scholarly journals The arachidonate-activatable, NADPH oxidase-associated H+ channel is contained within the multi-membrane-spanning N-terminal region of gp91-phox

1997 ◽  
Vol 325 (3) ◽  
pp. 701-705 ◽  
Author(s):  
Lydia M. HENDERSON ◽  
Stephen THOMAS ◽  
George BANTING ◽  
J. Brian CHAPPELL
Keyword(s):  
Amino Acids ◽  
Nadph Oxidase ◽  
Cell Line ◽  
Proton Motive Force ◽  
Transmembrane Domains ◽  
Terminal Region ◽  
Full Length ◽  
Protein Component ◽  
Cho Cell ◽  
Membrane Spanning

The generation of superoxide by the NADPH oxidase of neutrophils is accompanied by the efflux of H+ ions through a H+ channel. gp91-phox, a protein component of the oxidase, has been shown previously to function as a H+ channel [Henderson, Banting and Chappell (1995) J. Biol. Chem. 270, 5909–5916]. We have constructed a CHO cell line (CHO-N) that expresses an N-terminal fragment of gp91-phox containing the predicted multiple transmembrane domains of the protein. These cells exhibit H+ fluxes in response to an imposed proton motive force and in the presence of arachidonate (to open the channel). The H+ fluxes were indistinguishable from those observed in cells expressing full-length gp91-phox. Therefore the N-terminal 230 amino acids of gp91-phox contain all that is required to function as the NADPH oxidase-associated H+ channel.

The renal electrogenic Na+:HCO-3 cotransporter.

1997 ◽  
Vol 200 (2) ◽  
pp. 263-268 ◽  
Author(s):  
V F Boron ◽  
M A Hediger ◽  
E L Boulpaep ◽  
M F Romero
Keyword(s):  
Amino Acids ◽  
Cdna Library ◽  
Rat Kidney ◽  
Basolateral Membrane ◽  
Full Length ◽  
Xenopus Laevis Oocytes ◽  
New Family ◽  
All Solutions ◽  
Single Clone ◽  
Membrane Spanning

The electrogenic Na+:HCO3- cotransporter (symporter) is the major transporter for HCO3- reabsorption across the basolateral membrane of the renal proximal tubule and also contributes significantly to Na+ reabsorption. We expression-cloned the salamander renal electrogenic Na+:Bicarbonate Cotransporter (NBC) in Xenopus laevis oocytes. After injecting poly(A)+ RNA, fractionated poly(A)+ RNA or cRNA, we used microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi) All solutions contained ouabain to block the Na+/K+ pump (P-ATPase). After applying 1.5% CO2/10 mmol l-1 HCO3- (pH 7.5) and allowing pHi to stabilize from the CO2-induced acidification, we removed Na+. In native oocytes or water-injected controls, removing Na+ hyperpolarized the cell by -5 mV and had no effect on pHi. In oocytes injected with poly(A)+ RNA, removing Na+ transiently depolarized the cell by -10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. We enriched the signal by electrophoretic fractionation of the poly(A)+ RNA, and constructed a size-selected cDNA library in pSPORT1 using the optimal fraction. Screening the Ambystoma library yielded a single clone (aNBC). Expression was first obvious 3 days after injection of NBC cRNA. Adding CO2/HCO3- induced a large (> 50 mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Subsequent Na+ removal depolarized the cell by more than 40 mV and decreased pHi. aNBC is a full-length clone with a start Met and a poly(A)+ tail; it encodes a protein with 1025 amino acids and several putative membrane-spanning domains. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters. We used aNBC to screen a rat kidney cDNA library, and identified a full-length cDNA clone (rNBC) that encodes a protein of 1035 amino acids. rNBC is 86% identical to aNBC and can be functionally expressed in oocytes.

