scholarly journals 31P NMR Spectroscopy Demonstrates Large Amounts of Phosphohistidine in Mammalian Cells

2020 ◽  
Author(s):  
Mehul V. Makwana ◽  
Sandra van Meurs ◽  
Andrea M. Hounslow ◽  
Mike P. Williamson ◽  
Richard F. W. Jackson ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Bronchial Epithelial Cell ◽  
Phosphoryl Group ◽  
Accurate Information ◽  
31P Nmr Spectroscopy ◽  
Cellular Processes ◽  
Cell Lysate ◽  
Histidine Phosphorylation ◽  
In Cells

AbstractProtein phosphorylation plays a key role in many cellular processes but there is presently no accurate information or reliable procedure to determine the relative abundance of many phosphoamino acids in cells. At pH ≤ 8, phosphohistidine is unstable compared to the extensively studied phosphoserine, phosphothreonine and phosphotyrosine. This study reports the absolute quantitative analysis of histidine phosphorylation of proteins from a human bronchial epithelial cell (16HBE14o-) lysate using 31P NMR spectroscopic analysis. The method was designed to minimize loss of the phosphohistidine phosphoryl group. Phosphohistidine was determined on average to be approximately one third as abundant as phosphoserine and phosphothreonine combined (and thus roughly 20 times more abundant than phosphotyrosine). The amount of phosphohistidine, and phosphoserine/phosphothreonine per gram of protein from a cell lysate was determined to be 23 μmol/g and 68 μmol/g respectively. The amount of phosphohistidine, and phosphoserine/phosphothreonine per cell was determined to be 1.8 fmol/cell, and 5.8 fmol/cell respectively. After tryptic digest of proteins from the16HBE14o- cell lysate, the phosphohistidine signal was abolished and increasing phosphoserine/phosphothreonine signal was observed, which has implications for mass spectrometry investigations. The 31P NMR spectroscopic analysis not only highlights the abundance of phosphohistidine, which likely reflects its importance in mammalian cells, but also provides a way of measuring and comparing levels of phosphorylated amino acids in cells.

scholarly journals Lamin C regulates genome organization after mitosis

2020 ◽  
Author(s):  
X Wong ◽  
VE Hoskins ◽  
JC Harr ◽  
M Gordon ◽  
KL Reddy
Keyword(s):  
Genome Organization ◽  
Mammalian Cells ◽  
Regulation Of Gene Expression ◽  
Nuclear Periphery ◽  
Nuclear Lamina ◽  
Lamin A ◽  
3D Genome ◽  
Cellular Processes ◽  
Genomic Regions ◽  
In Cells

AbstractThe dynamic 3D organization of the genome is central to the regulation of gene expression and developmental progression, with its disruption being implicated in various diseases. The nuclear lamina, a proteinaceous meshwork underlying the nuclear envelope (NE), provides both structural and regulatory influences on genome organization through the tethering of large inactive genomic regions, called Lamina Associated Domains (LADs), to the nuclear periphery. Evidence suggests that the A type lamins, lamins A and C, are the predominant lamins involved in the peripheral association of LADs, with these two isotypes forming distinct networks and potentially involved in different cellular processes. Here we tested whether lamins A and C have distinct roles in genome organization by examining chromosome architecture in cells in which lamin C or lamin A are specifically down-regulated. We find that lamin C (not lamin A) is required for the 3D organization of LADs and overall chromosome organization in the cell nucleus. Striking differences in the localization of lamin A and lamin C are present as cells exit mitosis that persist through early G1. Whereas lamin A associates with the nascent NE during telophase, lamin C remains in the interior surrounding nucleoplasmic LAD clusters. Lamin C association with the NE is delayed until several hours into G1 and correlates temporally and spatially with the post-mitotic NE association of LADs. Post-mitotic LAD association with the NE, and consequently global 3D genome organization, is perturbed only in cells depleted of lamin C, and not in cells depleted of lamin A. We conclude that lamin C regulates LAD dynamics after mitosis and is a key regulator of genome organization in mammalian cells. These findings reveal an unexpectedly central role for lamin C in genome organization, including both inter-chromosomal LAD-LAD segregation and LAD scaffolding at the NE.

