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At least 19 recordsLinked to original sources
PubMed1992

Phosphorylatable and epitope-tagged human erythropoietins: utility and purification of native baculovirus-derived forms.

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.

Amino Acid Sequence↗
PubMed2027

Epitope Tagging and Coimmunoprecipitation to Identify Viral Protein Interactors.

Affinity purification-mass spectrometry (AP-MS) is a powerful proteomic approach for dissecting the interaction network between virus and host. Traditional AP-MS employs overexpression of viral proteins as baits to enrich host interactors. However, overexpressed viral proteins may mislocalize to inappropriate cellular compartments and trigger endoplasmic reticulum stress by overwhelming the protein-folding machinery, which leads to false identification of host factors. To overcome these limitations, we introduce an AP-MS strategy based on direct infection with an epitope-tagged chikungunya virus (CHIKV/myc-E2), which we used to successfully uncover two new antiviral factors in CHIKV cellular reservoirs-macrophages. In this protocol, we will describe this technique step by step: (1) design and construction of myc-tagged virus by advanced multi-fragment assembly, (2) in vitro transcription and preparation of infectious myc-tagged virus stocks, and (3) immunoprecipitation of myc-tagged viral protein and its interactome for mass spectrometry analysis. This strategy enables accurate identification of viral interactors in a physiologically relevant context, providing a framework for future proteomic studies using tagged viruses.

Chikungunya virus↗
PubMed1992

Molecular analysis of protein sorting during biogenesis of muscle cytoarchitecture.

Isolated, rod-shaped adult rat cardiomyocytes (ARC) were kept in long-term cell cultures and the changes of the cardiomyocyte structure were investigated by confocal microscopy. The cells round up and make contact with the substrate by very flat, foot-like structures. After prolonged culture the amorphous cells regenerate a cardiomyocyte-like cytoarchitecture and myofibrils reemerge. In the perinuclear region myofibrils form continuously while in other cells discontinuous myofibrillogenesis was observed, where short sarcomeric segments occur all over the cytoplasmic space. During the regeneration of myofibrils certain proteins like a smooth muscle actin sort to non sarcomeric region, while myomesin or heart C-protein localize on myofibrils with high specificity. This culture system combined with method of epitope-tagging of contractile proteins are ideally suited to monitor the intracellular localization sites of exogenously introduced constructs to different cytoskeletal, since ARC exhibit at the same time stress fiber-like filaments (SFLF) and nascent myofibrils. The molecular properties of the different members of the myosin light chain isoprotein family were investigated by transfection experiments using epitope-tagged myosin light chain (MLC) cDNA. The sorting of the different types of MLC was shown to be isoprotein specific and with chimeric constructs it was shown that the isoprotein-specific incorporation into myofibrils was dependent on the presence of the middle domain of MLC-1f/3f. These MLC isoproteins can be arranged into a sequence of increasing affinity to myofibrils. A hierarchical order of myofibrillar assembly is postulated based on the association affinity. Similar experiments with constructs containing alpha-cardiac, alpha-smooth muscle and gamma-cytoplasmic actins have shown that expression of epitope-tagged actins in ARC result in different epitope staining patterns. While the alpha-cardiac actin showed a marked preference for sarcomeres, the alpha-smooth muscle isoproteins had an intermediate specificity and could either be preferentially incorporated into stress fiber-like filaments (SFLF) and in some cells to a lesser extent into myofibrils as well. Most striking results were obtained with gamma-cytoplasmic actin carrying a 5 or 11-mer epitope. This actin gave rise to large cells, induced the formation of filopodia filled with the transfected actin and depletion of the transfected actin from the perinuclear myofibrillar region.

Actins↗
PubMed1992

Activation of the alpha subunit of Gs in intact cells alters its abundance, rate of degradation, and membrane avidity.

