scholarly journals Molecular Docking and In Silico Study of Denileukin Diftitox: Comparison of Wild Type With C519S Mutant

2020 ◽  
Vol 8 (2) ◽  
pp. 83-92
Author(s):  
Mohamad Najarasl ◽  
◽  
Mehdi Zeinoddini ◽  
Ali Reza Saeeidinia ◽  
Reza Hasan Sajedi ◽  
...  
Keyword(s):  
Fusion Protein ◽  
De Novo ◽  
Scale Up ◽  
Interleukin 2 ◽  
Cysteine Residue ◽  
Modeling Method ◽  
Site Directed Mutagenesis ◽  
Denileukin Diftitox ◽  
Recombinant Immunotoxin ◽  
Trade Name

Background: Denileukin diftitox (trade name, Ontak) is the first recombinant immunotoxin (IM), in which the binding domain of diphtheria toxin has been replaced by the amino acid sequence of human interleukin-2 (DT389IL-2) using genetic engineering. Purity, stability, and structural property of the protein are critical factors for the scale-up production of this fusion protein. In this IM, location 519 has free cysteine residue that leads to cross S-S bound formation in the refolding process and, as a result, misfolding/aggregation of the protein may occur. Materials and Methods: To inhibit misfolding/aggregation, we substituted cysteine 519 by a serine residue with site-directed mutagenesis, and then the ability of the mutated protein for binding to the IL-2 receptor was predicted and determined by bioinformatics tools. For this purpose, the sequence of the denileukin diftitox was adopted from Drugbank, and the mentioned substitution applied. Two methods determined the folding of the fusion protein: de novo modeling method (by utilizing the I-TASSER database) and homology modeling method (by using some databases and tools, including Swiss-Model, PHYRE2, M4T, ModWeb, RaptorX, and EasyModeller). Finally, the ability of the proteins for binding to the IL-2 receptor was investigated by pyDock and Zdock docking servers, as well as Hex software. Results: The result showed that the mutated form (C519S) of this protein folds appropriately, and the ΔG of the models, measured by STRUM, showed no significant variation. Also, docking analysis has shown that the protein can efficiently bind to the IL-2 receptor without any substantial changes in the binding energy. Conclusion: The present study shows that the suggested mutation of this protein can be an acceptable replacement for denileukin diftitox with a similar affinity and a more proper refolding process.

Lead Molecules as Novel Aromatase Inhibitors: In Silico De Novo Designing and Binding Affinity Studies

2020 ◽  
Vol 17 (5) ◽  
pp. 655-665 ◽  
Author(s):  
Laxmi Banjare ◽  
Sant Kumar Verma ◽  
Akhlesh Kumar Jain ◽  
Suresh Thareja
Keyword(s):  
Drug Design ◽  
Aromatase Inhibitors ◽  
De Novo ◽  
Scale Up ◽  
Geometry Optimization ◽  
Docking Study ◽  
Molecular Docking Study ◽  
De Novo Drug Design ◽  
Molegro Virtual Docker ◽  
Estrogen Production

Background:Aromatase inhibitors emerged as a pivotal moiety to selectively block estrogen production, prevention and treatment of tumour growth in breast cancer. De novo drug design is an alternative approach to blind virtual screening for successful designing of the novel molecule against various therapeutic targets.Objective:In the present study, we have explored the de novo approach to design novel aromatase inhibitors.Method:The e-LEA3D, a computational-aided drug design web server was used to design novel drug-like candidates against the target aromatase. For drug-likeness ADME parameters (molecular weight, H-bond acceptors, H-bond donors, LogP and number of rotatable bonds) of designed molecules were calculated in TSAR software package, geometry optimization and energy minimization was accomplished using Chem Office. Further, molecular docking study was performed in Molegro Virtual Docker (MVD).Results:Among 17 generated molecules using the de novo pathway, 13 molecules passed the Lipinski filter pertaining to their bioavailability characteristics. De novo designed molecules with drug-likeness were further docked into the mapped active site of aromatase to scale up their affinity and binding fitness with the target. Among de novo fabricated drug like candidates (1-13), two molecules (5, 6) exhibited higher affinity with aromatase in terms of MolDock score (-150.650, -172.680 Kcal/mol, respectively) while molecule 8 showed lowest target affinity (-85.588 Kcal/mol).Conclusion:The binding patterns of lead molecules (5, 6) could be used as a pharmacophore for medicinal chemists to explore these molecules for their aromatase inhibitory potential.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

