scholarly journals Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate

2020 ◽  
Author(s):  
Christiane Iserman ◽  
Christine Roden ◽  
Mark Boerneke ◽  
Rachel Sealfon ◽  
Grace McLaughlin ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Upper Airway ◽  
Dependent Manner ◽  
Rna Sequences ◽  
Concentration Dependent Manner ◽  
Health Crisis ◽  
Viral Packaging ◽  
N Protein ◽  
Human Body Temperature

AbstractA mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.

Cell surface interactions in conjugation: Tetrahymena ciliary membrane vesicles

1984 ◽  
Vol 4 (4) ◽  
pp. 681-687
Author(s):  
B Love ◽  
M B Rotheim
Keyword(s):  
Cell Surface ◽  
Mating Type ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Surface Interactions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ciliary Membrane ◽  
Adhesion Sites ◽  
Cell Surface Interactions

Tetrahymena ciliary membrane vesicles are shown to interact with preconjugant cells in a mating type-specific way. When cells are treated with vesicles of a different mating type before mixing for conjugation, cell pairing is enhanced, and the normal prepairing period is partially eliminated. This enhancement is mating type specific since it is not observed after pretreatment of cells with vesicles of their own mating type. In contrast, when vesicles are added at the time of mixing of two starved cultures, cell pairing is delayed in a concentration-dependent manner. By varying the conditions, we demonstrated enhancement or inhibition, or both. These results are interpreted in terms of two independent interactions of cells with vesicles. We suggest that first, vesicles substitute for another cell in cell-cell prepairing interaction and second, vesicles compete for adhesion sites produced during the prepairing period. Finally, the data presented are summarized within a speculative framework that calls attention to potential analogies with hormone-receptor signaling in mammalian cells.

scholarly journals Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

2010 ◽  
Vol 107 (5) ◽  
pp. 1870-1875 ◽  
Author(s):  
Alex K. Shalek ◽  
Jacob T. Robinson ◽  
Ethan S. Karp ◽  
Jin Seok Lee ◽  
Dae-Ro Ahn ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Silicon Nanowires ◽  
Mammalian Cells ◽  
Living Cells ◽  
Diverse Range ◽  
Viral Packaging ◽  
Neuronal Progenitor ◽  
Efficient Delivery ◽  
Surface Modified

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

scholarly journals TRAIN (Transcription of Repeats Activates INterferon) in response to chromatin destabilization induced by small molecules in mammalian cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Katerina Leonova ◽  
Alfiya Safina ◽  
Elimelech Nesher ◽  
Poorva Sandlesh ◽  
Rachel Pratt ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Genomic Stability ◽  
Cellular Responses ◽  
Dna Demethylation ◽  
Interferon Response ◽  
Dependent Manner ◽  
Dna Interactions ◽  
P53 Activation ◽  
Nf Kappab

Cellular responses to the loss of genomic stability are well-established, while how mammalian cells respond to chromatin destabilization is largely unknown. We previously found that DNA demethylation on p53-deficient background leads to transcription of repetitive heterochromatin elements, followed by an interferon response, a phenomenon we named TRAIN (Transcription of Repeats Activates INterferon). Here, we report that curaxin, an anticancer small molecule, destabilizing nucleosomes via disruption of histone/DNA interactions, also induces TRAIN. Furthermore, curaxin inhibits oncogene-induced transformation and tumor growth in mice in an interferon-dependent manner, suggesting that anticancer activity of curaxin, previously attributed to p53-activation and NF-kappaB-inhibition, may also involve induction of interferon response to epigenetic derepression of the cellular ‘repeatome’. Moreover, we observed that another type of drugs decondensing chromatin, HDAC inhibitor, also induces TRAIN. Thus, we proposed that TRAIN may be one of the mechanisms ensuring epigenetic integrity of mammalian cells via elimination of cells with desilenced chromatin.

scholarly journals Hydrogen sulfide upregulates acid-sensing ion channels via the MAPK-Erk1/2 signaling pathway

Function ◽  
2021 ◽  
Author(s):  
Zhong Peng ◽  
Stephan Kellenberger
Keyword(s):  
Hydrogen Sulfide ◽  
Ion Channels ◽  
Signaling Pathway ◽  
Cortical Neurons ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Membrane Expression ◽  
Concentration Dependent Manner ◽  
Acid Sensing Ion Channels

