Cell-free Formation Research Articles

Cell-free formation and interactome analysis of caveolae.

Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on Leishmania tarentolae (Lt) extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into Lt membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1Y14)-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1.

Material-based strategies to engineer fibronectin matrices for regenerative medicine

Engineering biomaterials for regenerative medicine involves a myriad of aspects to be considered for the successful design, interaction with cells and integration with living tissues (i.e. pore size, mechanical properties, degradation rate, biological activity). Among different technologies used to functionalise synthetic biomaterials and promote cell adhesion, cell growth and cell differentiation, this review focuses on strategies to organise extracellular matrix (ECM) proteins in a biomimetic way, as cells do in natural tissues in vivo (the ECM is a mesh of proteins that surrounds cells, and therefore, constitutes the scaffolding of a tissue), but using functional materials instead of living cells. The authors critically review material-based strategies to organise fibronectin (FN), a core component in the ECM of many tissues, and engineer microenvironments that recapitulate the structure and properties of the ECM. Material-driven organisation of FN in analogy with their natural cell-mediated assembly is a powerful route to engineer the network structure and biological activity of FN fibrillar matrices, seeking to develop biomimetic scaffolds for regenerative medicine. Here, the authors discuss different routes to promote the cell-free formation of FN fibrils as well as the biological impact of these engineered cellular microenvironments.

Cementomimetics-constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides.

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein–mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure–function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Cell-free Formation of RNA Granules: Bound RNAs Identify Features and Components of Cellular Assemblies

Cellular granules lacking boundary membranes harbor RNAs and their associated proteins and play diverse roles controlling the timing and location of protein synthesis. Formation of such granules was emulated by treatment of mouse brain extracts and human cell lysates with a biotinylated isoxazole (b-isox) chemical. Deep sequencing of the associated RNAs revealed an enrichment for mRNAs known to be recruited to neuronal granules used for dendritic transport and localized translation at synapses. Precipitated mRNAs contain extended 3' UTR sequences and an enrichment in binding sites for known granule-associated proteins. Hydrogels composed of the low complexity (LC) sequence domain of FUS recruited and retained the same mRNAs as were selectively precipitated by the b-isox chemical. Phosphorylation of the LC domain of FUS prevented hydrogel retention, offering a conceptual means of dynamic, signal-dependent control of RNA granule assembly.

Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Spatio-temporal variations in cell-free layer formation near bifurcations of small arterioles

Blood flow partitioning at an arteriolar bifurcation could lead to spatio-temporal variations in cell-free layer formation in the upstream and downstream vessels of the bifurcation. To investigate this effect, we quantitatively analyzed characteristics of the cell-free layer in the vicinity of an arteriolar bifurcation in the rat cremaster muscle in normal physiological flow conditions. To simulate hemorheological relevance to humans, red blood cell aggregation was elevated by infusion of Dextran 500 to levels seen in humans in normal states. Spatial variations of the layer width were observed in both the parent and larger daughter vessels. A more pronounced attenuation of the layer width was generally observed in the parent vessel at its wall adjacent to the side branch than at its opposite wall. A thicker layer width was consistently found at the opposite than adjacent wall of the larger daughter vessel. Accordingly, large asymmetries of the layer widths could be developed on opposite sides of the larger daughter vessel, which were significantly greater (P<0.01) than those observed in the parent vessel. A positive correlation was generally observed between mean layer widths in the downstream vessel and on the side of the parent vessel from which bulk of the flow enters the downstream vessel. The fraction of the downstream layer formation constituted by the side branch decreased with increasing flow fraction in this vessel. These findings confirmed the modulation of the cell-free layer formation near an arteriolar bifurcation, implicated by flow separation at the bifurcation.

Cell-Free Expression and Assembly of ATP Synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F1Fo-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α3β3γδɛab2c13. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.