Biochemical characterization of Plasmodium falciparum CTP:phosphoethanolamine cytidylyltransferase shows that only one of the two cytidylyltransferase domains is active

2013 ◽  
Vol 450 (1) ◽  
pp. 159-167 ◽  
Author(s):  
Sweta Maheshwari ◽  
Marina Lavigne ◽  
Alicia Contet ◽  
Blandine Alberge ◽  
Emilie Pihan ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Cellular Localization ◽  
Biochemical Characterization ◽  
De Novo ◽  
Membrane Lipids ◽  
Polyclonal Antibodies ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Recombinant Enzymes ◽  
Parasite Life Cycle

The intra-erythrocytic proliferation of the human malaria parasite Plasmodium falciparum requires massive synthesis of PE (phosphatidylethanolamine) that together with phosphatidylcholine constitute the bulk of the malaria membrane lipids. PE is mainly synthesized de novo by the CDP:ethanolamine-dependent Kennedy pathway. We previously showed that inhibition of PE biosynthesis led to parasite death. In the present study we characterized PfECT [P. falciparum CTP:phosphoethanolamine CT (cytidylyltransferase)], which we identified as the rate-limiting step of the PE metabolic pathway in the parasite. The cellular localization and expression of PfECT along the parasite life cycle were studied using polyclonal antibodies. Biochemical analyses showed that the enzyme activity follows Michaelis–Menten kinetics. PfECT is composed of two CT domains separated by a linker region. Activity assays on recombinant enzymes upon site-directed mutagenesis revealed that the N-terminal CT domain was the only catalytically active domain of PfECT. Concordantly, three-dimensional homology modelling of PfECT showed critical amino acid differences between the substrate-binding sites of the two CT domains. PfECT was predicted to fold as an intramolecular dimer suggesting that the inactive C-terminal domain is important for dimer stabilization. Given the absence of PE synthesis in red blood cells, PfECT represents a potential antimalarial target opening the way for a rational conception of bioactive compounds.

scholarly journals Repression of the pyr Operon in Lactobacillus plantarum Prevents Its Ability To Grow at Low Carbon Dioxide Levels

2005 ◽  
Vol 187 (6) ◽  
pp. 2093-2104 ◽  
Author(s):  
Hervé Nicoloff ◽  
Aram Elagöz ◽  
Florence Arsène-Ploetze ◽  
Benoît Kammerer ◽  
Jan Martinussen ◽  
...  
Keyword(s):  
Carbon Dioxide ◽  
Lactobacillus Plantarum ◽  
De Novo ◽  
Site Directed Mutagenesis ◽  
Low Carbon ◽  
Wild Type ◽  
Carbamoyl Phosphate ◽  
Phosphoribosyl Pyrophosphate ◽  
Pyrimidine Nucleotides ◽  
Transcription Attenuation

ABSTRACT Carbamoyl phosphate is a precursor for both arginine and pyrimidine biosynthesis. In Lactobacillus plantarum, carbamoyl phosphate is synthesized from glutamine, ATP, and carbon dioxide by two sets of identified genes encoding carbamoyl phosphate synthase (CPS). The expression of the carAB operon (encoding CPS-A) responds to arginine availability, whereas pyrAaAb (encoding CPS-P) is part of the pyrR1BCAaAbDFE operon coding for the de novo pyrimidine pathway repressed by exogenous uracil. The pyr operon is regulated by transcription attenuation mediated by a trans-acting repressor that binds to the pyr mRNA attenuation site in response to intracellular UMP/phosphoribosyl pyrophosphate pools. Intracellular pyrimidine triphosphate nucleoside pools were lower in mutant FB335 (carAB deletion) harboring only CPS-P than in the wild-type strain harboring both CPS-A and CPS-P. Thus, CPS-P activity is the limiting step in pyrimidine synthesis. FB335 is unable to grow in the presence of uracil due to a lack of sufficient carbamoyl phosphate required for arginine biosynthesis. Forty independent spontaneous FB335-derived mutants that have lost regulation of the pyr operon were readily obtained by their ability to grow in the presence of uracil and absence of arginine; 26 harbored mutations in the pyrR1-pyrB loci. One was a prototroph with a deletion of both pyrR1 and the transcription attenuation site that resulted in large amounts of excreted pyrimidine nucleotides and increased intracellular UTP and CTP pools compared to wild-type levels. Low pyrimidine-independent expression of the pyr operon was obtained by antiterminator site-directed mutagenesis. The resulting AE1023 strain had reduced UTP and CTP pools and had the phenotype of a high-CO2-requiring auxotroph, since it was able to synthesize sufficient arginine and pyrimidines only in CO2-enriched air. Therefore, growth inhibition without CO2 enrichment may be due to low carbamoyl phosphate pools from lack of CPS activity.

