scholarly journals State-dependent and site-directed photodynamic transformation of HCN2 channel by singlet oxygen

2014 ◽  
Vol 143 (5) ◽  
pp. 633-644 ◽  
Author(s):  
Weihua Gao ◽  
Zhuocheng Su ◽  
Qinglian Liu ◽  
Lei Zhou
Keyword(s):  
Singlet Oxygen ◽  
Basic Research ◽  
Laser Pulses ◽  
Inhibitory Effect ◽  
Biological Molecules ◽  
Hcn Channels ◽  
State Dependent ◽  
C Terminus ◽  
Instantaneous Current ◽  
Cardiac Pacemaking

Singlet oxygen (1O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels–which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain–are subject to modification by 1O2. To increase the site specificity of 1O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame 1O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves 1O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a 1O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, 1O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, 1O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for 1O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying 1O2 modifications at the molecular level.

scholarly journals Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel

2018 ◽  
Vol 150 (9) ◽  
pp. 1273-1286 ◽  
Author(s):  
Vinay Idikuda ◽  
Weihua Gao ◽  
Khade Grant ◽  
Zhuocheng Su ◽  
Qinglian Liu ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Molecular Level ◽  
Basic Research ◽  
Laser Pulses ◽  
Oxygen Species ◽  
Dependent Inactivation ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Short Laser Pulses ◽  
Working Distance

Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2. The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel’s pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

scholarly journals The Inhibitory Effect of a Novel Polypeptide Fraction fromArca subcrenataon Cancer-Related Inflammation in Human Cervical Cancer HeLa Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yu Wu ◽  
Xianjing Hu ◽  
Liyan Song ◽  
Jianhua Zhu ◽  
Rongmin Yu
Keyword(s):  
Cervical Cancer ◽  
Hela Cells ◽  
Molecular Mechanisms ◽  
Tumor Development ◽  
Basic Research ◽  
Inhibitory Effect ◽  
Fishery Resource ◽  
Proinflammatory Mediators ◽  
Raw264.7 Macrophage ◽  
Human Cervical Cancer

Inflammation is known to be closely associated with the development of cancer. The study was launched in human cervical cancer HeLa cells to investigate the antitumor and anti-inflammatory effects of P2, a marine polypeptide fraction from an important fishery resourceArca subcrenata. The basic research showed that P2 could suppress the production of nitric oxide in LPS-induced RAW264.7 macrophage cells as well as the secretion of inflammatory cytokines IL-6 and TNF-αin human cervical cancer HeLa cells. For the molecular mechanisms, P2 was shown to downregulate the gene expression of proinflammatory cytokines IL-6 and IL-8 and to inhibit the COX-2 and iNOS-related pathways in HeLa cells. In consequence, P2 might inhibit tumor development by blocking the interaction between tumor microenvironment and proinflammatory mediators. All findings indicate that P2 possesses the potential to be developed as a novel agent for cancer therapy.

Effect of guaianolides in the meiosis reinitiation of amphibian oocytes

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 10-16 ◽  
Author(s):  
J. Zapata-Martínez ◽  
G. Sánchez-Toranzo ◽  
F. Chaín ◽  
C.A.N. Catalán ◽  
M.I. Bühler
Keyword(s):  
Biological Activities ◽  
Wide Spectrum ◽  
Sesquiterpene Lactones ◽  
Inhibitory Effect ◽  
Biological Molecules ◽  
Plant Metabolites ◽  
Major Mechanism ◽  
Bufo Arenarum ◽  
Asteraceae Family

SummarySesquiterpene lactones (STLs) are a large and structurally diverse group of plant metabolites generally found in the Asteraceae family. STLs exhibit a wide spectrum of biological activities and it is generally accepted that their major mechanism of action is the alkylation of the thiol groups of biological molecules. The guaianolides is one of various groups of STLs. Anti-tumour and anti-migraine effects, an allergenic agent, an inhibitor of smooth muscle cells and of meristematic cell proliferation are only a few of the most commonly reported activities of STLs. In amphibians, fully grown ovarian oocytes are arrested at the beginning of meiosis I. Under stimulus with progesterone, this meiotic arrest is released and meiosis progresses to metaphase II, a process known as oocyte maturation. There are previous records of the inhibitory effect of dehydroleucodin (DhL), a guaianolide lactone, on the progression of meiosis. It has been also shown that DhL and its 11,13-dihydroderivative (2H-DhL; a mixture of epimers at C-11) act as blockers of the resumption of meiosis in fully grown ovarian oocytes from the amphibian Rhinella arenarum (formerly classified as Bufo arenarum). The aim of this study was to analyze the effect of four closely related guaianolides, i.e., DhL, achillin, desacetoxymatricarin and estafietin as possible inhibitors of meiosis in oocytes of amphibians in vitro and discuss some structure–activity relationships. It was found that the inhibitory effect on meiosis resumption is greater when the lactone has two potentially reactive centres, either a α,β–α′,β′-diunsaturated cyclopentanone moiety or an epoxide group plus an exo-methylene-γ-lactone function.

scholarly journals Allosteric potentiation of a ligand-gated ion channel is mediated by access to a deep membrane-facing cavity

2018 ◽  
Vol 115 (42) ◽  
pp. 10672-10677 ◽  
Author(s):  
Stephanie A. Heusser ◽  
Marie Lycksell ◽  
Xueqing Wang ◽  
Sarah E. McComas ◽  
Rebecca J. Howard ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Allosteric Modulation ◽  
Bacterial Protein ◽  
Detailed Model ◽  
Differential Motion ◽  
State Dependent ◽  
The Common ◽  
Pore Lining