Cell-free formation of misfolded prion protein with authentic prion infectivity

Prion propagation has been modeled in vitro; however, the low infectious titer of PrP(Sc) thus generated has cast doubt on the "protein-only" hypothesis. Here we show that prion delivery on suitable nitrocellulose carrier particles abrogates the apparent dissociation of PrP(Sc) and infectivity. Misfolded prion protein generated by protein misfolding cyclic amplification is as infectious as authentic brain-derived PrP(Sc) provided that confounding effects related to differences in the size distribution of prion protein aggregates generated in vitro and consecutive differences in regard to biological clearance are abolished.

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein.

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

Isolation and characterization of the principal ATPase associated with transitional endoplasmic reticulum of rat liver.

The transfer of membranes from the endoplasmic reticulum to the Golgi apparatus occurs via 50-70 nm transition vesicles which derive from part-rough, part-smooth transitional elements of the endoplasmic reticulum (TER). Vesicle budding from the TER is an ATP-dependent process both in vivo and in vitro. An ATPase with a monomer molecular weight of 100 kD by SDS-PAGE has been isolated from TER and designated as TER ATPase. The native TER ATPase has been characterized as a hexamer of six 100-kD subunits by gel filtration. The protein catalyzes the hydrolysis of [gamma 32-P]ATP and is phosphorylated in the presence of Mg2+. It is distinct from the classical transport ATPases based on pH optima, ion effects, and inhibitor specificity. Electron microscopy of negatively stained preparations revealed the TER ATPase to be a ring-shaped structure with six-fold rotational symmetry. A 19-amino acid sequence of TER ATPase having 84% identity with valosin-containing protein and 64% identity with a yeast cell-cycle control protein CDC48p was obtained. Anti-synthetic peptide antisera to a 15-amino acid portion of the sequence of TER ATPase recognized a 100-kD protein from TER. These antisera reduced the ATP-dependent cell-free formation of transition vesicles from isolated TER of rat liver. In a reconstituted membrane transfer system, TER ATPase antisera inhibited transfer of radiolabeled material from endoplasmic reticulum to Golgi apparatus, while preimmune sera did not. The results suggest that the TER ATPase is obligatorily involved in the ATP requirements for budding of transition vesicles from the TER. cDNA clones encoding TER ATPase were isolated by immunoscreening a rat liver cDNA library with the affinity-purified TER ATPase antibody. A computer search of deduced amino acid sequences revealed the cloned TER ATPase to be the rat equivalent of porcine valosin-containing protein, a member of a novel family of ATP binding, homo-oligomeric proteins including the N-ethylmaleimide-sensitive fusion protein.

Cell-free formation of disulfide-bonded multimer from isolated plasma fibronectin with a low concentration of SH reagent under physiological conditions

Cell-free formation of disulfide-bonded multimer from isolated plasma fibronectin with a low concentration of SH reagent under physiological conditions

Cell-free formation of protease-resistant prion protein.

The infectious agent (or 'prion') of the transmissible spongiform encephalopathies (TSEs) such as scrapie resembles a virus in that it replicates in vivo and has distinct strains, but it was postulated long ago to contain only protein. More recently, PrPSc, a pathogenic, scrapie-associated form of the host-encoded prion protein (PrP), was identified as a possible primary TSE agent protein. PrPSc is defined biochemically by its insolubility and resistance to proteases and is derived post-translationally from normal, protease-sensitive PrP (PrPc). The conversion seems to involve conformational change rather than covalent modification. However, the conversion mechanism and the relationship of PrPSc formation to TSE agent replication remain unclear. Here we report the conversion of PrPc to protease-resistant forms similar to PrPSc in a cell-free system composed of substantially purified constituents. This conversion was selective and required the presence of preexisting PrPSc, providing direct evidence that PrPSc derives from specific PrPc-PrPSc interactions.

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules

Non-hydrolysable analogues of GTP, such as GTPγS and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [AIF 4], a compound known to act on trimeric G-proteins. Addition of highly purified G-protein βγ subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein α subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.

Recent biosynthetic studies on beta-lactam antibiotics.

Recent experimental observations on the cell-free formation of penicillins and cephalosporins in protoplast lysates from C. acremonium are described. Conversion of radiolabelled penicillin N into deacetoxycephalosporin C is described. A proposal is made about the general types of mechanism involved in these conversions.