scholarly journals Mapping the BKCa Channel's “Ca2+ Bowl”

2004 ◽  
Vol 123 (5) ◽  
pp. 475-489 ◽  
Author(s):  
Lin Bao ◽  
Christina Kaldany ◽  
Ericka C. Holmstrand ◽  
Daniel H. Cox
Keyword(s):  
Fusion Protein ◽  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Membrane Domain ◽  
Intracellular Domain ◽  
High Affinity ◽  
Acidic Residues ◽  
The Relationship

There is controversy over whether Ca2+ binds to the BKCa channel's intracellular domain or its integral-membrane domain and over whether or not mutations that reduce the channel's Ca2+ sensitivity act at the point of Ca2+ coordination. One region in the intracellular domain that has been implicated in Ca2+ sensing is the “Ca2+ bowl”. This region contains many acidic residues, and large Ca2+-bowl mutations eliminate Ca2+ sensing through what appears to be one type of high-affinity Ca2+-binding site. Here, through site-directed mutagenesis we have mapped the residues in the Ca2+ bowl that are most important for Ca2+ sensing. We find acidic residues, D898 and D900, to be essential, and we find them essential as well for Ca2+ binding to a fusion protein that contains a portion of the BKCa channel's intracellular domain. Thus, much of our data supports the conclusion that Ca2+ binds to the BKCa channel's intracellular domain, and they define the Ca2+ bowl's essential Ca2+-sensing motif. Overall, however, we have found that the relationship between mutations that disrupt Ca2+ sensing and those that disrupt Ca2+ binding is not as strong as we had expected, a result that raises the possibility that, when examined by gel-overlay, the Ca2+ bowl may be in a nonnative conformation.

scholarly journals Avidin inhibits PHA-induced human peripheral blood mononuclear cell proliferation

2016 ◽  
Vol 25 (1) ◽  
pp. 19-24
Author(s):  
Cicia Firakania ◽  
Indra G. Mansur ◽  
Sri W.A. Jusman ◽  
Mohamad Sadikin
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Peripheral Blood Mononuclear Cell ◽  
Mononuclear Cell ◽  
Peripheral Blood ◽  
De Novo ◽  
Human Peripheral Blood ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Cell Proliferation Inhibition

Background: Cell proliferation occurs not only in normal but also in cancer cells. Most of cell proliferation inhibition can be done by inhibiting the DNA synthesis, notably by intervening the formation of purine or pyrimidine. In purine de novo synthesis, it was assumed that biotin plays a role as a coenzyme in carboxylation reaction, one of the pivotal steps in the purine de novo pathways. The aim of this study was to see the avidin potency to bind biotin and inhibit mitosis.Methods: Peripheral blood mononuclear cell (PBMC) was cultured in RPMI-1640 medium and stimulated by phytohemagglutinin (PHA) in the presence or absence of interleukin-2 (IL-2), with or without avidin. The effect of avidin addition was observed at 24, 48, and 72 hours for cell proliferation, viability, and cell cycle. Statistical analysis was done by one-way ANOVA.Results: Avidin inhibited cell proliferation and viability in culture under stimulation by PHA with and without IL-2. Cell cycle analysis showed that avidin arrested the progression of PBMC after 72 hours of culture. Most cells were found in G0/G1 phase.Conclusion: Inhibition of biotin utilization by avidin binding can halt cell proliferation.

