scholarly journals Chloride channel activity of ClC-2 is modified by the actin cytoskeleton

2000 ◽  
Vol 352 (3) ◽  
pp. 789-794 ◽  
Author(s):  
Najma AHMED ◽  
Mohabir RAMJEESINGH ◽  
Simeon WONG ◽  
Alison VARGA ◽  
Elizabeth GARAMI ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channel ◽  
Electrostatic Interactions ◽  
Osmotic Shock ◽  
Expression System ◽  
Cell Volume Regulation ◽  
Channel Activity ◽  
N Terminus ◽  
Inhibitory Domain

The chloride channel ClC-2has been implicated in essential physiological functions, including cell-volume regulation and fluid secretion by specific epithelial tissues. Although ClC-2 is known to be activated by hyperpolarization and hypo-osmotic shock, the molecular basis for the regulation of this channel remains unclear. Here we show in the Xenopus oocyte expression system that the chloride-channel activity of ClC-2 is enhanced after treatment with the actin-disrupting agents cytochalasin and latrunkulin. These findings suggest that the actin cytoskeleton normally exerts an inhibitory effect on ClC-2 activity. An inhibitory domain was previously defined in the N-terminus of ClC-2, so we sought to determine whether this domain might interact directly with actin in binding assays in vitro. We found that a glutathione S-transferase fusion protein containing the inhibitory domain was capable of binding actin in overlay and co-sedimentation assays. Further, the binding of actin to this relatively basic peptide (pI 8.4) might be mediated through electrostatic interactions because binding was inhibited at high concentrations of NaCl with a half-maximal decrease in signal at 180mM NaCl. This work suggests that electrostatic interactions between the N-terminus of ClC-2 and the actin cytoskeleton might have a role in the regulation of this channel.

scholarly journals Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

2008 ◽  
Vol 411 (3) ◽  
pp. 523-530 ◽  
Author(s):  
Gary S. Laco ◽  
Yves Pommier
Keyword(s):  
Amino Acids ◽  
Topoisomerase I ◽  
Electrostatic Interactions ◽  
Functional Domain ◽  
Site Mutation ◽  
Supercoiled Dna ◽  
Terminal Domain ◽  
Tryptophan Residues ◽  
N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

Cell volume regulation in rat thin ascending limb of Henle's loop

1992 ◽  
Vol 263 (3) ◽  
pp. F353-F362
Author(s):  
L. F. Onuchic ◽  
I. R. Arenstein ◽  
A. G. Lopes
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Osmotic Shock ◽  
Basolateral Membrane ◽  
Cell Volume Regulation ◽  
Tetraacetic Acid ◽  
Volume Regulatory Decrease ◽  
Henle's Loop ◽  
Volume Regulatory Increase

Thin ascending limb cells of Henle's loop from Wistar rats were studied with in vitro microperfusion and video-optical techniques to investigate their ability in regulating cell volume during osmotic shock and to identify mechanisms of ion transport involved in the process. These cells showed a clear volume regulatory decrease (VRD) response in hyposmotic medium, but no volume regulatory increase in hyperosmotic medium. The presence of barium in the bath abolished VRD. Removal of K+ from bath and perfusate also inhibited the VRD response. Reintroduction of K+ in hyposmotic conditions reestablished cell volume regulation. Introduction of anthracene-9-COOH to the basolateral medium blocked cell volume regulatory response. Cl- removal from perfusate and bath solutions also inhibited VRD, probably because of a significant intracellular Cl- depletion. Exposure of cells to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid in perfusate and bath solutions reduced significantly Ca2+ concentration and impaired VRD. Reintroduction of Ca2+ in hyposmotic conditions restored volume regulation. The presence of ouabain in basolateral medium also inhibited VRD. These data suggest that the following mechanisms in the basolateral membrane are involved in VRD response: K+ and Cl- conductive pathways, which might be Ca2+ dependent for activation, and an Na(+)-K(+)-adenosinetriphosphatase.

scholarly journals Chloride channel activity of ClC-2 is modified by the actin cytoskeleton

2000 ◽  
Vol 352 (3) ◽  
pp. 789 ◽  
Author(s):  
Najma AHMED ◽  
Mohabir RAMJEESINGH ◽  
Simeon WONG ◽  
Alison VARGA ◽  
Elizabeth GARAMI ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Chloride Channel ◽  
Channel Activity

scholarly journals Determinant of the extracellular location of the N-terminus of human multidrug-resistance-associated protein

2000 ◽  
Vol 348 (3) ◽  
pp. 597-606 ◽  
Author(s):  
Jian-Ting ZHANG
Keyword(s):  
Multidrug Resistance ◽  
Expression System ◽  
Critical Role ◽  
Signal Recognition Particle ◽  
Dependent Manner ◽  
Membrane Translocation ◽  
C Terminus ◽  
N Terminus ◽  
Polytopic Membrane Protein