Biosynthesis of ergot alkaloids cell-free formation of 4-(E-4′-hydroxy-3′-methyl-but-2′-enyl)-L-tryptophan

FEBS LettersVolume 82, Issue 1 p. 55-57 Full-length articleFree Access Biosynthesis of ergot alkaloids cell-free formation of 4-(E-4′-hydroxy-3′-methyl-but-2′-enyl)-L-tryptophan R.J. Petroski, R.J. Petroski School of Pharmacy, University of Connecticut, Storrs, Conn. 06268, USASearch for more papers by this authorW.J. Kelleher, W.J. Kelleher School of Pharmacy, University of Connecticut, Storrs, Conn. 06268, USASearch for more papers by this author R.J. Petroski, R.J. Petroski School of Pharmacy, University of Connecticut, Storrs, Conn. 06268, USASearch for more papers by this authorW.J. Kelleher, W.J. Kelleher School of Pharmacy, University of Connecticut, Storrs, Conn. 06268, USASearch for more papers by this author First published: October 01, 1977 https://doi.org/10.1016/0014-5793(77)80884-3Citations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 P.F. Heinstein, S.I. Lee, H.G. Floss, Biochem Biophys. Res. Commun., 44, (1971), 1244– 1251. 2 S.I. Lee, H.G. Floss, P.F. Heinstein, Arch. Biochem. Biophys., 177, (1976), 84– 94. 3 N.Y. Mary, W.J. Kelleher, A.E. Schwarting, Lloydia, 28, (1965), 218– 229. 4 J.A. Anderson, M.S. Saini, Tetrahedron Lett., 1974, (1974), 2107– 2108. 5 H. Plieninger, M. Hoebel, W. Liede, Ber. Dtsch. Chem. Ges., 96, (1963), 1618– 1629. 6 H. Plieninger, H. Sirowejl, Ber. Dtsch. Chem. Ges., 104, (1971), 2027– 2029. 7 H. Plieninger, C. Wagner, H. Immel, Justus Liebigs Ann. Chem., 743, (1971), 95– 111. 8 F. Arcamone, E.B. Chain, A. Ferretti, A. Minghetti, P. Pennella, A. Tonolo, L. Vero, Proc. R. Soc. London Ser. B, 155, (1961), 26– 54. 9 S. Brahmall, N.Wu. Noeck, J.R. Lowenberg, Anal. Biochem., 31, (1968), 146– 148. 10 L.E. Michelon, W.J. Kelleher, Lloydia, 26, (1963), 192– 201. 11 P. Pachlatko, C. Tobacik, W. Acklin, D. Arigoni, Chimia, 29, (1975), 526– 527. Citing Literature Volume82, Issue1October 01, 1977Pages 55-57 ReferencesRelatedInformation

The role of ribosomes in the cell-free formation of tryptophan synthetase in Escherichia coli

An increase in activity of B protein of tryptophan synthetase ( l-serine hydro-lyase (adding indole), EC 4.2.1.20) was observed when either of two ribosomal fractions from Escherichia coli was incubated with a supernatant fraction under appropriate conditions. In the “P 30 system” involving larger ribosomes, the activity increase was sensitive to inhibition by DNAase (EC 3.1.4.5), RNAase (EC 2.7.7.16), chloramphenicol, streptomycin and by l-tryptophan. In contrast, the “P 60–180 system”, containing smaller ribosomes, was insensitive to DNAase, chloramphenicol and streptomycin and partially sensitive to RNAase and l-tryptophan. Experiments in which a ribosomal or supernatant fraction from a deletion mutant was substituted for the corresponding wild-type fraction clearly demonstrated that the wild-type supernatant is essential for an activity increase in the P 30 system, whereas the wild-type ribosomes are required in the P 60–180 system. These results indicate the fundamental difference between the two systems with respect to the underlying mechanisms. The evidence suggests that the P 30 system involves a DNA-dependent formation of the enzyme, while the P 60–180 system exhibits an “activation-and-release” of nascent enzyme molecules from ribosomes. Furthermore, the latter reaction may represent a part of the mechanism leading to the increase in enzyme activity in the P 30 system. Implication of these results and the role of ribosomes in enzyme formation will be discussed.

