scholarly journals Molecular Architecture of the Essential Yeast Histone Acetyltransferase Complex NuA4 Redefines Its Multimodularity

2018 ◽  
Vol 38 (9) ◽  
Author(s):  
Dheva Setiaputra ◽  
Salar Ahmad ◽  
Udit Dalwadi ◽  
Anne-Lise Steunou ◽  
Shan Lu ◽  
...  
Keyword(s):  
Cell Fate ◽  
Histone Acetyltransferase ◽  
Structural Information ◽  
Regulation Of Gene Expression ◽  
Mass Spectrometry Analysis ◽  
Molecular Architecture ◽  
Catalytic Core ◽  
Spectrometry Analysis ◽  
Subunit Organization

ABSTRACT Conserved from yeast to humans, the NuA4 histone acetyltransferase is a large multisubunit complex essential for cell viability through the regulation of gene expression, genome maintenance, metabolism, and cell fate during development and stress. How the different NuA4 subunits work in concert with one another to perform these diverse functions remains unclear, and addressing this central question requires a comprehensive understanding of NuA4's molecular architecture and subunit organization. We have determined the structure of fully assembled native yeast NuA4 by single-particle electron microscopy. Our data revealed that NuA4 adopts a trilobal overall architecture, with each of the three lobes constituted by one or two functional modules. By performing cross-linking coupled to mass spectrometry analysis and in vitro protein interaction studies, we further mapped novel intermolecular interfaces within NuA4. Finally, we combined these new data with other known structural information of NuA4 subunits and subassemblies to construct a multiscale model to illustrate how the different NuA4 subunits and modules are spatially arranged. This model shows that the multiple chromatin reader domains are clustered together around the catalytic core, suggesting that NuA4's multimodular architecture enables it to engage in multivalent interactions with its nucleosome substrate.

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 Identification of DNAJA1 as a novel interacting partner and a substrate of human transglutaminase 2

2016 ◽  
Vol 473 (21) ◽  
pp. 3889-3901 ◽  
Author(s):  
Elvan Ergülen ◽  
Bálint Bécsi ◽  
István Csomós ◽  
László Fésüs ◽  
Kajal Kanchan
Keyword(s):  
Molecular Mechanisms ◽  
Transglutaminase 2 ◽  
Mass Spectrometry Analysis ◽  
Catalytic Core ◽  
Spectrometry Analysis ◽  
Inactive Form ◽  
In Situ Conditions ◽  
And Migration ◽  
Regulated Functions

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional member of the transglutaminase enzyme family. It has been implicated to have roles in many physiological and pathological processes such as differentiation, apoptosis, signal transduction, adhesion and migration, wound healing and inflammation. Previous studies revealed that TG2 has various intra- and extra-cellular interacting partners, which contribute to these processes. In the present study, we identified a molecular co-chaperone, DNAJA1, as a novel interacting partner of human TG2 using a GST pull-down assay and subsequent mass spectrometry analysis, and further confirmed this interaction via ELISA and surface plasmon resonance measurements. Interaction studies were also performed with domain variants of TG2 and results suggest that the catalytic core domain of TG2 is essential for the TG2–DNAJA1 interaction. Cross-linking activity was not essential for the interaction since DNAJA1 was also found to interact with the catalytically inactive form of TG2. Furthermore, we have showed that DNAJA1 interacts with the open form of TG2 and regulates its transamidation activity under both in vitro and in situ conditions. We also found that DNAJA1 is a glutamine donor substrate of TG2. Since DNAJA1 and TG2 are reported to regulate common pathological conditions such as neurodegenerative disorders and cancer, the findings in the present paper open up possibilities to explore molecular mechanisms behind TG2-regulated functions.

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 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.

Coronatin-2 from the entomopathogenic fungus Conidiobolus coronatus kills Galleria mellonella larvae and incapacitates hemocytes

2016 ◽  
Vol 107 (1) ◽  
pp. 66-76 ◽  
Author(s):  
M.I. Boguś ◽  
W. Wieloch ◽  
M. Ligęza-Żuber
Keyword(s):  
Entomopathogenic Fungus ◽  
Galleria Mellonella ◽  
Elongation Factor ◽  
Peptide Sequence ◽  
Mass Spectrometry Analysis ◽  
Sequence Coverage ◽  
Spectrometry Analysis ◽  
Conidiobolus Coronatus ◽  
Liquid Chromatography Tandem Mass

AbstractCoronatin-2, a 14.5 kDa protein, was isolated from culture filtrates of the entomopathogenic fungus Conidiobolus coronatus (Costantin) Batko (Entomophthoramycota: Entomophthorales). After LC–MS/MS (liquid chromatography tandem mass spectrometry) analysis of the tryptic peptide digest of coronatin-2 and a mass spectra database search no orthologs of this protein could be found in fungi. The highest homology was observed to the partial translation elongation factor 1a from Sphaerosporium equinum (protein sequence coverage, 21%), with only one peptide sequence, suggesting that coronatin-2 is a novel fungal protein that has not yet been described. In contrast to coronatin-1, an insecticidal 36 kDa protein, which shows both elastolytic and chitinolytic activity, coronatin-2 showed no enzymatic activity. Addition of coronatin-2 into cultures of hemocytes taken from larvae of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), resulted in progressive disintegration of nets formed by granulocytes and plasmatocytes due to rapid degranulation of granulocytes, extensive vacuolization of plasmatocytes accompanied by cytoplasm expulsion, and cell disintegration. Spherulocytes remained intact, while oenocytes rapidly disintegrated. Coronatin-2 produced 80% mortality when injected into G. mellonella at 5 µg larva−1. Further study is warranted to determine the relevance of the acute toxicity of coronatin-2 and its effects on hemocytes in vitro to virulence of C. coronatus against its hosts.

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