Membrane Electrostatics Sensed by Tryptophan Anchors in Hydrophobic Model Peptides Depends on Non-Aromatic Interfacial Amino Acids: Implications in Hydrophobic Mismatch

2020 ◽  
Author(s):  
Sreetama Pal ◽  
Roger E. Koeppe II ◽  
Amitabha Chattopadhyay
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Transmembrane Domains ◽  
Hydrophobic Mismatch ◽  
Consensus Motif ◽  
Membrane Electrostatics ◽  
Membrane Spanning ◽  
Model Peptides

WALPs are synthetic α-helical membrane-spanning peptides that constitute a well-studied system for exploring hydrophobic mismatch. These peptides represent a simplified consensus motif for transmembrane domains of intrinsic membrane proteins due...

scholarly journals Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR

2005 ◽  
Vol 387 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Daniel MONLEÓN ◽  
Vicent ESTEVE ◽  
Helena KOVACS ◽  
Juan J. CALVETE ◽  
Bernardo CELDA
Keyword(s):  
Amino Acids ◽  
Terminal Region ◽  
Full Length ◽  
Binding Motif ◽  
Recognition Sequence ◽  
Functional Regions ◽  
Nuclear Overhauser ◽  
Nuclear Overhauser Effects ◽  
Backbone Atoms ◽  
Binding Loop

Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30°. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin–αvβ3 docking model suggests that echistatin's C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function correlations.

Mammalian cells have two functional RCC1 proteins produced by alternative splicing

1994 ◽  
Vol 107 (8) ◽  
pp. 2203-2208 ◽  
Author(s):  
J. Miyabashira ◽  
T. Sekiguchi ◽  
T. Nishimoto
Keyword(s):  
Amino Acids ◽  
Alternative Splicing ◽  
Nucleotide Sequence ◽  
Cell Line ◽  
Mammalian Cells ◽  
Genomic Sequence ◽  
Terminal Region ◽  
Noncoding Region ◽  
Coding Region

Previously we cloned two human RCC1 cDNAs that differed in their noncoding region. In this study, we have found new human and hamster RCC1 cDNAs, which have an even more different coding region from that of the previously cloned RCC1 cDNAs yet can complement the RCC1 mutation in the tsBN2 cell line. The newly found RCC1 cDNAs encode a protein (designated as RCC1-I) that has an insertion of 31 (human) and 13 (hamster) amino acids at valine25 in the N-terminal region outside the RCC1-seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence that had already been determined, and was found to be located between the 6th and 7th exons, designated as the 6′ exon. Both the 5′ and 3′ ends of the 6′ exon correspond to the GT-AG rules for splicing, indicating that human RCC1-I mRNAs are produced by alternative splicing. The finding that both humans and hamsters have the insertion at the same RCC1 site suggests that the pattern of alternative splicing in the RCC1 gene has been conserved through evolution.

Leveraging a CHO cell line toolkit to accelerate biotherapeutics into the clinic

2017 ◽  
Vol 33 (6) ◽  
pp. 1468-1475 ◽  
Author(s):  
Chapman Wright ◽  
Christina Alves ◽  
Rashmi Kshirsagar ◽  
John Pieracci ◽  
Scott Estes
Keyword(s):  
Cell Line ◽  
Cho Cell ◽  
Cho Cell Line

Multiple pathways for cationic amino acid transport in rat thyroid epithelial cell line PC Cl3

2005 ◽  
Vol 288 (2) ◽  
pp. C290-C303 ◽  
Author(s):  
Tiziano Verri ◽  
Cinzia Dimitri ◽  
Sonia Treglia ◽  
Fabio Storelli ◽  
Stefania De Micheli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Amino Acid Transport ◽  
Transport Systems ◽  
Residual Fraction ◽  
Acid Transport ◽  
Thyroid Cells ◽  
Cationic Amino Acid ◽  
Rat Thyroid