The cell-free formation of tryptophan synthetase in Escherichia coli. Studies with crude extracts

The cell-free formation of tryptophan synthetase ( l-serine hydro-lyase (adding indole), EC 4.2.1.20) in Escherichia coli was studied in an effort to investigate the genetic control of specific enzyme formation at the subcellular level. Activities of A and B component proteins of this enzyme and that of indole-3-glycerol phosphate synthetase (indole-3-glycerol phosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.8) increased when crude extracts prepared from partially derepressed wild-type cells were incubated with the supplements. A detailed characterization of the activity increase with respect to the B protein of tryptophan synthetase showed that the increase in enzyme activity depended on the presence of ATP and its generating system and was stimulated by a mixture of other ribonucleoside triphosphates and by that of l-amino acids. The system was partially sensitive to inhibition by DNAase (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5), RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16), chloramphenicol, puromycin and notably by l-tryptophan. In a parallel experiment, amino acid incorporation into protein in the present system was completely inhibited by RNAase. The newly developed enzyme activity was found entirely in the supernatant fraction, and the increase in enzyme activity seems to be accompanied by the corresponding increase in protein immunologically reactive with anti-enzyme serum. Crude extracts of various mutant strains have also been examined for their capacity to form the enzymes.

The cell-free formation of tryptophan synthetase in Escherichia coli. Studies with crude extracts

The cell-free formation of tryptophan synthetase (l-serine hydro-lyase (adding indole), EC 4.2.1.20) in Escherichia coli was studied in an effort to investigate the genetic control of specific enzyme formation at the subcellular level. Activities of A and B component proteins of this enzyme and that of indole-3-glycerol phosphate synthetase (indole-3-glycerol phosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.8) increased when crude extracts prepared from partially derepressed wild-type cells were incubated with the supplements. A detailed characterization of the activity increase with respect to the B protein of tryptophan synthetase showed that the increase in enzyme activity depended on the presence of ATP and its generating system and was stimulated by a mixture of other ribonucleoside triphosphates and by that of l-amino acids. The system was partially sensitive to inhibition by DNAase (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5), RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16), chloramphenicol, puromycin and notably by l-tryptophan. In a parallel experiment, amino acid incorporation into protein in the present system was completely inhibited by RNAase. The newly developed enzyme activity was found entirely in the supernatant fraction, and the increase in enzyme activity seems to be accompanied by the corresponding increase in protein immunologically reactive with anti-enzyme serum. Crude extracts of various mutant strains have also been examined for their capacity to form the enzymes.

The cell-free formation of tryptophan synthetase in Escherichia coli. Studies with crude extracts

The cell-free formation of tryptophan synthetase (l-serine hydro-lyase (adding indole), EC 4.2.1.20) in Escherichia coli was studied in an effort to investigate the genetic control of specific enzyme formation at the subcellular level. Activities of A and B component proteins of this enzyme and that of indole-3-glycerol phosphate synthetase (indole-3-glycerol phosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.8) increased when crude extracts prepared from partially derepressed wild-type cells were incubated with the supplements. A detailed characterization of the activity increase with respect to the B protein of tryptophan synthetase showed that the increase in enzyme activity depended on the presence of ATP and its generating system and was stimulated by a mixture of other ribonucleoside triphosphates and by that of l-amino acids. The system was partially sensitive to inhibition by DNAase (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.5), RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16), chloramphenicol, puromycin and notably by l-tryptophan. In a parallel experiment, amino acid incorporation into protein in the present system was completely inhibited by RNAase. The newly developed enzyme activity was found entirely in the supernatant fraction, and the increase in enzyme activity seems to be accompanied by the corresponding increase in protein immunologically reactive with anti-enzyme serum. Crude extracts of various mutant strains have also been examined for their capacity to form the enzymes.

The cell-free formation of tryptophan synthetase enzyme in Escherichia coli

The cell-free formation of tryptophan synthetase enzyme in Escherichia coli