scholarly journals Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

1996 ◽  
Vol 7 (4) ◽  
pp. 595-611 ◽  
Author(s):  
J P Incardona ◽  
T L Rosenberry
Keyword(s):  
Catalytic Activity ◽  
Cell Surface ◽  
Signal Peptide ◽  
Cell Biology ◽  
Quaternary Structure ◽  
Cultured Cells ◽  
Biochemical Properties ◽  
Wild Type ◽  
In Vivo Experiments

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

scholarly journals Reovirus Binding to Cell Surface Sialic Acid Potentiates Virus-Induced Apoptosis

2001 ◽  
Vol 75 (9) ◽  
pp. 4029-4039 ◽  
Author(s):  
Jodi L. Connolly ◽  
Erik S. Barton ◽  
Terence S. Dermody
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
Cultured Cells ◽  
Cell Types ◽  
Biochemical Properties ◽  
Viral Protein Synthesis ◽  
Efficient Induction ◽  
Critical Requirement ◽  
Induced Apoptosis

ABSTRACT Reovirus induces apoptosis in cultured cells and in vivo. Genetic studies indicate that the efficiency with which reovirus strains induce apoptosis is determined by the viral S1 gene, which encodes attachment protein ς1. However, the biochemical properties of ς1 that influence apoptosis induction are unknown. To determine whether the capacity of ς1 to bind cell surface sialic acid determines the magnitude of the apoptotic response, we used isogenic reovirus mutants that differ in the capacity to engage sialic acid. We found that T3SA+, a virus capable of binding sialic acid, induces high levels of apoptosis in both HeLa cells and L cells. In contrast, non-sialic-acid-binding strain T3SA− induces little or no apoptosis in these cell types. Differences in the capacity of T3SA− and T3SA+ to induce apoptosis are not due to differences in viral protein synthesis or production of viral progeny. Removal of cell surface sialic acid with neuraminidase abolishes the capacity of T3SA+ to induce apoptosis. Similarly, incubation of T3SA+ with sialyllactose, a trisaccharide comprised of lactose and sialic acid, blocks apoptosis. These findings demonstrate that reovirus binding to cell surface sialic acid is a critical requirement for the efficient induction of apoptosis and suggest that virus receptor utilization plays an important role in regulating cell death.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals Vaccinia Virus Envelope H3L Protein Binds to Cell Surface Heparan Sulfate and Is Important for Intracellular Mature Virion Morphogenesis and Virus Infection In Vitro and In Vivo

2000 ◽  
Vol 74 (7) ◽  
pp. 3353-3365 ◽  
Author(s):  
Chi-Long Lin ◽  
Che-Sheng Chung ◽  
Hans G. Heine ◽  
Wen Chang
Keyword(s):  
Cell Surface ◽  
Vaccinia Virus ◽  
Heparan Sulfate ◽  
Mutant Virus ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Virion Morphogenesis

ABSTRACT An immunodominant antigen, p35, is expressed on the envelope of intracellular mature virions (IMV) of vaccinia virus. p35 is encoded by the viral late gene H3L, but its role in the virus life cycle is not known. This report demonstrates that soluble H3L protein binds to heparan sulfate on the cell surface and competes with the binding of vaccinia virus, indicating a role for H3L protein in IMV adsorption to mammalian cells. A mutant virus defective in expression of H3L (H3L−) was constructed; the mutant virus has a small plaque phenotype and 10-fold lower IMV and extracellular enveloped virion titers than the wild-type virus. Virion morphogenesis is severely blocked and intermediate viral structures such as viral factories and crescents accumulate in cells infected with the H3L− mutant virus. IMV from the H3L− mutant virus are somewhat altered and less infectious than wild-type virions. However, cells infected by the mutant virus form multinucleated syncytia after low pH treatment, suggesting that H3L protein is not required for cell fusion. Mice inoculated intranasally with wild-type virus show high mortality and severe weight loss, whereas mice infected with H3L− mutant virus survive and recover faster, indicating that inactivation of the H3L gene attenuates virus virulence in vivo. In summary, these data indicate that H3L protein mediates vaccinia virus adsorption to cell surface heparan sulfate and is important for vaccinia virus infection in vitro and in vivo. In addition, H3L protein plays a role in virion assembly.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

scholarly journals A cellular endolysosome-modulating pore-forming protein from a toad is negatively regulated by its paralog under oxidizing conditions