Abstract Hydrogen sulfide (H2S) emerged recently as a new gasotransmitter and was shown to exert cellular effects by interacting with proteins, among them many ion channels. Acid-sensing ion channels (ASICs) are neuronal voltage-insensitive Na+ channels activated by extracellular protons. ASICs are involved in many physiological and pathological processes, such as fear conditioning, pain sensation and seizures. We characterize here the regulation of ASICs by H2S. In transfected mammalian cells, the H2S donor NaHS increased the acid-induced ASIC1a peak currents in a time- and concentration-dependent manner. Similarly, NaHS potentiated also the acid-induced currents of ASIC1b, ASIC2a and ASIC3. An upregulation induced by the H2S donors NaHS and GYY4137 was also observed with the endogenous ASIC currents of cultured hypothalamus neurons. In parallel with the effect on function, the total and plasma membrane expression of ASIC1a was increased by GYY4137, as determined in cultured cortical neurons. H2S also enhanced the phosphorylation of extracellular signal-regulated kinase, which belongs to the family of mitogen-activated protein kinases (MAPKs). Pharmacological blockade of the MAPK signaling pathway prevented the GYY4137-induced increase of ASIC function and expression, indicating that this pathway is required for ASIC regulation by H2S. Our study demonstrates that H2S regulates ASIC expression and function, and identifies the involved signaling mechanism. Since H2S shares several roles with ASICs, as e.g. facilitation of learning and memory, protection during seizure activity and modulation of nociception, it may be possible that H2S exerts some of these effects via a regulation of ASIC function.

scholarly journals Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue

2006 ◽  
Vol 397 (2) ◽  
pp. 369-375 ◽  
Author(s):  
Fiona E. Baird ◽  
Jorge J. Pinilla-Tenas ◽  
William L. J. Ogilvie ◽  
Vadival Ganapathy ◽  
Harinder S. Hundal ◽  
...  
Keyword(s):  
Amino Acid ◽  
Mammalian Cells ◽  
Ph Sensitivity ◽  
Amino Acid Transporters ◽  
Dependent Manner ◽  
Histidine Residue ◽  
Concentration Dependent Manner ◽  
Histidine Residues ◽  
System A ◽  
External Ph

System A and N amino acid transporters are key effectors of movement of amino acids across the plasma membrane of mammalian cells. These Na+-dependent transporters of the SLC38 gene family are highly sensitive to changes in pH within the physiological range, with transport markedly depressed at pH 7.0. We have investigated the possible role of histidine residues in the transporter proteins in determining this pH-sensitivity. The histidine-modifying agent DEPC (diethyl pyrocarbonate) markedly reduces the pH-sensitivity of SNAT2 and SNAT5 transporters (representative isoforms of System A and N respectively, overexpressed in Xenopus oocytes) in a concentration-dependent manner but does not completely inactivate transport activity. These effects of DEPC were reversed by hydroxylamine and partially blocked in the presence of excess amino acid substrate. DEPC treatment also blocked a reduction in apparent affinity for Na+ (K0.5Na+) of the SNAT2 transporter at low external pH. Mutation of the highly conserved C-terminal histidine residue to alanine in either SNAT2 (H504A) or SNAT5 (H471A) produced a transport phenotype exhibiting reduced, DEPC-resistant pH-sensitivity with no change in K0.5Na+ at low external pH. We suggest that the pH-sensitivity of these structurally related transporters results at least partly from a common allosteric mechanism influencing Na+ binding, which involves an H+-modifier site associated with C-terminal histidine residues.

scholarly journals SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

PLoS Biology ◽  
2021 ◽  
Vol 19 (10) ◽  
pp. e3001425
Author(s):  
Amanda Jack ◽  
Luke S. Ferro ◽  
Michael J. Trnka ◽  
Eddie Wehri ◽  
Amrut Nadgir ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Genomic Rna ◽  
N Protein ◽  
Infected Cells ◽  
Rna Genome ◽  
Viral Genomic Rna