Binding of GTP induces alpha subunits of heterotrimeric G proteins to take on an active conformation, capable of regulating effector molecules. We expressed epitope-tagged versions of the alpha subunit (alpha s) of Gs in genetically alpha s-deficient S49 cyc- cells. Addition of a hemagglutinin (HA) epitope did not alter the ability of wild type alpha s to mediate hormonal stimulation of adenylyl cyclase or to attach to cell membranes. The HA epitope did, however, allow a mAb to immunoprecipitate the recombinant protein (HA-alpha s) quantitatively from cell extracts. We activated the epitope-tagged alpha s in intact cells by: (a) exposure of cells to cholera toxin, which activates alpha s by covalent modification; (b) mutational replacement of arginine-201 in HA-alpha s by a cysteine residue, to create HA-alpha s-R201C; like the cholera toxin-catalyzed modification, this mutation activates alpha s by slowing its intrinsic GTPase activity; and (c) treatment of cells with the beta-adrenoceptor agonist, isoproterenol, which promotes binding of GTP to alpha s, thereby activating adenylyl cyclase. Both cholera toxin and the R201C mutation accelerated the rate of degradation of alpha s (0.03 h-1) by three- to fourfold and induced a partial shift of the protein from a membrane bound to a soluble compartment. At steady state, 80% of HA-alpha s- R201C was found in the soluble fraction, as compared to 10% of wild type HA-alpha s. Isoproterenol rapidly (in < 2 min) caused 20% of HA-alpha s to shift from the membrane-bound to the soluble compartment. Cholera toxin induced a 3.5-fold increase in the rate of degradation of a second mutant, HA-alpha s-G226A, but did not cause it to move into the soluble fraction; this observation shows that loss of membrane attachment is not responsible for the accelerated degradation of alpha s in response to activation. Taken together, these findings show that activation of alpha s induces a conformational change that loosens its attachment to membranes and increases its degradation rate.

Adenylyl Cyclases↗
PubMed1992

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.

Animals↗
PubMed1992

Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.

The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.

Affinity Labels↗
PubMed2025

Generation of Hoxa11-3XFLAG and Hoxd11-3XFLAG alleles to investigate Hox11 genome-wide binding.

Hox genes encode for evolutionary conserved transcription factors that direct the proper development of the body plan. Despite decades of research, little is known regarding their downstream target genes, especially in vertebrates. The strong evolutionary conservation of their DNA-binding homeodomain, their generic AT-rich binding sites, and the lack of specific antibodies has precluded rigorous examination. To circumvent these limitations, we have generated two mouse models in which a 3XFLAG epitope tag has been inserted into the 5' end of the coding sequence of both Hoxa11 and Hoxd11 loci via Cas9/CRISPR. The alleles have been validated by sequencing, PCR genotyping, western blotting, and protein expression analyses, demonstrating proper targeting and expression. Breeding these alleles in combination produces viable and fertile Hoxa11FLAG/FLAG; Hoxd11FLAG/FLAG animals, with no overt patterning defects unlike Hoxa11/Hoxd11 mutants that are infertile and have severe kidney and limb defects. By performing CUT&RUN and CUT&Tag analyses, we have confirmed DNA binding to a known Six2 enhancer in the developing kidney. These novel alleles will allow characterization of the genome-wide binding profile of Hox11 proteins in vivo.

Animals↗
PubMed1992

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.

Animals↗
PubMed1992

TFC3: gene encoding the B-block binding subunit of the yeast transcription factor IIIC.

Yeast transcription factor IIIC (TFIIIC) is a multisubunit protein complex that interacts with two control elements of class III promoters called the A and B blocks. Here we describe the gene encoding the 138-kDa subunit (tau 138), which is involved in B-block binding. From the DNA sequence, the open reading frame, interrupted by an intron with an unusual 3' splice acceptor site, is in agreement with all the microsequencing data for peptides within tau 138. TFC3 is a single-copy gene located on chromosome I; it is essential for cell viability as shown by a gene disruption experiment. Epitope-tagging of the TFC3 gene product and DNA binding experiments are consistent with the presence of one copy of tau 138 in TFIIIC-DNA complexes.

Amino Acid Sequence↗
PubMed2025

Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.

Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.

Gene Editing↗
PubMed2026

Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network.

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro) development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by data-independent acquisition and label free quantitation mass spectrometry. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-&#x3b5;-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7's activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

Ubiquitin-Specific Peptidase 7↗
PubMed2019

Estrogen-independent molecular actions of mutant estrogen receptor 1 in endometrial cancer.