Information regarding cationic amino acid transport systems in thyroid is limited to Northern blot detection of y+LAT1 mRNA in the mouse. This study investigated cationic amino acid transport in PC cell line clone 3 (PC Cl3 cells), a thyroid follicular cell line derived from a normal Fisher rat retaining many features of normal differentiated follicular thyroid cells. We provide evidence that in PC Cl3 cells plasmalemmal transport of cationic amino acids is Na+ independent and occurs, besides diffusion, with the contribution of high-affinity, carrier-mediated processes. Carrier-mediated transport is via y+, y+L, and b0,+ systems, as assessed by l-arginine uptake and kinetics, inhibition of l-arginine transport by N-ethylmaleimide and neutral amino acids, and l-cystine transport studies. y+L and y+ systems account for the highest transport rate (with y+L > y+) and b0,+ for a residual fraction of the transport. Uptake data correlate to expression of the genes encoding for CAT-1, CAT-2B, 4F2hc, y+LAT1, y+LAT2, rBAT, and b0,+AT, an expression profile that is also shown by the rat thyroid gland. In PC Cl3 cells cationic amino acid uptake is under TSH and/or cAMP control (with transport increasing with increasing TSH concentration), and upregulation of CAT-1, CAT-2B, 4F2hc/y+LAT1, and rBAT/b0,+AT occurs at the mRNA level under TSH stimulation. Our results provide the first description of an expression pattern of cationic amino acid transport systems in thyroid cells. Furthermore, we provide evidence that extracellular l-arginine is a crucial requirement for normal PC Cl3 cell growth and that long-term l-arginine deprivation negatively influences CAT-2B expression, as it correlates to reduction of CAT-2B mRNA levels.

scholarly journals A Region Within the Third Extracellular Loop of Rat AQP6 Precludes Its Trafficking to Plasma Membrane in a Heterologous Cell Line.

2021 ◽  
Author(s):  
David Soler ◽  
Thomas Kowatz ◽  
Andrew Sloan ◽  
Thomas McCormick ◽  
Kevin Cooper ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Membrane Protein ◽  
Cell Line ◽  
Membrane Trafficking ◽  
Extracellular Loop ◽  
Reporter System ◽  
The Third ◽  
Heterologous Cell ◽  
Retention Signal

Abstract The inability to over-express AQP6 in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail. Using the AGR reporter system we have identified a region within loop C of AQP6 that is responsible for severely hampering its plasma membrane localization. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous cells.

scholarly journals Plasma Membrane Effects of Sphingolipid-Synthesis Inhibition by Myriocin in CHO Cells: A Biophysical and Lipidomic Study

2021 ◽  
Author(s):  
Bingen G. Monasterio ◽  
Noemi Jiménez-Rojo ◽  
Aritz B. García-Arribas ◽  
Howard Riezman ◽  
Félix M. Goñi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cho Cells ◽  
Plasma Membranes ◽  
Mechanical Resistance ◽  
Serine Palmitoyltransferase ◽  
Cho Cell ◽  
Cellular Effects ◽  
Inhibitory Compound ◽  
Membrane Effects ◽  
Biophysical Changes

Abstract Two main strategies for establishing the cellular effects of a given enzyme activity suppression are (a) the use of a stably mutated cell line that lacks a functional gene, or (b) treating the wild type with an inhibitory compound that affects the same gene-product protein. In this work, myriocin was used to block the serine palmitoyltransferase (SPT) enzyme of CHO cells and the subsequent biophysical changes in membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) happened to be lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated glycerophospholipids than the non-treated ones, although they achieved it only partially, their plasma membranes remaining more fluid and less penetrable than those from the control cells.

scholarly journals The Capsid (ORF2) Protein of Hepatitis E Virus in Feces Is C-Terminally Truncated

Pathogens ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 24
Author(s):  
Takashi Nishiyama ◽  
Koji Umezawa ◽  
Kentaro Yamada ◽  
Masaharu Takahashi ◽  
Satoshi Kunita ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Structure Prediction ◽  
Hepatitis E Virus ◽  
Culture Media ◽  
Molecular Size ◽  
Hepatitis E ◽  
Terminal Region ◽  
Translation Product ◽  
Orf2 Protein

The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.

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