Uptake of Polynucleotides by Mammalian Cells XV. Properties and Function of a DNA-Protein Complex Situated in the Outer Membrane of Ehrlich Ascites Tumor Cells

1978 ◽  
Vol 33 (1-2) ◽  
pp. 96-104 ◽  
Author(s):  
P. L. Schell
Keyword(s):  
Protein Complex ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Protein Structures ◽  
Uptake Capacity ◽  
Viable Cells ◽  
Ehrlich Ascites ◽  
Protease Treatment ◽  
And Function

Abstract 1) DNA-protein complexes are supposed to be original constituents of the membrane of Ehrlich ascites tum or cells. These complexes can be attacked at the surface of viable cells by DNase or protease. The DNA is partially embedded in protein structures.2) The net charge of this complex is of major importance for the RNA uptake capacity of the cells. Negatively charged DNA which is situated at the surface hinders RNA uptake. This is the explanation for the stimulation of RNA uptake by DNase or the decrease in RNA uptake after protease treatment.3) Upon treatment of DNA-dcficient complexes with homologous or heterologous DNA the original RNA uptake capacity of the cells is restored but the original conformation of the complex cannot be regained.4) The DNase action on the complex is tem perature dependent in a sigmoidal fashion. It is m arkedly slowed down at tem peratures below 12 °C. This implies that structural dianges in the complex occur at this transition tem perature which make surface DNA susceptible to DNase. This effect can only be observed in original structures but not in reconstituted ones.5) Polyanion treatment of the cells [poly (L-lysine) ] which increases their RNA uptake capacity, most probably does not interact with the DNA-protein complex. Poly (L-lysine) appears to act at other m em brane sites.6) The DNA-protein complex has been investigated entirely in situ , i. e. situated in the membrane of viable cells.

scholarly journals In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices

2020 ◽  
Vol 21 (1) ◽  
pp. 308
Author(s):  
Yichen Zhang ◽  
Shu Wang ◽  
Zongchao Jia
Keyword(s):  
Protein Complexes ◽  
Complex Molecule ◽  
Protein Structures ◽  
Hexagonal Lattice ◽  
Atomic Displacement ◽  
Asymmetric Unit ◽  
Orthorhombic Lattice ◽  
Wide Range ◽  
Crystallization Solution

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality

Regulation of microRNA biogenesis and function

2012 ◽  
Vol 107 (04) ◽  
pp. 605-610 ◽  
Author(s):  
Thomas Treiber ◽  
Nora Treiber ◽  
Gunter Meister
Keyword(s):  
Mammalian Cells ◽  
Untranslated Region ◽  
Protein Complexes ◽  
Mature Mirnas ◽  
Review Article ◽  
Cellular Functions ◽  
Target Sites ◽  
Cellular Pathways ◽  
And Function ◽  
Site Binding

SummaryMicroRNAs (miRNAs) are considered as key regulators of literally all cellular pathways. Therefore, miRNA biosynthesis and their individual cellular functions must be tightly regulated as well. MiRNAs are transcribed as primary transcripts, which are processed to mature miRNAs in two consecutive maturation steps. Finally, the mature miRNA is incorporated into a miRNA-protein complex, where it directly interacts with a member of the Argonaute (Ago) protein family. The miRNA guides such protein complexes to partial complementary target sites, which are typically located in the 3’ untranslated region (UTR) of mRNAs leading to inhibition of gene expression. MiRNA activity and abundance is regulated on various levels ranging from transcription and processing to target site binding and miRNA stability. Recent advances in our understanding of how miRNA activity is regulated in mammalian cells are summarised and discussed in this review article.

scholarly journals Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system.

1985 ◽  
Vol 5 (2) ◽  
pp. 342-351 ◽  
Author(s):  
J R Greenberg ◽  
E Carroll
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Protein Complexes ◽  
Micrococcal Nuclease ◽  
Translation System ◽  
Cytoplasmic Process ◽  
Free Translation ◽  
Associated Proteins ◽  
A Cell ◽  
And Function

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

scholarly journals Trapping mammalian protein complexes in viral particles

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Sven Eyckerman ◽  
Kevin Titeca ◽  
Emmy Van Quickelberghe ◽  
Eva Cloots ◽  
Annick Verhee ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Complexes ◽  
Gag Protein ◽  
Bait Protein ◽  
Protein Partners ◽  
Complementary Approach ◽  
Viral Particles ◽  
Interaction Partners ◽  
Enrichment Protocol

Abstract Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes.

Reconstitution of functional mRNA-protein complexes in a rabbit reticulocyte cell-free translation system

1985 ◽  
Vol 5 (2) ◽  
pp. 342-351
Author(s):  
J R Greenberg ◽  
E Carroll
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Protein Complexes ◽  
Micrococcal Nuclease ◽  
Translation System ◽  
Cytoplasmic Process ◽  
Free Translation ◽  
Associated Proteins ◽  
A Cell ◽  
And Function

A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.