2020 ◽  
Vol 295 (30) ◽  
pp. 10293-10306 ◽  
Author(s):  
Qiquan Wang ◽  
Xianling Bian ◽  
Lin Zeng ◽  
Fei Pan ◽  
Lingzhen Liu ◽  
...  
Keyword(s):  
Disulfide Bond ◽  
Cell Biology ◽  
Biochemical Properties ◽  
Bacterial Toxin ◽  
Cell Physiology ◽  
Dependent Manner ◽  
Membrane Pore ◽  
Intracellular Vesicles

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular targets of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example of a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore-forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and the elimination of biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity of an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

scholarly journals α-Tocomonoenol Is Bioavailable in Mice and May Partly Be Regulated by the Function of the Hepatic α-Tocopherol Transfer Protein

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4803
Author(s):  
Andrea Irías-Mata ◽  
Nadine Sus ◽  
Maria-Lena Hug ◽  
Marco Müller ◽  
Walter Vetter ◽  
...  
Keyword(s):  
Tissue Distribution ◽  
Room Temperature ◽  
Systemic Circulation ◽  
Future Research ◽  
Wild Type ◽  
Transfer Protein ◽  
In Vivo Experiments ◽  
And Storage

Tocomonoenols are vitamin E derivatives present in foods with a single double bond at carbon 11’ in the sidechain. The α-tocopherol transfer protein (TTP) is required for the maintenance of normal α-tocopherol (αT) concentrations. Its role in the tissue distribution of α-11′-tocomonoenol (αT1) is unknown. We investigated the tissue distribution of αT1 and αT in wild-type (TTP+/+) and TTP knockout (TTP−/−) mice fed diets with either αT or αT1 for two weeks. αT1 was only found in blood, not tissues. αT concentrations in TTP+/+ mice were in the order of adipose tissue > brain > heart > spleen > lungs > kidneys > small intestine > liver. Loss of TTP function depleted αT in all tissues. αT1, contrary to αT, was still present in the blood of TTP−/− mice (16% of αT1 in TTP+/+). Autoclaving and storage at room temperature reduced αT and αT1 in experimental diets. In conclusion, αT1 is bioavailable, reaches the blood in mice, and may not entirely depend on TTP function for secretion into the systemic circulation. However, due to instability of the test compounds in the experimental diets, further in vivo experiments are required to clarify the role of TTP in αT1 secretion. Future research should consider compound stability during autoclaving of rodent feed.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

scholarly journals Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2242 ◽  
Author(s):  
Jacob L. Bouchard ◽  
Taylor C. Davey ◽  
Todd M. Doran
Keyword(s):  
Self Assembly ◽  
Quaternary Structure ◽  
Biological Activities ◽  
Amyloid Β ◽  
Long Term Potentiation ◽  
Wild Type ◽  
Molecular Weights ◽  
Term Potentiation ◽  
Quaternary Structures

Amyloid-β oligomers (AβOs) self-assemble into polymorphic species with diverse biological activities that are implicated causally to Alzheimer’s disease (AD). Synaptotoxicity of AβO species is dependent on their quaternary structure, however, low-abundance and environmental sensitivity of AβOs in vivo have impeded a thorough assessment of structure–function relationships. We developed a simple biochemical assay to quantify the relative abundance and morphology of cross-linked AβOs. We compared oligomers derived from synthetic Aβ40 (wild-type (WT) Aβ40) and a recombinant source, called Aβ(M1–40). Both peptides assemble into oligomers with common sizes and morphology, however, the predominant quaternary structures of Aβ(M1–40) oligomeric states were more diverse in terms of dispersity and morphology. We identified self-assembly conditions that stabilize high-molecular weight oligomers of Aβ(M1–40) with apparent molecular weights greater than 36 kDa. Given that mixtures of AβOs derived from both peptides have been shown to be potent neurotoxins that disrupt long-term potentiation, we anticipate that the diverse quaternary structures reported for Aβ(M1–40) oligomers using the assays reported here will facilitate research efforts aimed at isolating and identifying common toxic species that contribute to synaptic dysfunction.

scholarly journals In Vivo Dimerization of Types 1, 2, and 3 Iodothyronine Selenodeiodinases

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 937-946 ◽  
Author(s):  
Cyntia Curcio-Morelli ◽  
Balazs Gereben ◽  
Ann Marie Zavacki ◽  
Brian W. Kim ◽  
Stephen Huang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Western Blot ◽  
Relative Molecular Mass ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Cell Lysate ◽  
Sds Page ◽  
Catalytically Active

The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of 75Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2–5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (Mr) of 55,000 for D1, Mr 62,000 for D2, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.

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