Theories of general anesthesia have shifted in focus from bulk lipid effects to specific interactions with membrane proteins. Target receptors include several subtypes of pentameric ligand-gated ion channels; however, structures of physiologically relevant proteins in this family have yet to define anesthetic binding at high resolution. Recent cocrystal structures of the bacterial protein GLIC provide snapshots of state-dependent binding sites for the common surgical agent propofol (PFL), offering a detailed model system for anesthetic modulation. Here, we combine molecular dynamics and oocyte electrophysiology to reveal differential motion and modulation upon modification of a transmembrane binding site within each GLIC subunit. WT channels exhibited net inhibition by PFL, and a contraction of the cavity away from the pore-lining M2 helix in the absence of drug. Conversely, in GLIC variants exhibiting net PFL potentiation, the cavity was persistently expanded and proximal to M2. Mutations designed to favor this deepened site enabled sensitivity even to subclinical concentrations of PFL, and a uniquely prolonged mode of potentiation evident up to ∼30 min after washout. Dependence of these prolonged effects on exposure time implicated the membrane as a reservoir for a lipid-accessible binding site. However, at the highest measured concentrations, potentiation appeared to be masked by an acute inhibitory effect, consistent with the presence of a discrete, water-accessible site of inhibition. These results support a multisite model of transmembrane allosteric modulation, including a possible link between lipid- and receptor-based theories that could inform the development of new anesthetics.

Both products of the fosB gene, FosB and its short form, FosB/SF, are transcriptional activators in fibroblasts

1991 ◽  
Vol 11 (11) ◽  
pp. 5470-5478
Author(s):  
P Dobrazanski ◽  
T Noguchi ◽  
K Kovary ◽  
C A Rizzo ◽  
P S Lazo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Cycle Progression ◽  
Short Form ◽  
Constitutive Expression ◽  
Inhibitory Effect ◽  
Negative Regulator ◽  
3T3 Cells ◽  
C Terminus ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We demonstrate that a member of the fos family, the fosB gene, gives rise to two transcripts by alternative splicing of exon 4, generating two proteins, FosB of 338 amino acids and a short form, FosB/SF, which contains the DNA binding and dimerization domains but not the 101 amino acids of the C terminus. FosB/SF activates an AP-1-chloramphenicol acetyltransferase construct in NIH 3T3 cells, as determined by transient and stable transfections, although more weakly than does FosB. In contrast to FosB, FosB/SF has lost its ability to repress the dyad symmetry element of the c-fos gene. FosB/SF when expressed in excess to FosB can downmodulate the activity of FosB. Constitutive expression of high levels of FosB/SF in NIH 3T3 cells has no significant inhibitory effect in the induction of cell proliferation or cell cycle progression, indicating that FosB/SF is not a negative regulator of cell growth. This conclusion is further confirmed by the observation that the majority of the Jun molecules are complexed with FosB/SF in the FosB/SF-overexpressing cells.

scholarly journals A Conserved Regulatory Module at the C Terminus of the Papillomavirus E1 Helicase Domain Controls E1 Helicase Assembly

2014 ◽  
Vol 89 (2) ◽  
pp. 1129-1142 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Inhibitory Effect ◽  
Dna Helicase ◽  
Regulatory Sequence ◽  
Helicase Domain ◽  
E1 Protein ◽  
C Terminus ◽  
Regulatory Module ◽  
Origin Of Dna Replication ◽  
Coding Capacity ◽  
Regulatory Challenges

ABSTRACTViruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase. However, E1 also has the more unusual ability to generate local melting by forming a double trimer (DT) complex that can untwist the double-stranded origin of DNA replication (ori) DNA in preparation for DH formation. Here we describe a switching mechanism that allows the papillomavirus E1 protein to form these two different kinds of oligomers and to transition between them. We show that a conserved regulatory module attached to the E1 helicase domain blocks hexamer and DH formation and promotes DT formation. In the presence of the appropriate trigger, the inhibitory effect of the regulatory module is relieved and the transition to DH formation can occur.IMPORTANCEThis study provides a mechanistic understanding into how a multifunctional viral polypeptide can provide different, seemingly incompatible activities. A conserved regulatory sequence module attached to the AAA+helicase domain in the papillomavirus E1 protein allows the formation of different oligomers with different biochemical activities.

scholarly journals Prostaglandin E1 inhibits endocytosis in the β-cell endocytosis

2016 ◽  
Vol 229 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Ying Zhao ◽  
Qinghua Fang ◽  
Susanne G Straub ◽  
Manfred Lindau ◽  
Geoffrey W G Sharp
Keyword(s):  
Inhibitory Effect ◽  
Prostaglandin E ◽  
Β Cell ◽  
Α Subunit ◽  
Capacitance Measurements ◽  
Readily Releasable Pool ◽  
Whole Cell Patch Clamp ◽  
C Terminus ◽  
High Level

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca2+ influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca2+ influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

Withaferin A binds covalently to the N-terminal domain of annexin A2

2012 ◽  
Vol 393 (10) ◽  
pp. 1151-1163 ◽  
Author(s):  
Gabriel Ozorowski ◽  
Christopher M. Ryan ◽  
Julian P. Whitelegge ◽  
Hartmut Luecke
Keyword(s):  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Annexin A2 ◽  
Actin Dynamics ◽  
Withaferin A ◽  
Core Domain ◽  
Cancer Pathways ◽  
Terminal Domain ◽  
C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

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