Multidrug-resistance-associated protein (MRP) is a member of the ATP-binding cassette (ABC) membrane-transport superfamily and is responsible for multidrug resistance in cancer cells. Distinct from other members of the ABC superfamily, MRP has three membrane-spanning domains (MSDs) and the N-terminus is located extracellularly. It has been shown that the first MSD (MSD1) with an extracellular N-terminus is important for MRP function. To address what ensures the generation of this structural organization of MRP and to understand in general the molecular mechanism of membrane folding of polytopic proteins with extracellular N-termini, the biogenesis of MSD1 in human MRP1 was examined using an in vitro expression system. Surprisingly, the second transmembrane segment (TM2) in MSD1 was found to play a critical role in the correct membrane translocation and folding of MSD1 in human MRP1. TM2 not only plays an essential role to ensure the N-terminus-outside/C-terminus-inside orientation of TM1 with an extracellular N-terminus, it can also translocate into membranes post-translationally in a signal-recognition particle and ribosome-dependent manner to provide an additional insurance for correct folding of MSD1 in MRP. These findings suggest that TM2 in a polytopic membrane protein with an extracellular N-terminus may play a critical role in controlling correct membrane translocation and folding of the protein in general.

Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα

1998 ◽  
Vol 80 (08) ◽  
pp. 310-315 ◽  
Author(s):  
Marie-Christine Bouton ◽  
Christophe Thurieau ◽  
Marie-Claude Guillin ◽  
Martine Jandrot-Perrus
Keyword(s):  
Platelet Activation ◽  
Electrostatic Interactions ◽  
Platelet Glycoprotein ◽  
Thrombin Receptor ◽  
Central Position ◽  
Amidolytic Activity ◽  
N Terminus ◽  
Hydrolysis Of ◽  
Chemical Cross Linking ◽  
Positively Charged

SummaryThe interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Protein Analysis of Pollen Tubes after the Treatments of Membrane Trafficking Inhibitors Gains Insights on Molecular Mechanism Underlying Pollen Tube Polar Growth

2021 ◽  
Vol 40 (2) ◽  
pp. 205-222
Author(s):  
Monica Scali ◽  
Alessandra Moscatelli ◽  
Luca Bini ◽  
Elisabetta Onelli ◽  
Rita Vignani ◽  
...  
Keyword(s):  
Pollen Tube ◽  
Actin Cytoskeleton ◽  
Membrane Trafficking ◽  
Tip Growth ◽  
Male Gametophyte ◽  
Pollen Tubes ◽  
Structural Features ◽  
Two Dimensional Gel Electrophoresis ◽  
Depth Analysis

AbstractPollen tube elongation is characterized by a highly-polarized tip growth process dependent on an efficient vesicular transport system and largely mobilized by actin cytoskeleton. Pollen tubes are an ideal model system to study exocytosis, endocytosis, membrane recycling, and signaling network coordinating cellular processes, structural organization and vesicular trafficking activities required for tip growth. Proteomic analysis was applied to identifyNicotiana tabacumDifferentially Abundant Proteins (DAPs) after in vitro pollen tube treatment with membrane trafficking inhibitors Brefeldin A, Ikarugamycin and Wortmannin. Among roughly 360 proteins separated in two-dimensional gel electrophoresis, a total of 40 spots visibly changing between treated and control samples were identified by MALDI-TOF MS and LC–ESI–MS/MS analysis. The identified proteins were classified according to biological processes, and most proteins were related to pollen tube energy metabolism, including ammino acid synthesis and lipid metabolism, structural features of pollen tube growth as well modification and actin cytoskeleton organization, stress response, and protein degradation. In-depth analysis of proteins corresponding to energy-related pathways revealed the male gametophyte to be a reliable model of energy reservoir and dynamics.

scholarly journals RNA Helicase A Regulates the Replication of RNA Viruses

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 361
Author(s):  
Rui-Zhu Shi ◽  
Yuan-Qing Pan ◽  
Li Xing
Keyword(s):  
Virus Replication ◽  
Rna Binding ◽  
Rna Viruses ◽  
Rna Helicase ◽  
Rna Helicase A ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
N Terminus

The RNA helicase A (RHA) is a member of DExH-box helicases and characterized by two double-stranded RNA binding domains at the N-terminus. RHA unwinds double-stranded RNA in vitro and is involved in RNA metabolisms in the cell. RHA is also hijacked by a variety of RNA viruses to facilitate virus replication. Herein, this review will provide an overview of the role of RHA in the replication of RNA viruses.

scholarly journals Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Vikas A. Tillu ◽  
James Rae ◽  
Ya Gao ◽  
Nicholas Ariotti ◽  
Matthias Floetenmeyer ◽  
...  
Keyword(s):  
Electrostatic Interactions ◽  
Membrane Lipid ◽  
Membrane Curvature ◽  
Caveolin 1 ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Solution Generation ◽  
Endosomal System ◽  
Disordered Regions

AbstractCaveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.

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