scholarly journals Burkholderia cenocepacia transcriptome during the early contacts with giant plasma membrane vesicles derived from live bronchial epithelial cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Andreia I. Pimenta ◽  
Nuno Bernardes ◽  
Marta M. Alves ◽  
Dalila Mil-Homens ◽  
Arsenio M. Fialho
Keyword(s):  
Membrane Vesicles ◽  
Bronchial Epithelial Cell ◽  
Host Cells ◽  
Burkholderia Cenocepacia ◽  
Transport Systems ◽  
Epithelial Cell Line ◽  
Plasma Membrane Vesicles ◽  
Bronchial Epithelial ◽  
Cellular Processes ◽  
Associated Functions

AbstractBurkholderia cenocepacia is known for its capacity of adherence and interaction with the host, causing severe opportunistic lung infections in cystic fibrosis patients. In this work we produced Giant Plasma Membrane Vesicles (GPMVs) from a bronchial epithelial cell line and validated their use as a cell-like alternative to investigate the steps involved in the adhesion process of B. cenocepacia. RNA-sequencing was performed and the analysis of the B. cenocepacia K56-2 transcriptome after the first contacts with the surface of host cells allowed the recognition of genes implicated in bacterial adaptation and virulence-associated functions. The sensing of host membranes led to a transcriptional shift that caused a cascade of metabolic and physiological adaptations to the host specific environment. Many of the differentially expressed genes encode proteins related with central metabolic pathways, transport systems, cellular processes, and virulence traits. The understanding of the changes in gene expression that occur in the early steps of infection can uncover new proteins implicated in B. cenocepacia-host cell adhesion, against which new blocking agents could be designed to control the progression of the infectious process.

scholarly journals Miga-mediated endoplasmic reticulum–mitochondria contact sites regulate neuronal homeostasis

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lingna Xu ◽  
Xi Wang ◽  
Jia Zhou ◽  
Yunyi Qiu ◽  
Weina Shang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Mitochondrial Dynamics ◽  
Molecular Organization ◽  
Lipid Transport ◽  
Cellular Processes ◽  
Contact Sites ◽  
Neuronal Homeostasis ◽  
Higher Eukaryotes ◽  
Lateral Sclerosis

Endoplasmic reticulum (ER)–mitochondria contact sites (ERMCSs) are crucial for multiple cellular processes such as calcium signaling, lipid transport, and mitochondrial dynamics. However, the molecular organization, functions, regulation of ERMCS, and the physiological roles of altered ERMCSs are not fully understood in higher eukaryotes. We found that Miga, a mitochondrion located protein, markedly increases ERMCSs and causes severe neurodegeneration upon overexpression in fly eyes. Miga interacts with an ER protein Vap33 through its FFAT-like motif and an amyotrophic lateral sclerosis (ALS) disease related Vap33 mutation considerably reduces its interaction with Miga. Multiple serine residues inside and near the Miga FFAT motif were phosphorylated, which is required for its interaction with Vap33 and Miga-mediated ERMCS formation. The interaction between Vap33 and Miga promoted further phosphorylation of upstream serine/threonine clusters, which fine-tuned Miga activity. Protein kinases CKI and CaMKII contribute to Miga hyperphosphorylation. MIGA2, encoded by the miga mammalian ortholog, has conserved functions in mammalian cells. We propose a model that shows Miga interacts with Vap33 to mediate ERMCSs and excessive ERMCSs lead to neurodegeneration.

scholarly journals Methylation of deoxyribonucleic acid in cultured mammalian cells by N-methyl-N′-nitro-N-nitrosoguanidine. The influence of cellular thiol concentrations on the extent of methylation and the 6-oxygen atom of guanine as a site of methylation

1970 ◽  
Vol 116 (4) ◽  
pp. 693-707 ◽  
Author(s):  
P. D. Lawley ◽  
Carolyn J. Thatcher
Keyword(s):  
Oxygen Atom ◽  
Nucleic Acids ◽  
Mammalian Cells ◽  
Dimethyl Sulphate ◽  
Biological Effects ◽  
Methylation Of Dna ◽  
Thiol Content ◽  
Cellular Thiol ◽  
In Cells

