scholarly journals Rapid Modulation of Aromatase Activity in the Vertebrate Brain

2013 ◽  
Vol 7 ◽  
pp. JEN.S11268 ◽  
Author(s):  
Thierry D. Charlier ◽  
Charlotte A. Cornil ◽  
Jacques Balthazart
Keyword(s):  
Amino Acids ◽  
Enzymatic Activity ◽  
Kinase Inhibitors ◽  
Cell Types ◽  
Protein Kinase Inhibitors ◽  
Site Directed Mutagenesis ◽  
General Mechanism ◽  
Aromatase Activity ◽  
Vertebrate Brain ◽  
Rapid Modulation

Numerous steroid hormones, including 17β-estradiol (E2), activate rapid and transient cellular, physiological, and behavioral changes in addition to their well-described genomic effects. Aromatase is the key-limiting enzyme in the production of estrogens, and the rapid modulation of this enzymatic activity could produce rapid changes in local E2 concentrations. The mechanisms that might mediate such rapid enzymatic changes are not fully understood but are currently under intense scrutiny. Recent studies in our laboratory indicate that brain aromatase activity is rapidly inhibited by an increase in intracellular calcium concentration resulting from potassium-induced depolarization or from the activation of glutamatergic receptors. Phosphorylating conditions also reduce aromatase activity within minutes, and this inhibition is blocked by the addition of multiple protein kinase inhibitors. This rapid modulation of aromatase activity by phosphorylating conditions is a general mechanism observed in different cell types and tissues derived from a variety of species, including human aromatase expressed in various cell lines. Phosphorylation processes affect aromatase itself and do not involve changes in aromatase protein concentration. The control of aromatase activity by multiple kinases suggests that several amino acids must be concomitantly phosphorylated to modify enzymatic activity but site-directed mutagenesis of several amino acids alone or in combination has not to date revealed the identity of the targeted residue(s). Altogether, the phosphorylation processes affecting aromatase activity provide a new general mechanism by which the concentration of estrogens can be rapidly altered in the brain.

scholarly journals Human and Quail Aromatase Activity Is Rapidly and Reversibly Inhibited by Phosphorylating Conditions

Endocrinology ◽  
2011 ◽  
Vol 152 (11) ◽  
pp. 4199-4210 ◽  
Author(s):  
Thierry D. Charlier ◽  
Nobuhiro Harada ◽  
Jacques Balthazart ◽  
Charlotte A. Cornil
Keyword(s):  
Enzymatic Activity ◽  
Bioinformatic Analysis ◽  
Site Directed Mutagenesis ◽  
General Mechanism ◽  
Aromatase Activity ◽  
Potential Implication ◽  
Hek 293 ◽  
Calcium Dependent ◽  
293 Cells ◽  
Rapid Changes

Besides their slow genomic actions, estrogens also induce rapid physiological responses. To be functionally relevant, these effects must be associated with rapid changes in local concentrations of estrogens. Rapid changes in aromatase activity (AA) controlled by calcium-dependent phosphorylations of the enzyme can alter in a rapid manner local estrogen concentrations, but so far this mechanism was identified only in the avian (quail) brain. We show here that AA is also rapidly down-regulated by phosphorylating conditions in quail ovary homogenates and in various cell lines transfected with human aromatase (HEK 293, Neuro2A, and C6). Enzymatic activity was also rapidly inhibited after depolarization of aromatase-expressing HEK 293 cells with 100 mm KCl, and activity was fully restored when cells returned to control conditions. Western blot analysis demonstrated that the reduction of enzymatic activity is not due to protein degradation. We next investigated by site-directed mutagenesis the potential implication in the control of AA of specific aromatase residues identified by bioinformatic analysis. Mutation of the amino acids S118, S247, S267, T462, T493, or S497 to alanine, alone or in combination, did not block the rapid inhibition of enzymatic activity induced by phosphorylating conditions, but basal AA was markedly decreased in the S118A mutant. Altogether, these results demonstrate that the rapid inhibition of AA is a widespread and fully reversible process and that phosphorylation of specific residues modulate AA. These processes provide a new general mechanism by which local estrogen concentration can be rapidly altered in the brain and other tissues.