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection causes Coronavirus Disease 2019 (COVID-19), a pandemic that seriously threatens global health. SARS-CoV-2 propagates by packaging its RNA genome into membrane enclosures in host cells. The packaging of the viral genome into the nascent virion is mediated by the nucleocapsid (N) protein, but the underlying mechanism remains unclear. Here, we show that the N protein forms biomolecular condensates with viral genomic RNA both in vitro and in mammalian cells. While the N protein forms spherical assemblies with homopolymeric RNA substrates that do not form base pairing interactions, it forms asymmetric condensates with viral RNA strands. Cross-linking mass spectrometry (CLMS) identified a region that forms interactions between N proteins in condensates, and truncation of this region disrupts phase separation. We also identified small molecules that alter the formation of N protein condensates and inhibit the proliferation of SARS-CoV-2 in infected cells. These results suggest that the N protein may utilize biomolecular condensation to package the SARS-CoV-2 RNA genome into a viral particle.

scholarly journals STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Evaristus C. Mbanefo ◽  
Ming Yan ◽  
Minkyung Kang ◽  
Sahar A. Alhakeem ◽  
Yingyos Jittayasothorn ◽  
...  
Keyword(s):  
T Cells ◽  
Mammalian Cells ◽  
Therapeutic Potential ◽  
Active Immunization ◽  
Chain Molecule ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Regulate Cell Growth ◽  
V Region

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

scholarly journals Cell surface interactions in conjugation: Tetrahymena ciliary membrane vesicles.

1984 ◽  
Vol 4 (4) ◽  
pp. 681-687 ◽  
Author(s):  
B Love ◽  
M B Rotheim
Keyword(s):  
Cell Surface ◽  
Mating Type ◽  
Mammalian Cells ◽  
Membrane Vesicles ◽  
Surface Interactions ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ciliary Membrane ◽  
Adhesion Sites ◽  
Cell Surface Interactions

Tetrahymena ciliary membrane vesicles are shown to interact with preconjugant cells in a mating type-specific way. When cells are treated with vesicles of a different mating type before mixing for conjugation, cell pairing is enhanced, and the normal prepairing period is partially eliminated. This enhancement is mating type specific since it is not observed after pretreatment of cells with vesicles of their own mating type. In contrast, when vesicles are added at the time of mixing of two starved cultures, cell pairing is delayed in a concentration-dependent manner. By varying the conditions, we demonstrated enhancement or inhibition, or both. These results are interpreted in terms of two independent interactions of cells with vesicles. We suggest that first, vesicles substitute for another cell in cell-cell prepairing interaction and second, vesicles compete for adhesion sites produced during the prepairing period. Finally, the data presented are summarized within a speculative framework that calls attention to potential analogies with hormone-receptor signaling in mammalian cells.

scholarly journals ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

2021 ◽  
Author(s):  
Agnès Marchio ◽  
Christophe Batejat ◽  
Jessica Vanhomwegen ◽  
Maxence Feher ◽  
Quentin Grassin ◽  
...  
Keyword(s):  
Upper Airway ◽  
Digital Pcr ◽  
N Gene ◽  
Rna Sequences ◽  
Subgenomic Rna ◽  
N Protein ◽  
Viral Loads ◽  
Gene Coding ◽  
Diagnosis Method

AbstractRT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

Chemical validation and optimization of pharmacoperones targeting vasopressin type 2 receptor mutant

2018 ◽  
Vol 475 (18) ◽  
pp. 2941-2953 ◽  
Author(s):  
Jo Ann Janovick ◽  
Timothy P. Spicer ◽  
Thomas D. Bannister ◽  
Emery Smith ◽  
Vadivel Ganapathy ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput Screening ◽  
Therapeutic Potential ◽  
Molecular Probe ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Toxicity ◽  
Future Studies ◽  
Receptor Mutant

A series of compounds formerly identified by high-throughput screening was studied for their ability to serve as pharmacoperones for the vasopressin type 2 receptor (V2R) mutant L83Q, which is known to cause nephrogenic diabetes insipidus (NDI). Three compounds were particularly effective in rerouting the mutant receptor in a concentration-dependent manner, were neither agonists nor antagonists, and displayed low cellular toxicity. Compound 1 was most effective and can be used as a molecular probe for future studies of how small molecules may affect NDI caused by mutant V2R. These compounds, however, failed to rescue the V2R Y128S mutant, indicating that the compounds described may not work in the rescue of all known mutants of V2R. Taken collectively, the present studies have now identified a promising lead compound that could function as a pharmacoperone to correct the trafficking defect of the NDI-associated mutant V2R L83Q and thus has the therapeutic potential for the treatment of NDI.

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