scholarly journals Structure of SPH (self-incompatibility protein homologue) proteins: a widespread family of small, highly stable, secreted proteins

2019 ◽  
Vol 476 (5) ◽  
pp. 809-826
Author(s):  
Karthik V. Rajasekar ◽  
Shuangxi Ji ◽  
Rachel J. Coulthard ◽  
Jon P. Ride ◽  
Gillian L. Reynolds ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Analytical Ultracentrifugation ◽  
De Novo ◽  
Learning Algorithm ◽  
Sequence Similarity ◽  
Homology Modelling ◽  
3D Structure ◽  
Large Family ◽  
Secreted Proteins ◽  
Self Incompatibility

Abstract SPH (self-incompatibility protein homologue) proteins are a large family of small, disulfide-bonded, secreted proteins, initially found in the self-incompatibility response in the field poppy (Papaver rhoeas), but now known to be widely distributed in plants, many containing multiple members of this protein family. Using the Origami strain of Escherichia coli, we expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin fusion protein and purified it from the cytosol. The fusion protein was cleaved and characterised by analytical ultracentrifugation, circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. This showed that SPH15 is monomeric and temperature stable, with a β-sandwich structure. The four strands in each sheet have the same topology as the unrelated proteins: human transthyretin, bacterial TssJ and pneumolysin, with no discernible sequence similarity. The NMR-derived structure was compared with a de novo model, made using a new deep learning algorithm based on co-evolution/correlated mutations, DeepCDPred, validating the method. The DeepCDPred de novo method and homology modelling to SPH15 were then both used to derive models of the 3D structure of the three known PrsS proteins from P. rhoeas, which have only 15–18% sequence homology to SPH15. The DeepCDPred method gave models with lower discreet optimised protein energy scores than the homology models. Three loops at one end of the poppy structures are postulated to interact with their respective pollen receptors to instigate programmed cell death in pollen tubes.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

2018 ◽  
Vol 69 (9) ◽  
pp. 873
Author(s):  
Xin Ma ◽  
Xuye Du ◽  
Cunyao Bo ◽  
Hongwei Wang ◽  
Anfei Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Wheat Flour ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Dough Strength ◽  
Typical Structure ◽  
Dough Quality

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

scholarly journals Construction of Recombinant Lactococcus lactis Strain Expressing VP1 Fusion Protein of Duck Hepatitis A Virus Type 1 and Evaluation of Its Immune Effect

Vaccines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1479
Author(s):  
Xiaoting Zhang ◽  
Ruihua Zhang ◽  
Jingyu Wang ◽  
Nana Sui ◽  
Guige Xu ◽  
...  
Keyword(s):  
Immune System ◽  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Virus Type ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Interleukin 2 ◽  
Interleukin 4 ◽  
Duck Hepatitis

With the continuous development of duck farming and the increasing breeding density, the incidence of duck hepatitis A virus type 1 (DHAV-1) has been on the rise, seriously endangering the development of duck farming. To reduce the use of antibiotics in duck breeding, susceptibility risks and mortality, and avoid virulence recovery and immune failure risk, this study aims to develop a new type of mucosal immune probiotics and make full use of molecular biology techniques, on the level of genetic engineering, to modify Lactococcus lactis (L. lactis). In this study, a secretory recombinant L. lactis named MG1363-VP1 with an enhanced Green Fluorescent Protein (eGFP) and translation enhancer T7g10L was constructed, which could express the VP1-eGFP fusion protein of DHAV-1. The animal experiment in ducklings was performed to detect the immune response and protection effect of oral microecologics by recombinant L. lactis. The results showed that oral L. lactis MG1363-VP1 significantly induced the body’s humoral immune system and mucosal immune system to produce specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) for DHAV-1 in ducklings, and cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon gamma (IFN-γ). The mortality rate was monitored simultaneously by the natural infestation in the process of production and breeding; notably, the ducklings vaccinated with L. lactis MG1363-VP1 were effectively protected against the nature infection of DHAV-1. The recombinant L. lactis MG1363-VP1 constructed in this study provides a new means of preventing and controlling DHAV-1 infection in the future.

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