RNA regulation have been uncovered leading to the emerging field of epitranscriptomics dealing with RNA modifications. Among those post-transcriptional modifications, N6-methyladenosine (m6A) is the most prevalent in messenger RNAs (mRNAs). This mark can either prevent or stimulate the formation of RNA-protein complexes, thereby influencing mRNA-related mechanisms and cellular processes. This review focuses on proteins containing a YTH domain (for YT521-B Homology), a small building block, that selectively detects the m6A nucleotide embedded within a consensus motif. Thereby, it contributes to the recruitment of various effectors involved in the control of mRNA fates through adjacent regions present in the different YTH-containing proteins.
Assuntos
Adenosina/análogos & derivados , Proteínas do Tecido Nervoso/metabolismo , Processamento Pós-Transcricional do RNA , Fatores de Processamento de RNA/metabolismo , Adenosina/metabolismo , Animais , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismoRESUMO
Organic pollutants, like phenol, along with heavy metals, like chromium, are present in various industrial effluents that pose serious health hazard to humans. The present study looked at removal of chromium (VI) in presence of phenol in a counter-current continuous packed bed reactor packed with E. coli cells immobilized on clay chips. The cells removed 85% of 500 mg/L of chromium (VI) from MS media containing glucose. Glucose was then replaced by 500 mg/L phenol. Temperature and pH of the MS media prior to addition of phenol were 30°C and 7, respectively. Hydraulic retention times of phenol- and chromium (VI)-containing synthetic media and air flow rates were varied to study the removal efficiency of the reactor system. Then temperature conditions of the reactor system were varied from 10°C to 50°C, the optimum being 30°C. The pH of the media was varied from pH 1 to pH 12, and the optimum pH was found to be 7. The maximum removal efficiency of 77.7% was achieved for synthetic media containing phenol and chromium (VI) in the continuous reactor system at optimized conditions, namely, hydraulic retention time at 4.44 hr, air flow rate at 2.5 lpm, temperature at 30°C, and pH at 7.