scholarly journals Non-Mammalian Prdx6 Enzymes (Proteins with 1-Cys Prdx Mechanism) Display PLA2 Activity Similar to the Human Orthologue

Antioxidants ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 52 ◽  
Author(s):  
Renata Bannitz-Fernandes ◽  
Rogério Aleixo-Silva ◽  
João Silva ◽  
Chandra Dodia ◽  
Jose Vazquez-Medina ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Amino Acid Sequences ◽  
Future Research ◽  
Structure Modeling ◽  
Pla2 Activity ◽  
Bifunctional Enzymes ◽  
Human Orthologue ◽  
Neutral Conditions ◽  
Layer Chromatography ◽  
Sequences Alignment

Mammalian peroxiredoxin class 6 (Prdx6) are bifunctional enzymes. Non-mammalian Prdx6 enzymes display Cys-based peroxidase activity, but to date their putative phospholipase A2 (PLA2 activities) has not been experimentally investigated. Initially, we observed that five non-mammalian Prdx6 enzymes (enzymes from Arabidopsis thaliana (AtPER1), Triticum aestivum (TaPER1), Pseudomonas aeruginosa (PaLsfA) and Aspergillus fumigatus (AfPrx1 and AfPrxC)) present features compatible with PLA2 activities in mammalian Prdx6 by amino acid sequences alignment and tertiary structure modeling. Employing unilamellar liposomes with tracer amounts of [3H]-1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and thin layer chromatography, all the tested non-mammalian Prdx6 enzymes displayed PLA2 activities, with values ranging from 3.4 to 6.1 nmol/min/mg protein. It was previously shown that Thr177 phosphorylation of human Prdx6 increases its PLA2 activity, especially at neutral pH. Therefore, we investigated if human Erk2 kinase could also phosphorylate homologous Thr residues in non-mammalian Prdx6 proteins. We observed phosphorylation of the conserved Thr in three out of the five non-mammalian Prdx enzymes by mass spectrometry. In the case of the mitochondrial Prdx6 from A. fumigatus (AfPrxC), we also observed phosphorylation by western blot, and as a consequence, the PLA2 activity was increased in acidic and neutral conditions by the human Erk2 kinase treatment. The possible physiological meanings of these PLA2 activities described open new fields for future research.

scholarly journals In-silicoprediction and modeling of theEntamoeba histolyticaproteins: Serine-richEntamoeba histolyticaprotein and 29 kDa Cysteine-rich protease

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3160 ◽  
Author(s):  
Kumar Manochitra ◽  
Subhash Chandra Parija
Keyword(s):  
Amino Acid ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Protein Structures ◽  
Amino Acid Sequences ◽  
Treatment Modalities ◽  
Bioinformatic Tools ◽  
Complex Protein ◽  
A Cell ◽  
Quaternary Structures

BackgroundAmoebiasis is the third most common parasitic cause of morbidity and mortality, particularly in countries with poor hygienic settings. There exists an ambiguity in the diagnosis of amoebiasis, and hence there arises a necessity for a better diagnostic approach. Serine-richEntamoeba histolyticaprotein (SREHP), peroxiredoxin and Gal/GalNAc lectin are pivotal inE. histolyticavirulence and are extensively studied as diagnostic and vaccine targets. For elucidating the cellular function of these proteins, details regarding their respective quaternary structures are essential. However, studies in this aspect are scant. Hence, this study was carried out to predict the structure of these target proteins and characterize them structurally as well as functionally using appropriatein-silicomethods.MethodsThe amino acid sequences of the proteins were retrieved from National Centre for Biotechnology Information database and aligned using ClustalW. Bioinformatic tools were employed in the secondary structure and tertiary structure prediction. The predicted structure was validated, and final refinement was carried out.ResultsThe protein structures predicted by i-TASSER were found to be more accurate than Phyre2 based on the validation using SAVES server. The prediction suggests SREHP to be an extracellular protein, peroxiredoxin a peripheral membrane protein while Gal/GalNAc lectin was found to be a cell-wall protein. Signal peptides were found in the amino-acid sequences of SREHP and Gal/GalNAc lectin, whereas they were not present in the peroxiredoxin sequence. Gal/GalNAc lectin showed better antigenicity than the other two proteins studied. All the three proteins exhibited similarity in their structures and were mostly composed of loops.DiscussionThe structures of SREHP and peroxiredoxin were predicted successfully, while the structure of Gal/GalNAc lectin could not be predicted as it was a complex protein composed of sub-units. Also, this protein showed less similarity with the available structural homologs. The quaternary structures of SREHP and peroxiredoxin predicted from this study would provide better structural and functional insights into these proteins and may aid in development of newer diagnostic assays or enhancement of the available treatment modalities.

ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Phospholipase A2 ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Human Platelets ◽  
Phospholipid Bilayer ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Enzyme Substrate ◽  
Membrane Bound ◽  
Layer Chromatography

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

scholarly journals De Novo Computational Protein Tertiary Structure Modeling Pipeline for Cryo-EM Maps of Intermediate Resolution

2020 ◽  
Vol 118 (3) ◽  
pp. 292a
Author(s):  
Daisuke Kihara ◽  
Genki Terashi ◽  
Sai Raghavendra Maddhuri Venkata Subramaniya
Keyword(s):  
Tertiary Structure ◽  
De Novo ◽  
Structure Modeling ◽  
Protein Tertiary Structure

scholarly journals Analysis of a nucleotide-binding site of 5-lipoxygenase by affinity labelling: binding characteristics and amino acid sequences

2000 ◽  
Vol 351 (3) ◽  
pp. 697-707 ◽  
Author(s):  
Ying-Yi ZHANG ◽  
Tove HAMMARBERG ◽  
Olof RADMARK ◽  
Bengt SAMUELSSON ◽  
Carol F. NG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Tertiary Structure ◽  
Nucleotide Binding ◽  
Peptide Sequencing ◽  
Amino Acid Sequences ◽  
Nucleotide Binding Site ◽  
Atp Binding ◽  
Binding Characteristics ◽  
Atp Binding Site

5-Lipoxygenase (5LO) catalyses the first two steps in the biosynthesis of leukotrienes, which are inflammatory mediators derived from arachidonic acid. 5LO activity is stimulated by ATP; however, a consensus ATP-binding site or nucleotide-binding site has not been found in its protein sequence. In the present study, affinity and photoaffinity labelling of 5LO with 5′-p-fluorosulphonylbenzoyladenosine (FSBA) and 2-azido-ATP showed that 5LO bound to the ATP analogues quantitatively and specifically and that the incorporation of either analogue inhibited ATP stimulation of 5LO activity. The stoichiometry of the labelling was 1.4mol of FSBA/mol of 5LO (of which ATP competed with 1mol/mol) or 0.94mol of 2-azido-ATP/mol of 5LO (of which ATP competed with 0.77mol/mol). Labelling with FSBA prevented further labelling with 2-azido-ATP, indicating that the same binding site was occupied by both analogues. Other nucleotides (ADP, AMP, GTP, CTP and UTP) also competed with 2-azido-ATP labelling, suggesting that the site was a general nucleotide-binding site rather than a strict ATP-binding site. Ca2+, which also stimulates 5LO activity, had no effect on the labelling of the nucleotide-binding site. Digestion with trypsin and peptide sequencing showed that two fragments of 5LO were labelled by 2-azido-ATP. These fragments correspond to residues 73–83 (KYWLNDDWYLK, in single-letter amino acid code) and 193–209 (FMHMFQSSWNDFADFEK) in the 5LO sequence. Trp-75 and Trp-201 in these peptides were modified by the labelling, suggesting that they were immediately adjacent to the C-2 position of the adenine ring of ATP. Given the stoichiometry of the labelling, the two peptide sequences of 5LO were probably near each other in the enzyme's tertiary structure, composing or surrounding the ATP-binding site of 5LO.

scholarly journals MULTICOM2: an open-source protein structure prediction system powered by deep learning and distance prediction

2021 ◽  
Author(s):  
Tianqi Wu ◽  
Jian Liu ◽  
Zhiye Guo ◽  
Jie Hou ◽  
Jianlin Cheng
Keyword(s):  
Deep Learning ◽  
Protein Structure ◽  
Open Source ◽  
Protein Structure Prediction ◽  
Structure Prediction ◽  
Tertiary Structure ◽  
Modeling Method ◽  
Structure Modeling ◽  
Prediction System ◽  
Template Free

Abstract Protein structure prediction is an important problem in bioinformatics and has been studied for decades. However, there are still few open-source comprehensive protein structure prediction packages publicly available in the field. In this paper, we present our latest open-source protein tertiary structure prediction system - MULTICOM2, an integration of template-based modeling (TBM) and template-free modeling (FM) methods. The template-based modeling uses sequence alignment tools with deep multiple sequence alignments to search for structural templates, which are much faster and more accurate than MULTICOM1. The template-free (ab initio or de novo) modeling uses the inter-residue distances predicted by DeepDist to reconstruct tertiary structure models without using any known structure as template. In the blind CASP14 experiment, the average TM-score of the models predicted by our server predictor based on the MULTICOM2 system is 0.720 for 58 TBM (regular) domains and 0.514 for 38 FM and FM/TBM (hard) domains, indicating that MULTICOM2 is capable of predicting good tertiary structures across the board. It can predict the correct fold for 76 CASP14 domains (95% regular domains and 55% hard domains) if only one prediction is made for a domain. The success rate is increased to 3% for both regular and hard domains if five predictions are made per domain. Moreover, the prediction accuracy of the pure template-free structure modeling method on both TBM and FM targets is very close to the combination of template-based and template-free modeling methods. This demonstrates that the distance-based template-free modeling method powered by deep learning can largely replace the traditional template-based modeling method even on TBM targets that TBM methods used to dominate and therefore provides a uniform structure modeling approach to any protein. Finally, on the 38 CASP14 FM and FM/TBM hard domains, MULTICOM2 server predictors (MULTICOM-HYBRID, MULTICOM-DEEP, MULTICOM-DIST) were ranked among the top 20 automated server predictors in the CASP14 experiment. After combining multiple predictors from the same research group as one entry, MULTICOM-HYBRID was ranked no. 5. The source code of MULTICOM2 is freely available at https://github.com/multicom-toolbox/multicom/tree/multicom_v2.0.