Conditional fast expression and function of multimeric TRPV5 channels using Shield-1

2009 ◽  
Vol 296 (1) ◽  
pp. F204-F211 ◽  
Author(s):  
Joost P. H. Schoeber ◽  
Stan F. J. van de Graaf ◽  
Kyu Pil Lee ◽  
Hanneke G. M. Wittgen ◽  
Joost G. J. Hoenderop ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Fk506 Binding Protein ◽  
Synthetic Cell ◽  
Conditional Expression ◽  
Model Protein ◽  
Dose Dependent Fashion ◽  
Regulate Protein ◽  
And Function

A recently described novel controllable method to regulate protein expression is based on a mutated FK506-binding protein-12 (mtFKBP) that is unstable and rapidly degraded in mammalian cells. This instability can be conferred to other proteins directly fused to mtFKBP. Binding of a synthetic cell-permeant ligand (Shield-1) to mtFKBP reverses the instability, allowing conditional expression of mtFKBP-fused proteins. We adapted this strategy to study multimeric plasma membrane proteins using the ion channel TRPV5 as model protein. mtFKBP-TRPV5 forms functional ion channels and its expression can be controlled in a time- and dose-dependent fashion using Shield-1. Moreover, in the presence of Shield-1, mtFKBP-TRPV5 formed heteromultimeric channels with untagged TRPV5, which were codegraded upon washout of Shield-1, providing a strategy to study multimeric plasma membrane protein complexes without the need to destabilize all individual subunits.

scholarly journals In-Cell Sensitivity-Enhanced NMR of Intact Living Mammalian Cells

2021 ◽  
Author(s):  
Rupam Ghosh ◽  
Yiling Xiao ◽  
Jaka Kragelj ◽  
Kendra K Frederick
Keyword(s):  
Mammalian Cells ◽  
Nuclear Polarization ◽  
Protein Structures ◽  
Cellular Viability ◽  
Complex Environments ◽  
Experimental Conditions ◽  
Structural Investigations ◽  
Viable Cells ◽  
Sample Composition ◽  
Before And After

NMR has the resolution and specificity to determine atomic-level protein structures of isotopically-labeled proteins in complex environments and, with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

Transfection of a DNA/protein complex into nuclei of mammalian cells using polyoma capsids and electroporation

1987 ◽  
Vol 7 (2) ◽  
pp. 107-112 ◽  
Author(s):  
Wolf Bertling
Keyword(s):  
Protein Complex ◽  
Mammalian Cells ◽  
Protein Complexes ◽  
Physical Method ◽  
Dna Binding Protein ◽  
Dna Fragments ◽  
Biological Method ◽  
Dna Transfection ◽  
Viral Capsids ◽  
Transfected Cells

We used empty capsids ofpolyoma virus to transfer DNA fragments and DNA/protein complexes into human cells. We encapsulated labeled and unlabeled single stranded DNA fragments by viral capsids. A complex of DNA with a DNA binding protein, recA, will also be taken up by the capsids, whereas the free protein is not incorporated. We further compared this gentle biological method of DNA transfection with a well-established physical method, electroporation. Electroporation also allows the transfer of DNA as well as protein into cells, although there is no proof that a DNA/protein complex can survive the procedure functionally. Whereas the viability of capsid transfected cells is unaffected (100%), electroporation reduces the viability to 90–95%. On the other hand, the amount of DNA found in the nucleus of electroporated cells is higher than for cells treated with loaded viral capsids.

Interaction proteomics: characterization of protein complexes using tandem affinity purification–mass spectrometry

2010 ◽  
Vol 38 (4) ◽  
pp. 883-887 ◽  
Author(s):  
Pamela Völkel ◽  
Perrine Le Faou ◽  
Pierre-Olivier Angrand
Keyword(s):  
Protein Complex ◽  
Mammalian Cells ◽  
Protein Identification ◽  
Biochemical Characterization ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein A ◽  
High Sensitivity ◽  
Tandem Affinity Purification

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP–calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP–MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.

Structure of contact sites between the outer and inner mitochondrial membranes investigated by HVEM tomography

1996 ◽  
Vol 54 ◽  
pp. 966-967
Author(s):  
C.A. Mannella ◽  
K.F. Buttle ◽  
K.A. O‘Farrell ◽  
A. Leith ◽  
M. Marko
Keyword(s):  
Liver Mitochondrion ◽  
Adenine Nucleotide ◽  
Protein Complexes ◽  
Mitochondrial Membranes ◽  
Electron Microscopic ◽  
Contact Sites ◽  
Transmission Electron ◽  
Precursor Proteins ◽  
Membrane Contacts ◽  
And Function

Early transmission electron microscopy of plastic-embedded, thin-sectioned mitochondria indicated that there are numerous junctions between the outer and inner membranes of this organelle. More recent studies have suggested that the mitochondrial membrane contacts may be the site of protein complexes engaged in specialized functions, e.g., import of mitochondrial precursor proteins, adenine nucleotide channeling, and even intermembrane signalling. It has been suggested that the intermembrane contacts may be sites of membrane fusion involving non-bilayer lipid domains in the two membranes. However, despite growing interest in the nature and function of intramitochondrial contact sites, little is known about their structure.We are using electron microscopic tomography with the Albany HVEM to determine the internal organization of mitochondria. We have reconstructed a 0.6-μm section through an isolated, plasticembedded rat-liver mitochondrion by combining 123 projections collected by tilting (+/- 70°) around two perpendicular tilt axes. The resulting 3-D image has confirmed the basic inner-membrane organization inferred from lower-resolution reconstructions obtained from single-axis tomography.

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