scholarly journals Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1106 ◽  
Author(s):  
Mira Laajala ◽  
Minna M. Hankaniemi ◽  
Juha A. E. Määttä ◽  
Vesa P. Hytönen ◽  
Olli H. Laitinen ◽  
...  
Keyword(s):  
Mass Spectrometry Analysis ◽  
Structural Proteins ◽  
Calpain Inhibitor ◽  
Nonstructural Proteins ◽  
Vitro Assay ◽  
Spectrometry Analysis ◽  
Viral Proteases ◽  
Infected Cells ◽  
Calpain 2

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

Modeling of drug-drug interactions between omeprazole and erythromycin with cytochrome P450 3A4 in vitro assay

2020 ◽  
Vol 66 (3) ◽  
pp. 241-249
Author(s):  
P.I. Koroleva ◽  
A.V. Kuzikov ◽  
R.A. Masamrekh ◽  
D.A. Filimonov ◽  
A.V. Dmitriev ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Drug Interactions ◽  
Eradication Therapy ◽  
Mass Spectrometry Analysis ◽  
Cytochrome P450 3A4 ◽  
Vitro Assay ◽  
Spectrometry Analysis ◽  
Omeprazole Sulfone ◽  
Demethylase Activity

In the present study the electrochemical system based on recombinant cytochrome P450 3A4 (CYP3A4) was used for the investigation of potential drug-drug interaction between medicinal preparations employed for Helicobacter pylori eradication therapy. Drug interactions were demonstrated in association of omeprazole as a proton pump inhibitor (PPI) and macrolide antibiotic erythromycin during cytochrome P450 3A4-mediated metabolism. It was shown that in the presence of omeprazole the rate of N-demethylase activity of CYP3A4 to erythromycin measured by means of product (formaldehyde) formation decreased. Mass-spectrometry analysis of omeprazole sulfone as a CYP3A4-mediated metabolite demonstrated the absence of erythromycin influence on CYP3A4-dependent omeprazole metabolism. This phenomenon may be explained by lower spectral dissociation constant of CYP3A4-omeprazole complex (Kd = 18±2 μM) than that of CYP3A4-erythromycin complex (Kd = 52 μM). Using the electrochemical model of electrochemically-driven drug metabolism it is possible to register CYP3A4-mediated catalytic conversion of certain drugs. In vitro experiments of potential CYP3A4-mediated drug-drug interactions are in accordance with in silico modeling with program PASS and PoSMNA descriptors in the case of omeprazole/erythromycin combinations.

scholarly journals Identification of Nrl1 Domains Responsible for Interactions with RNA-Processing Factors and Regulation of Nrl1 Function by Phosphorylation

2021 ◽  
Vol 22 (13) ◽  
pp. 7011
Author(s):  
Barbora Mikolaskova ◽  
Matus Jurcik ◽  
Ingrid Cipakova ◽  
Tomas Selicky ◽  
Jan Jurcik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Processing ◽  
Affinity Purification ◽  
Regulation Of Gene Expression ◽  
Terminal Region ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modification ◽  
Vitro Assay ◽  
Spectrometry Analysis

Pre-mRNA splicing is a key process in the regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, Nrl1 regulates splicing and expression of several genes and non-coding RNAs, and also suppresses the accumulation of R-loops. Here, we report analysis of interactions between Nrl1 and selected RNA-processing proteins and regulation of Nrl1 function by phosphorylation. Bacterial two-hybrid system (BACTH) assays revealed that the N-terminal region of Nrl1 is important for the interaction with ATP-dependent RNA helicase Mtl1 while the C-terminal region of Nrl1 is important for interactions with spliceosome components Ctr1, Ntr2, and Syf3. Consistent with this result, tandem affinity purification showed that Mtl1, but not Ctr1, Ntr2, or Syf3, co-purifies with the N-terminal region of Nrl1. Interestingly, mass-spectrometry analysis revealed that in addition to previously identified phosphorylation sites, Nrl1 is also phosphorylated on serines 86 and 112, and that Nrl1-TAP co-purifies with Cka1, the catalytic subunit of casein kinase 2. In vitro assay showed that Cka1 can phosphorylate bacterially expressed Nrl1 fragments. An analysis of non-phosphorylatable nrl1 mutants revealed defects in gene expression and splicing consistent with the notion that phosphorylation is an important regulator of Nrl1 function. Taken together, our results provide insights into two mechanisms that are involved in the regulation of the spliceosome-associated factor Nrl1, namely domain-specific interactions between Nrl1 and RNA-processing proteins and post-translational modification of Nrl1 by phosphorylation.

scholarly journals Phytochemicals of Minthostachys diffusa Epling and Their Health-Promoting Bioactivities

