scholarly journals New RNA Structural Elements Identified in the Coding Region of the Coxsackie B3 Virus Genome

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1232
Author(s):  
Mariola Dutkiewicz ◽  
Jakub Kuczynski ◽  
Michal Jarzab ◽  
Aleksandra Stachowiak ◽  
Agata Swiatkowska
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Magnesium Concentration ◽  
Dependent Manner ◽  
Structural Elements ◽  
Coding Region ◽  
Virus Propagation ◽  
Concentration Dependent Manner ◽  
Computer Prediction ◽  
Coding Regions

Here we present a set of new structural elements formed within the open reading frame of the virus, which are highly probable, evolutionarily conserved and may interact with host proteins. This work focused on the coding regions of the CVB3 genome (particularly the V4-, V1-, 2C-, and 3D-coding regions), which, with the exception of the cis-acting replication element (CRE), have not yet been subjected to experimental analysis of their structures. The SHAPE technique, chemical modification with DMS and RNA cleavage with Pb2+, were performed in order to characterize the RNA structure. The experimental results were used to improve the computer prediction of the structural models, whereas a phylogenetic analysis was performed to check universality of the newly identified structural elements for twenty CVB3 genomes and 11 other enteroviruses. Some of the RNA motifs turned out to be conserved among different enteroviruses. We also observed that the 3′-terminal region of the genome tends to dimerize in a magnesium concentration-dependent manner. RNA affinity chromatography was used to confirm RNA–protein interactions hypothesized by database searches, leading to the discovery of several interactions, which may be important for virus propagation.

scholarly journals Effect of the divalent cations zinc and calcium on the structure and mechanics of reconstituted vimentin intermediate filaments

2019 ◽  
Author(s):  
Huayin Wu ◽  
Yinan Shen ◽  
Dianzhuo Wang ◽  
Harald Herrmann ◽  
Robert D. Goldman ◽  
...  
Keyword(s):  
Intermediate Filaments ◽  
Network Structure ◽  
Protein Interactions ◽  
Divalent Cations ◽  
Dependent Manner ◽  
Structural Elements ◽  
Concentration Dependent Manner ◽  
Hofmeister Effect ◽  
Ion Species

AbstractDivalent cations in a concentration-dependent manner behave as effective crosslinkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here we investigate the effects of two biologically important divalent cations, Zn2+ and Ca2+, on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn2+ concentrations, but strong bundle formation is observed at a concentration of 100 μM. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca2+, but remarkably, a concentration of Ca2+ that is two orders higher is needed to achieve the same effect as with Zn2+, which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics.SignificanceIntermediate filaments are key structural elements within cells; they are known to form networks that can be crosslinked by divalent cations, but the interactions between the ions and the filaments are not well understood. By measuring the effects that two divalent cations, zinc and calcium, have on the structure and mechanics of vimentin intermediate filaments (VIFs), we show that although both have concentration-dependent effects on VIFs, much more calcium is needed to achieve the same effect as a small amount of zinc. Furthermore, when mixtures of the ions are present, the results suggest that there is binding competition. Thus, cells may use the presence of different cation species to precisely control their internal mechanical properties.

Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery

1993 ◽  
Vol 13 (1) ◽  
pp. 399-407
Author(s):  
I J McEwan ◽  
A P Wright ◽  
K Dahlman-Wright ◽  
J Carlstedt-Duke ◽  
J A Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Protein Interactions ◽  
Core Promoter ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

scholarly journals Amino Acid Starvation Enhances Programmed Ribosomal Frameshift in Metavirus Ty3 of Saccharomyces cerevisiae

2016 ◽  
Vol 2016 ◽  
pp. 1-6
Author(s):  
Sezai Türkel
Keyword(s):  
Amino Acid ◽  
Amino Acid Starvation ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Coding Region ◽  
Coding Regions ◽  
In The Wild ◽  
Amino Acid Depletion ◽  
A Site ◽  
Wild Type Yeast

Ty3 is a retroviral-like element and propagates with a retroviral-like mechanism within the yeast cells. Ty3 mRNA contains two coding regions, which are GAG3 and POL3. The coding region POL3 is translated as a GAG3-POL3 fusion protein by a +1 programmed frameshift. In this study, it was shown that the Ty3 frameshift frequency is significantly increased by amino acid starvation in a Gcn2p complex dependent manner. When the yeast cells were subjected to amino acid starvation, the frameshift frequency of Ty3 increased more than 2-fold in the wild-type yeast cells, mostly independent of Gcn4p. However, Ty3 frameshift frequency remained at basal level in the gcn1, gcn20, or gcn2 mutant yeast cells in amino acid starved yeasts. Gcn1p forms a complex with Gcn2p and Gcn20p and is involved in the sensing of uncharged tRNAs on the ribosomal A-site during translation. Increases in uncharged tRNA levels due to amino acid depletion lead to ribosomal pauses. These ribosomal pauses are significant actors in the regulation of Ty3 frameshift frequency. Results of this research revealed that frameshift frequency in Ty3 is regulated by the Gcn2p complex in response to amino acid starvation in yeast.