Estrogen receptor 1 (ESR1) mutations have been identified in hormone therapy-resistant breast cancer and primary endometrial cancer. Analyses in breast cancer suggest that mutant ESR1 exhibits estrogen-independent activity. In endometrial cancer, ESR1 mutations are associated with worse outcomes and less obesity, however, experimental investigation of these mutations has not been performed. Using a unique CRISPR/Cas9 strategy, we introduced the D538G mutation, a common endometrial cancer mutation that alters the ligand binding domain of ESR1, while epitope tagging the endogenous locus. We discovered estrogen-independent mutant ESR1 genomic binding that is significantly altered from wild-type ESR1. The D538G mutation impacted expression, including a large set of nonestrogen-regulated genes, and chromatin accessibility, with most affected loci bound by mutant ESR1. Mutant ESR1 is distinct from constitutive ESR1 activity because mutant-specific changes are not recapitulated with prolonged estrogen exposure. Overall, the D538G mutant ESR1 confers estrogen-independent activity while causing additional regulatory changes in endometrial cancer cells that are distinct from breast cancer cells.

CRISPR-Cas Systems↗
PubMed1992

Attempts to convert the cellular prion protein into the scrapie isoform in cell-free systems.

The scrapie prion protein (PrPSc) is derived from a cellular isoform (PrPC) that acquires protease resistance posttranslationally. We have used several different experimental approaches in attempts to reconstitute in vitro the processes leading to protease-resistant PrPSc molecules. In the first study, we performed mixing experiments by adding mouse PrP 27-30 (MoPrP27-30), the protease-resistant core of PrPSc, to PrPC and then incubating the mixture to investigate the possibility of heterodimer formation as a first step in prion replication. We used epitopically tagged PrP molecules, synthesized in murine neuroblastoma (N2a) cells transfected with the chimeric mouse/Syrian hamster MHM2 PrP construct, which are recognized by the Syrian hamster-specific monoclonal antibody 3F4. After as long as 24 h of incubation, the reaction mixture was assayed for heterodimeric intermediates of MHM2 PrPC and MoPrPSc and for protease-resistant 3F4-reactive PrP. We were unable to identify any aggregates of MHM2 PrPC and MoPrPSc on immunoblots; furthermore, we did not observe de novo formation of protease-resistant MHM2 PrP. In a second study, MoPrPC was metabolically radiolabeled in scrapie prion-infected N2a cultured cells, and then the cell extract was homogenized and incubated under various conditions to allow for the formation of protease-resistant MoPrPSc. We observed no radiolabeled MoPrPSc by immunoprecipitation after as long as 24 h of in vitro incubation. In a third approach, Syrian hamster PrP (SHaPrP) was synthesized in a cell-free translation system supplemented with microsomal membranes derived from either normal or scrapie prion-infected cultured cells. We found that all SHaPrP species translocated across microsomal membranes from scrapie prion-infected cells were protease sensitive in the presence of detergents and displayed the same topology as those generated by microsomes from normal cells or from dog pancreas. We also studied PrP molecules that encode the codon 102 mutation that causes the rare human prion disease Gerstmann-Sträussler-Scheinker (GSS) syndrome. On the basis of our data, GSSPrP appears to yield topological forms similar to those of the wild-type PrP when processed by either normal or scrapie prion-derived microsomes.

Animals↗
PubMed2025

Subcellular proteomics of the protist Paradiplonema papillatum reveals the digestive capacity of the cell membrane and the plasticity of peroxisomes across euglenozoans.

Diplonemids are among the most diverse and abundant protists in the deep ocean, have extremely complex and ancient cellular systems, and exhibit unique metabolic capacities. Despite this, we know very little about this major group of eukaryotes. To establish a model organism for comprehensive investigation, we performed subcellular proteomics on Paradiplonema papillatum and localized 4,870 proteins to 22 cellular compartments. We additionally confirmed the predicted location of several proteins by epitope tagging and fluorescence microscopy. To probe the metabolic capacities of P. papillatum, we explored the proteins predicted to the cell membrane compartment in our subcellular proteomics dataset. Our data revealed an accumulation of many carbohydrate-degrading enzymes (CDZymes). Our predictions suggest that these CDZymes are exposed to the&#xa0;extracellular space, supporting proposals that diplonemids may specialize in breaking down carbohydrates in plant and algal cell walls. Further exploration of carbohydrate metabolism revealed an evolutionary divergence in the function of glycosomes (modified peroxisomes) in diplonemids versus kinetoplastids. Our subcellular proteome provides a resource for future investigations into the unique cell biology of diplonemids.