Foods ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 144
Author(s):  
Immacolata Faraone ◽  
Daniela Russo ◽  
Lucia Chiummiento ◽  
Eloy Fernandez ◽  
Alka Choudhary ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Radical Scavenging ◽  
Ethyl Acetate Fraction ◽  
Mass Spectrometry Analysis ◽  
Antioxidant Power ◽  
Spectrometry Analysis ◽  
In Silico Docking ◽  
Aerial Parts ◽  
Health Promoting

The genus Minthostachys belonging to the Lamiaceae family, and is an important South American mint genus used commonly in folk medicine as an aroma in cooking. The phytochemical-rich samples of the aerial parts of Minthostachys diffusa Epling. were tested for pharmacological and health-promoting bioactivities using in vitro chemical and enzymatic assays. A range of radical scavenging activities of the samples against biological radicals such as nitric oxide and superoxide anion and against synthetic 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radicals, the ferric reducing antioxidant power and the lipid peroxidation inhibition were determined and ranked using the ‘relative antioxidant capacity index’ (RACI). The ethyl acetate fraction showed the highest RACI of +1.12. Analysis of the various fractions’ inhibitory ability against enzymes involved in diabetes (α-amylase and α-glucosidase), and against enzymes associated with Parkinson’s or Alzheimer’s diseases (acetylcholinesterase and butyrylcholinesterase) also suggested that the ethyl acetate fraction was the most active. Liquid chromatography–tandem mass spectrometry analysis of the ethyl acetate fraction showed more than 30 polyphenolic compounds, including triterpenes. The inhibitory cholinesterase effects of the triterpenes identified from M. diffusa were further analysed by in silico docking of these compounds into 3D-structures of acetylcholinesterase and butyrylcholinesterase. This is the first study on pharmacological activities and phytochemical profiling of the aerial parts of M. diffusa, showing that this plant, normally used as food in South America, is also rich in health-promoting phytochemicals.

scholarly journals A Comparative Study of the Antioxidative Effects of Helichrysum italicum and Helichrysum arenarium Infusions

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 380
Author(s):  
Katja Kramberger ◽  
Zala Jenko Pražnikar ◽  
Alenka Baruca Arbeiter ◽  
Ana Petelin ◽  
Dunja Bandelj ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
High Performance ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Morphological Type ◽  
Mass Spectrometry Analysis ◽  
In Vitro Assays ◽  
Spectrometry Analysis ◽  
Stress Related Genes

Helichrysum arenarium (L.) Moench (abbrev. as HA) has a long tradition in European ethnomedicine and its inflorescences are approved as a herbal medicinal product. In the Mediterranean part of Europe, Helichrysum italicum (Roth) G. Don (abbrev. as HI) is more common. Since infusions from both plants are traditionally used, we aimed to compare their antioxidative potential using in vitro assays. Two morphologically distinct HI plants, HIa and HIb, were compared to a commercially available HA product. Genetic analysis using microsatellites confirmed a clear differentiation between HI and HA and suggested that HIb was a hybrid resulting from spontaneous hybridization from unknown HI subspecies. High-performance liquid chromatography–mass spectrometry analysis showed the highest amounts of hydroxycinnamic acids and total arzanol derivatives in HIa, whereas HIb was richest in monohydroxybenzoic acids, caffeic acids, and coumarins, and HA contained the highest amounts of flavonoids, especially flavanones. HIa exhibited the highest radical scavenging activity; it was more efficient in protecting different cell lines from induced oxidative stress and in inducing oxidative stress-related genes superoxide dismutase 1, catalase, and glutathione reductase 1. The antioxidative potential of HI was not only dependent on the morphological type of the plant but also on the harvest date, revealing important information for obtaining the best possible product. Considering the superior properties of HI compared to HA, the evaluation of HI as a medicinal plant could be recommended.

scholarly journals Phytotoxicity of Essential Oils on Selected Weeds: Potential Hazard on Food Crops

Plants ◽  
2018 ◽  
Vol 7 (4) ◽  
pp. 79 ◽  
Author(s):  
María Ibáñez ◽  
María Blázquez
Keyword(s):  
Seed Germination ◽  
Essential Oil ◽  
Essential Oils ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Food Crops ◽  
Potential Hazard ◽  
Spectrometry Analysis