Nuclear receptor action involved with sex differentiation

2003 ◽  
Vol 75 (11-12) ◽  
pp. 1771-1784 ◽  
Author(s):  
I. A. Hughes ◽  
Howard Martin ◽  
Jarmo Jääskeläinen ◽  
C. L. Acerini
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Reproductive Tract ◽  
Sex Differentiation ◽  
Disorders Of Sex Development ◽  
Nuclear Hormone Receptor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Sex Development ◽  
Sequence Of Events

Sex determination and differentiation in the male is an orderly sequence of events coordinated by genetic and hormonal factors operating in a time- and concentration-dependent manner. The constitutive sex in mammals is female. Disorders of fetal sex development have provided the means to identify testis-determining genes and the molecular mechanisms of hormone action. Thus, the androgen receptor, a nuclear hormone receptor critical for androgen-induced male sex differentiation, displays unique intra-receptor and protein-protein interactions which, when disturbed, can result in extreme forms of sex reversal. Polymorphic variants are associated with milder disorders of sex development. Against this genetic background, endocrine active substances may further contribute to the underlying causes of an increase in male reproductive tract disorders.

scholarly journals Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

2020 ◽  
Vol 21 (20) ◽  
pp. 7605
Author(s):  
Pavel V. Ershov ◽  
Alexander V. Veselovsky ◽  
Yuri V. Mezentsev ◽  
Evgeniy O. Yablokov ◽  
Leonid A. Kaluzhskiy ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Wide Spectrum ◽  
Three Dimensional ◽  
3D Models ◽  
Bioinformatic Analysis ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Interacting Protein ◽  
Enhancing Effect

Isatin (indole-2, 3-dione) is a non-peptide endogenous bioregulator exhibiting a wide spectrum of biological activity, realized in the cell via interactions with numerous isatin-binding proteins, their complexes, and (sub) interactomes. There is increasing evidence that isatin may be involved in the regulation of complex formations by modulating the affinity of the interacting protein partners. Recently, using Surface Plasmon Resonance (SPR) analysis, we have found that isatin in a concentration dependent manner increased interaction between two human mitochondrial proteins, ferrochelatase (FECH), and adrenodoxine reductase (ADR). In this study, we have investigated the affinity-enhancing effect of isatin on the FECH/ADR interaction. The SPR analysis has shown that FECH forms not only homodimers, but also FECH/ADR heterodimers. The affinity-enhancing effect of isatin on the FECH/ADR interaction was highly specific and was not reproduced by structural analogues of isatin. Bioinformatic analysis performed using three dimensional (3D) models of the interacting proteins and in silico molecular docking revealed the most probable mechanism involving FECH/isatin/ADR ternary complex formation. In this complex, isatin is targeted to the interface of interacting FECH and ADR monomers, forming hydrogen bonds with both FECH and ADR. This is a new regulatory mechanism by which isatin can modulate protein–protein interactions (PPI).

scholarly journals Methylene Blue Inhibits the SARS-CoV-2 Spike–ACE2 Protein-Protein Interaction–a Mechanism that can Contribute to its Antiviral Activity Against COVID-19

2021 ◽  
Vol 11 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Peter Buchwald
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viral Attachment ◽  
Protein Protein Interaction

Due to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction. Methylene blue inhibited the entry of a SARS-CoV-2 spike bearing pseudovirus into ACE2-expressing cells with similar IC50 (3.5 μM). Hence, this PPI inhibitory activity could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.

scholarly journals Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery.

1993 ◽  
Vol 13 (1) ◽  
pp. 399-407 ◽  
Author(s):  
I J McEwan ◽  
A P Wright ◽  
K Dahlman-Wright ◽  
J Carlstedt-Duke ◽  
J A Gustafsson
Keyword(s):  
Glucocorticoid Receptor ◽  
Protein Interactions ◽  
Core Promoter ◽  
Specific Interaction ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Concentration Dependent Manner

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.

scholarly journals Detection of Envelope Glycoprotein Assembly from Old-World Hantaviruses in the Golgi Apparatus of Living Cells

2020 ◽  
Author(s):  
R. A. Petazzi ◽  
A. A. Koikkarah ◽  
N.D. Tischler ◽  
S. Chiantia
Keyword(s):  
Golgi Apparatus ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Living Cells ◽  
Viral Envelope ◽  
Fluctuation Analysis ◽  
Dependent Manner ◽  
Emerging Pathogens ◽  
Concentration Dependent Manner ◽  
Cellular Models