1. In neutral aqueous solution N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) yields salts of nitrocyanamide as u.v.-absorbing products. With cysteine, as found independently by Schulz & McCalla (1969), the principal product is 2-nitràminothiazoline-4-carboxylic acid. Both these reactions liberate the methylating species; thiols enhance the rate markedly at neutral pH values. An alternative reaction with thiols gives cystine, presumably via the unstable S-nitrosocysteine. 2. Thiols (glutathione or N-acetylcysteine) in vitro at about the concentration found in mammalian cells enhance the rate of methylation of DNA markedly over that in neutral solution. 3. Treatment of cultured mammalian cells with MNNG results in rapid methylation of nucleic acids, the extent being greater the higher the thiol content of the cells. Rodent embryo cells are more extensively methylated than mouse L-cells of the same thiol content. Cellular thiol concentrations are decreased by MNNG. Proteins are less methylated by MNNG than are nucleic acids. 4. Methylation of cells by dimethyl sulphate does not depend on cellular thiol content and protein is not less methylated than nucleic acids. Methylation by MNNG may therefore be thiol-stimulated in cells. 5. Both in vitro and in cells about 7% of the methylation of DNA by MNNG occurs at the 6-oxygen atom of guanine. The major products 7-methylguanine and 3-methyladenine are given by both MNNG and dimethyl sulphate, but dimethyl sulphate does not yield O6-methylguanine. Possible reaction mechanisms to account for this difference between these methylating agents and its possible significance as a determinant of their biological effects are discussed.

scholarly journals Diffusion in the aqueous compartment.

1984 ◽  
Vol 99 (1) ◽  
pp. 180s-187s ◽  
Author(s):  
A M Mastro ◽  
A D Keith
Keyword(s):  
Rotational Motion ◽  
Spin Label ◽  
Mammalian Cells ◽  
Translational Motion ◽  
Diffusion Constant ◽  
Rotational Correlation Time ◽  
Model Systems ◽  
Quiescent Cells ◽  
Spin Resonance ◽  
In Cells

Measurements of diffusion of molecules in cells can provide information about cytoplasmic viscosity and structure. In a series of studies electron-spin resonance was used to measure the diffusion of a small spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity of the cytoplasm from both rotational and translational motion would distinguish between the effects of viscosity and structure on diffusion. The diffusion constant measured in several cell lines averaged 3.3 X 10(-6) cm2/s. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion constant or the rotational correlation time was 2.0-3.0 centipoise, about two to three times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiologic conditions. However, when cells were subjected to hypertonic conditions, the translational motion of the spin label decreased threefold, whereas the rotational motion changed by less than 20%. These data suggest that the decrease in cell volume under hypertonic conditions is accompanied by an increase in cytoplasmic barriers and a decrease in the space between existing cytoplasmic components without a significant increase in viscosity in the aqueous phase. In addition, a comparison of reported diffusion values of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.

Phosphotransfer and Nucleotidyltransfer

2007 ◽  
Author(s):  
Perry A. Frey ◽  
Adrian D. Hegeman
Keyword(s):  
Transition State ◽  
Molecular Mechanisms ◽  
Enzymatic Reaction ◽  
Covalent Bond ◽  
Phosphoryl Group ◽  
Dissociative Mechanism ◽  
Chemical Mechanisms ◽  
Cellular Processes ◽  
Nucleic Acid Biosynthesis ◽  
Transfer Mechanisms