Peroxisomes↗
PubMed1992

The alternative-splice isoforms of the PDGF A-chain differ in their ability to associate with the extracellular matrix and to bind heparin in vitro.

Platelet-derived growth factor (PDGF) consists of disulfide-linked homo- or heterodimers of A and B chains. mRNA encoding the A chain (PDGF-A) occurs in two versions that differ by the presence or absence of a single short exon. These alternatively-spliced mRNAs encode polypeptides that differ in length by fifteen amino acids. The longer isoform (PDGF-AL) possesses a highly basic carboxy-terminal extension that is responsible for retaining PDGF-AL homodomers at the cell surface after secretion, while homodimers of the shorter isoform (PDGF-AS) are released into the extracellular medium. We have investigated the mechanism by which PDGF-AL remains in association with the cells that produce it. We expressed epitope-tagged versions of PDGF-AL and PDGF-AS in Cos cells and compared their intra- and extracellular distributions by immunofluorescence microscopy. PDGF-AL, but not PDGF-AS, was detected on and around cells in a diffuse pattern suggesting associated with the extracellular matrix (ECM). Metabolically radiolabelled PDGF-AL, but not PDGF-AS, could be eluted from ECM preparations by washing in high salt. Moreover, PDGF-AL bound reversibly to heparin-Sepharose in vitro at physiological salt concentrations, eluting at a salt concentration around 0.5 M. PDGF-AS did not bind to heparin under the same conditions. Thus, PDGF dimers that contain PDGF-AL may remain immobilized near the cells that secrete them by virtue of binding to heparin-like constituents of the ECM.

3T3 Cells↗
PubMed1992

Expression of a ubiquitin derivative that conjugates to protein irreversibly produces phenotypes consistent with a ubiquitin deficiency.

Ubiquitin (Ub) exists in a dynamic equilibrium between the free form and the conjugated form. This equilibrium is maintained and regulated through the antagonistic actions of the conjugation system and a class of enzymes referred to collectively as the Ub-protein hydrolases. Using a previously described epitope-tagged Ub approach (Ellison, M., and Hochstrasser, M. (1991) J. Biol. Chem. 266, 21150-21157) we show here that a single amino acid substitution at the carboxyl terminus of Ub (Gly-76 to Ala-76) results in a derivative of Ub (UbA-76) that becomes irreversibly conjugated to protein when expressed in the yeast Saccharomyces cerevisiae, producing a profound effect on the Ub-conjugate equilibrium. The major target of UbA-76 conjugation is itself (and presumably wild-type Ub) producing unanchored chains at the expense of the free form. Unsurprisingly, the expression of UbA-76 results in yeast phenotypes that would be expected in situations of Ub deprivation. Such cells show slow growth characteristics and sensitivity to various forms of environmental stress and to ultraviolet light. In view of these findings, the expression of UbA-76 in higher organisms may represent a convenient epigenetic strategy for examining the physiological consequences of Ub deprivation or Ub-protein hydrolase disfunction in living cells without the need for gene disruption or replacement. The observation that UbA-76 couples to itself irreversibly also provides an effective tool for elucidating the role of Ub as the proteolytic signal.

Amino Acids↗
PubMed1992

Constraints on models for the folding of the Na,K-ATPase.

We have attempted to bring together in graphic fashion the available evidence on the structure of the Na,K-ATPase and the H,K-ATPase. There appears to be much room for modification of the existing models for transmembrane folding. More sites on each side of the membrane need to be identified. Whether these will be antibody epitopes, sites of covalent modification, or tags inserted by mutagenesis is less important than that there be many of them and that each be verified by alternative approaches. If any single principle has emerged from the study of the topography of membrane proteins, it is that it is easy to reach conclusions too soon.

Amino Acid Sequence↗
PubMed1975

Presence of "receptors" for growth hormone on membranes of rat thymocytes.

Growth hormone conjugated to Sepharose-4 B was found to mimic the action of the free hormone in the stimulation of the uptake and incorporation of radioactive uridine into RNA in thymocytes in vitro. The integrity of the cellular structure was essential for the stimulating effect of the hormone. The bonding of the immunologically reactive determinants of the hormone on membranes of thymocytes was demonstrated by electron micrographs of preparations incubated sequentially with rabbit antigrowth hormone globulins and monospecific sheep anti-rabbit globulins tagged with horse-radish peroxidase.

Animals↗