The chemical composition of winter savory, peppermint, and anise essential oils, and in vitro and in vivo phytotoxic activity against weeds (Portulaca oleracea, Lolium multiflorum, and Echinochloa crus-galli) and food crops (maize, rice, and tomato), have been studied. Sixty-four compounds accounting for between 97.67–99.66% of the total essential oils were identified by Gas Chromatography-Mass Spectrometry analysis. Winter savory with carvacrol (43.34%) and thymol (23.20%) as the main compounds produced a total inhibitory effect against the seed germination of tested weed. Menthol (48.23%), menthone (23.33%), and iso-menthone (16.33%) from peppermint only showed total seed germination inhibition on L. multiflorum, whereas no significant effects were observed with trans-anethole (99.46%) from anise at all concentrations (0.125–1 µL/mL). Low doses of peppermint essential oil could be used as a sustainable alternative to synthetic agrochemicals to control L. multiflorum. The results corroborate that in vivo assays with a commercial emulsifiable concentrate need higher doses of the essential oils to reproduce previous in vitro trials. The higher in vivo phytotoxicity of winter savory essential oil constitutes an eco-friendly and less pernicious alternative to weed control. It is possible to achieve a greater in vivo phytotoxicity if less active essential oil like peppermint is included with other active excipients.

scholarly journals Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories

2018 ◽  
Vol 115 (51) ◽  
pp. E12015-E12023 ◽  
Author(s):  
Jeanette M. Criglar ◽  
Ramakrishnan Anish ◽  
Liya Hu ◽  
Sue E. Crawford ◽  
Banumathi Sankaran ◽  
...  
Keyword(s):  
Lattice Structure ◽  
Crystallographic Analysis ◽  
Kinase Assay ◽  
Nonstructural Proteins ◽  
Cytoplasmic Inclusions ◽  
Protein Levels ◽  
Dual Specificity ◽  
Infected Cells ◽  
Phosphorylation Cascade

The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication.

scholarly journals Data on peptides identified by mass spectrometry analysis of in vitro DYRK1A-mediated phosphorylation sites on GLI1

Data in Brief ◽  
2017 ◽  
Vol 15 ◽  
pp. 577-583 ◽  
Author(s):  
Ben K. Ehe ◽  
David R. Lamson ◽  
Michael Tarpley ◽  
Rob U. Onyenwoke ◽  
Lee M. Graves ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Analysis ◽  
Phosphorylation Sites ◽  
Spectrometry Analysis

scholarly journals Deubiquitination and Stabilization of PD-L1 by USP21

2021 ◽  
Author(s):  
Shuying Yang ◽  
Youqian Wu ◽  
Huanhuan Yan ◽  
Bing Shan ◽  
Dongheng Zhou ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Lung Squamous Cell Carcinoma ◽  
Mass Spectrometry Analysis ◽  
Polyubiquitin Chains ◽  
Spectrometry Analysis ◽  
Protein Levels ◽  
Programmed Death ◽  
Programmed Death Protein 1 ◽  
Novel Strategy

Abstract Background: The immunotherapy for different types of cancers that targeting programmed death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) has highlighted the importance of suppressing specific T cell responses. Recently, several studies have shown that the expression level of PD-L1 in tumor cells is positively correlated with tumor metastasis as well as recurrence rate. The potent effects of post-translational modifications (PTMs) for PD-L1, such as ubiquitination, glycosylation, phosphorylation and palmitoylation, have been reported to be related to immunosuppression. However, the regulation of PD-L1 degradation in cancers is still not well understood. In this paper, we mainly investigate the deubiquitination regulation of PD-L1. Methods: The protein levels of PD-L1 and USP21 were detected by Immunoblotting and immunohistochemistry. The interaction between PD-L1 and USP21 was determined by co-immunoprecipitation. The deubiquitination of PD-L1 was determined by in vitro deubiquitination assay. The deubiquitination sites of PD-L1 were identified by mass spectrometry analysis. The expression of mRNA in target tissues was presented by bioinformatics analysis.Results: Overexpression of USP21 significantly increased PD-L1 abundance and knockdown of USP21 induced degradation of PD-L1. In vitro deubiquitination assay showed that USP21-WT reduced polyubiquitin chains from PD-L1 while USP21-C221A did not. Furthermore, five lysines in intracellular segment of PD-L1 are potential deubiquitin sites and cancer-derived mutations of PD-L1 in Asp276 have the ability to enhance the deubiquitination of PD-L1 mediated by USP21. Finally, we found that USP21 is the frequently amplified deubiquitinase in lung cancer, especially in lung squamous cell carcinoma, and its amplification co-occurs with the upregulation of PD-L1 levels. Moreover, IHC analysis showed stronger staining of PD-L1 and USP21 in lung cancer samples than adjacent tissues. Conclusion: We identified USP21 as a novel deubiquitinase of PD-L1. Hopefully, targeting PD-L1 by inhibiting USP21 might be a potentially novel strategy for the treatment of lung cancer.

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