scholarly journals Tolerance of nonsynonymous variation is closely correlated between human and mouse orthologues

2019 ◽  
Author(s):  
George Powell ◽  
Michelle Simon ◽  
Sara Pulit ◽  
Ann-Marie Mallon ◽  
Cecilia M. Lindgren
Keyword(s):  
Mouse Models ◽  
Negative Selection ◽  
Future Research ◽  
Mendelian Disease ◽  
Pathogenic Variants ◽  
Genome Wide ◽  
Human Genes ◽  
Human Orthologue ◽  
Nonsynonymous Variation ◽  
Human And Mouse

ABSTRACTGenic constraint describes how tolerant a gene is of nonsynonymous variation before it is removed from the population by negative selection. Here, we provide the first estimates of intraspecific constraint for mouse genes genome-wide, and show constraint is positively correlated between human and mouse orthologues (r = 0.806). We assess the relationships between mouse gene constraint and knockout phenotypes, showing gene constraint is positively associated with pleiotropy (ie an increased number of phenotype associations (R2 = 0.65)), in addition to an enrichment in lethal, developmental, and craniofacial knockout phenotypes amongst the most constrained genes. Finally, we show mouse constraint can be used to predict human genes associated with Mendelian disease, and is positively correlated with an increase in the number of known pathogenic variants in the human orthologue (R2 = 0.23). Our metrics of mouse and human constraint are available to inform future research using mouse models.

In Silico Design of Chimeric and Immunogenic Protein-Containing IpaB and IpaD as a Vaccine Candidate against Shigella dysenteriae

2020 ◽  
Vol 17 (4) ◽  
pp. 333-341
Author(s):  
Seyed Akbar Arianzad ◽  
Mehdi Zeinoddini ◽  
Azam Haddadi ◽  
Shahram Nazarian ◽  
Reza Hasan Sajedi
Keyword(s):  
Tertiary Structure ◽  
Vaccine Candidate ◽  
Minimum Energy ◽  
Chimeric Protein ◽  
Severe Form ◽  
Amino Acid Sequences ◽  
Shigella Dysenteriae ◽  
Intestinal Epithelial ◽  
Suitable Target ◽  
Immunogenic Properties

Background: S. dysenteriaeis the causative agent of shigellosis, a severe form of bacillary dysentery and this infectious disease is still a health problem worldwide, especially in children. The most important proteins of the Shigellatype III secretion system are IpaB and IpaD, which attach to the intestinal epithelial cells and provide the possibility of invasion and disease. These two proteins with immunogenic properties can be a suitable target to design and manufacture subunit recombinant vaccines. Objective: The aim of this study is to design an immunogenic chimeric protein against IpaB and IpaD as a subunit vaccine candidate through an in silicostudy. Methods: Firstly, the immunogenic epitopes of amino acid sequences, physico-chemical parameters, and the allergenicity of the chimeric protein were determined. Then the tertiary structure and the potential ability of the chimeric protein were predicted and evaluated in terms of inducing B cells’ immune responses with effective epitopes. Finally, the optimization of the chimeric protein was examined as the index affecting the protein expression. Results: Data showed an instability index of 37.18 and a well-established predicted third structure for the chimeric protein, with a z-score of -6.11. Also, more than 99% of its amino acids were in the optimal range. Minimum energy for mRNA structure increased to -317.9 and the Codon Adaptive Index (CAI) rose to 88%. The designed protein had no IgE specific B cell epitopes. Conclusion: Overall, the results of this study show that the designed protein can be considered as an immunogen vaccine candidate against S. dysenteriae.

Synopses from the poster exhibition organized by I. M. Kerr, 1. Interferon: a tertiary structure predicted from amino acid sequences

1982 ◽  
Vol 299 (1094) ◽  
pp. 125-127 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Dimensional Structure ◽  
X Ray ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Functional Studies

Functional studies on interferon would be helped by a three-dimensional structure for the molecule. However, it may be several years before the structure of the protein is determined by X-ray crystallography. We have therefore used available methods for predicting the secondary - and the tertiary - structure of a protein from its amino acid sequence to propose a tertiary model involving the packing of four a-helices. Details of this work have been published elsewhere (Sternberg & Cohen 1982).

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