Phosphotransferases, phosphatases, and nucleotidyltransferases catalyze nucleophilic substitution at phosphorus. They constitute a dominant class of enzymes in intermediary metabolism, energy transduction, nucleic acid biosynthesis and processing, and regulation of many cellular processes, including replication, cellular development, and apoptosis. The mechanisms of the action of these enzymes have been studied intensively at several levels, ranging from the biosynthesis of metabolites and nucleic acids to unmasking signaling networks to elucidating the molecular mechanisms of catalysis. We focus on the chemical mechanisms of the reactions of biological phosphates. More than 40 years of research on this chemistry reveals that the mechanisms can be grouped into two classes: the phosphoryl group (PO3−) transfer mechanisms and the nucleotidyl or alkylphosphoryl group (ROPO2−) transfer mechanisms. Because the fundamental chemical mechanisms of these reactions are not treated in textbooks, we begin by considering this chemistry and then move on to the enzymatic reaction mechanisms. Phosphomonoesters, phosphoanhydrides, and phosphoramidates undergo substitution at phosphorus by transfer of the phosphoryl (PO3–) group, that is, by P—O and P—N cleavage. The current description of a typical phosphoryl group transfer mechanism is one in which the phosphoryl donor and acceptor interact weakly with the phosphoryl group in flight in a transition state in which the total bonding to phosphorus is decreased relative to the ground state. The bonding is weak between phosphorus and the leaving group R–X and between phosphorus and the accepting group Y in the transition state of. Because of decreased bonding to phosphorus, this is a loose transition state that has been described as dissociative. The latter should not be confused with the dissociative mechanism, which is considered later. To avoid confusion, we use the term loose transition state. Detailed studies indicate that the bonding denoted by the dashed lines in represents partial covalency on the order of 10% to 20% of the strength of a full covalent bond, or a bond order of 0.1 to 0.2.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals MipTec Proceedings: Transcriptional Assay Screens Using Renilla Luciferase as an Internal Control

1998 ◽  
Vol 3 (4) ◽  
pp. 41-44
Author(s):  
Rita R. Hannah ◽  
Martha L. Jennens-Clough ◽  
Keith V. Wood
Keyword(s):  
Internal Control ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Imaging System ◽  
Test Sample ◽  
Firefly Luciferase ◽  
Renilla Luciferase ◽  
Cell Lysate ◽  
Biology Research ◽  
Assay Conditions

In cell biology research and pharmaceutical discovery, physiological responses of mammalian cells are commonly screened using transcriptional assays. Although firefly luciferase is widely used because of its rapid and simple assay, greater precision can be achieved using a second reporter as an internal control. Renilla luciferase serves as an efficient internal control because it can be measured as easily and rapidly using the same instrument. The Dual-Luciferase™ Reporter (DLR™) Assay developed at Promega measures both reporters sequentially within each sample, which eliminates the need to separate the test sample into aliquots for each assay. The expression of each reporter is independently quantitated using selective assay conditions based on their distinctive chemical characteristics. The firefly luciferase is initiated first by the addition of LAR II to the sample or cell lysate. Following measurement of the luminescent output, the firefly reaction is rapidly quenched and the Renilla reaction is simultaneously activated by addition of Stop & Glo™ Reagent, and the luminescence is measured a second time. The DLR™ Assay allows quantitation of both reporters within 4 seconds per well using a 96-well luminometer equipped with two reagent injectors. Multi-well plates may be processed even more rapidly using a CCD-based imaging system.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

scholarly journals Natural Products Attenuating Biosynthesis, Processing, and Activity of Ras Oncoproteins: State of the Art and Future Perspectives

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1535
Author(s):  
Renata Tisi ◽  
Vadim Gaponenko ◽  
Marco Vanoni ◽  
Elena Sacco
Keyword(s):  
Natural Products ◽  
Mammalian Cells ◽  
Developmental Disorders ◽  
Health Gain ◽  
Point Mutations ◽  
Ras Signaling ◽  
Cellular Processes ◽  
Oncogenic Ras ◽  
Ras Genes ◽  
Critical Issues

RAS genes encode signaling proteins, which, in mammalian cells, act as molecular switches regulating critical cellular processes as proliferation, growth, differentiation, survival, motility, and metabolism in response to specific stimuli. Deregulation of Ras functions has a high impact on human health: gain-of-function point mutations in RAS genes are found in some developmental disorders and thirty percent of all human cancers, including the deadliest. For this reason, the pathogenic Ras variants represent important clinical targets against which to develop novel, effective, and possibly selective pharmacological inhibitors. Natural products represent a virtually unlimited resource of structurally different compounds from which one could draw on for this purpose, given the improvements in isolation and screening of active molecules from complex sources. After a summary of Ras proteins molecular and regulatory features and Ras-dependent pathways relevant for drug development, we point out the most promising inhibitory approaches, the known druggable sites of wild-type and oncogenic Ras mutants, and describe the known natural compounds capable of attenuating Ras signaling. Finally, we highlight critical issues and perspectives for the future selection of potential Ras inhibitors from natural sources.

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