AbstractHantaviruses are emerging pathogens that occasionally cause deadly outbreaks in the human population. While the structure of the viral envelope has been characterized with high precision, the protein-protein interactions leading to the formation of new virions in infected cells are not fully understood yet. In this work, we use quantitative fluorescence microscopy (i.e. Number&Brightness analysis and fluorescence fluctuation spectroscopy) to quantify the interactions that lead to oligomeric spike complex formation in the physiological context of living cells. To this aim, we have analyzed proteins from Puumala and Hantaan orthohantaviruses in several cellular models. For the first time, we quantified the oligomerization state of each protein in relation to its subcellular localization, concentration and the concentration of its interaction partner. Our results indicate that when expressed separately, both glycoproteins can form homo-multimers in a concentration-dependent manner. Fluorescence fluctuation analysis was applied to prove that Gc:Gc contacts observed on virions are also relevant for Gc-Gc interactions in living cells, in the absence of Gn. Furthermore, we proved that the membrane-distal lobes of Gn are not necessary for Gn homo-multimerization. In cells co-expressing both glycoproteins, we observe clear indication of Gn-Gc interactions and the formation of protein complexes with different sizes, while using various labelling schemes to minimize the influence of the fluorescent tags. Our data are compatible with an assembly model according to which hantavirus spikes are formed via the assembly of Gn-Gc hetero-dimers. Furthermore, our results indicate the interconnection of large Gn-Gc hetero-multimers in the Golgi apparatus. Such large glycoprotein multimers may be identified as multiple interacting viral spikes and provide a possible first evidence for the initial assembly steps of the viral envelope, within this organelle, directly in living cells.

scholarly journals A novel luminescence-based β-arrestin membrane recruitment assay for unmodified GPCRs

2020 ◽  
Author(s):  
Maria Hauge Pedersen ◽  
Jennifer Pham ◽  
Helena Mancebo ◽  
Asuka Inoue ◽  
Jonathan A. Javitch
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Receptor Activation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Membrane Recruitment ◽  
Terminal Fragment ◽  
Downstream Effectors ◽  
Agonist Concentration ◽  
Membrane Tether

AbstractG protein-coupled receptors (GPCRs) signal through activation of G proteins and subsequent modulation of downstream effectors. More recently, G protein-independent signaling via the arrestin pathway has also been implicated in important physiological functions. This has led to great interest in the identification of biased ligands that favor either the G protein or arrestin-signaling pathways. Currently available screening techniques that measure arrestin recruitment have required C-terminal receptor modifications that can in principle alter protein interactions and thus signaling. Here, we have developed a novel luminescence-based assay to measure arrestin recruitment to any unmodified receptor.NanoLuc, an engineered luciferase from ophlorus gracilirostris (deep sea shrimp), is smaller and brighter than other well-established luciferases. Recently, several publications have explored functional NanoLuc split sites for use in complementation assays. Here, we have identified a novel split site and have fused the N-terminal fragment to a membrane tether and the C-terminal fragment to the N-terminus of either β-arrestin 1 or 2. Upon receptor activation, arrestin is recruited to the plasma membrane in an agonist concentration-dependent manner and the two NanoLuc fragments complement to reconstitute functional luciferase, which allows quantification of recruitment with a single luminescence signal. Our assay avoids potential artifacts related to C-terminal receptor modification. The split NanoLuc arrestin recruitment assay has promise as a new generic assay for measuring arrestin recruitment to diverse GPCR types in heterologous or native cells.

scholarly journals Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

2020 ◽  
Author(s):  
Damir Bojadzic ◽  
Oscar Alcazar ◽  
Peter Buchwald
Keyword(s):  
Methylene Blue ◽  
Antiviral Activity ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Organic Dyes ◽  
Chemical Space ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viral Attachment

AbstractDue to our interest in the chemical space of organic dyes to identify potential small-molecule inhibitors (SMIs) for protein-protein interactions (PPIs), we initiated a screen of such compounds to assess their inhibitory activity against the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this coronavirus responsible for the ongoing COVID-19 pandemic. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved by the FDA for the treatment of methemoglobinemia and used for other medical applications (including the inactivation of viruses in blood products prior to transfusion when activated by light), inhibits this interaction. We confirmed that it does so in a concentration-dependent manner with a low micromolar half-maximal inhibitory concentration (IC50 = 3 μM) in our protein-based ELISA-type setup, while chloroquine, siramesine, and suramin showed no inhibitory activity in this assay. Erythrosine B, which we have shown before to be a promiscuous SMI of PPIs, also inhibited this interaction with an activity similar, possibly slightly higher, than those found for it for other PPIs. This PPI inhibitory activity of methylene blue could contribute to its antiviral activity against SARS-CoV-2 even in the absence of light by blocking its attachment to ACE2-expressing cells and making this inexpensive and widely available drug potentially useful in the prevention and treatment of COVID-19 as an oral or inhaled medication.

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