Calcium-activated sodium and chloride fluxes modulate platelet volume: role of Ca2+ stores

1994 ◽  
Vol 267 (5) ◽  
pp. C1435-C1441 ◽  
Author(s):  
B. P. Fine ◽  
E. S. Marques ◽  
K. A. Hansen
Keyword(s):  
Protein Kinases ◽  
Volume Change ◽  
Kinase Inhibitors ◽  
Cell Types ◽  
Protein Kinase Inhibitors ◽  
Disulfonic Acid ◽  
Platelet Volume ◽  
Ionic Fluxes ◽  
Dependent Protein

An increase in cytosolic ionized Ca2+ concentration ([Ca2+]i) initiates volume changes in various types of cells. In response to increases in [Ca2+]i most cell types contract by efflux of K+ and Cl-, whereas platelets expand in response to rises in [Ca2+]i. This study examined the importance of the source of Ca2+, the flux of ions responsible for the volume change, and the role of Ca(2+)-dependent protein kinases in regulating these ionic fluxes. The baseline platelet volume was independent of extracellular Ca2+ but when stimulated by the Ca2+ ionophore A-23187 (50 nM) the volume increased in both the presence and absence of extracellular Ca2+ (1.18 +/- 0.08 vs. 0.83 +/- 0.06 fl, respectively). The increased volume was caused by the gain of Na+ and Cl-. Na+ entered through both conductive and nonconductive (Na+/H+ exchange) pathways, whereas the influx of Cl- was conductive and inhibited by the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The Ca(2+)-induced volume change was blocked by both calmodulin and protein kinase inhibitors. Thus the activation of Ca(2+)-dependent protein kinases promotes platelet swelling by stimulating Na+ and Cl- influx.

scholarly journals Arf-1 (ADP-ribosylation factor-1) is involved in the activation of a mammalian Na+-selective current

2004 ◽  
Vol 377 (2) ◽  
pp. 539-544 ◽  
Author(s):  
Sebastian STRAUBE ◽  
Anant B. PAREKH
Keyword(s):  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Brefeldin A ◽  
Cell Types ◽  
Protein Kinase Inhibitors ◽  
Na Current ◽  
Camp Phosphodiesterase ◽  
Pipette Solution ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Stimulation of mammalian cells often results in an increase in the intracellular Na+ concentration, brought about by Na+ influx into the cell via Na+-permeable ion channels. In some cell types, particularly renal epithelia and mast cells, non-hydrolysable analogues of GTP, such as GTP[S] (guanosine 5´-[γ-thio]triphosphate), activate a non-voltage-activated Na+-selective current. We have carried out whole-cell patch–clamp experiments to examine how GTP[S] activates the Na+ current in a rat mast cell line. The ability of GTP[S] to activate Na+ influx was prevented by including GTP in the pipette solution, indicating the involvement of small G-proteins. Brefeldin A and Arf-1-(2–17), inhibitors of Arf-1 (ADP-ribosylation factor-1) proteins, suppressed the activation of Na+ entry by GTP[S]. However, non-active succinylated Arf-1-(2–17) or an N-terminal myristoylated peptide directed towards Arf-5 were ineffective. Arf proteins modulate the cytoskeleton, and disruption of the cytoskeleton with cytochalasin D or its stabilization with phalloidin impaired the development of the Na+ current. Disaggregation of microtubules was without effect. Dialysis with cAMP or inhibition of cAMP phosphodiesterase with caffeine both decreased the extent of Na+ entry, and this was not prevented by pre-treatment with broad-spectrum protein kinase inhibitors. Collectively, our results suggest that the mechanism of activation of a mammalian non-voltage-activated Na+-selective current requires an Arf small G-protein, most probably Arf-1.

scholarly journals Improving the Thermostability of Raw-Starch-Digesting Amylase from a Cytophaga sp. by Site-Directed Mutagenesis

2003 ◽  
Vol 69 (4) ◽  
pp. 2383-2385 ◽  
Author(s):  
Rong-Jen Shiau ◽  
Hui-Chen Hung ◽  
Chii-Ling Jeang
Keyword(s):  
Amino Acids ◽  
Enzymatic Activity ◽  
Half Life ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Raw Starch ◽  
Heat Stable ◽  
Independent Mechanism

ABSTRACT A heat-stable raw-starch-digesting amylase (RSDA) was generated through PCR-based site-directed mutagenesis. At 65°C, the half-life of this mutant RSDA, which, compared with the wild-type RSDA, lacks amino acids R178 and G179, was increased 20-fold. While the wild type was inactivated completely at pH 3.0, the mutant RSDA still retained 41% of its enzymatic activity. The enhancement of RSDA thermostability was demonstrated to be via a Ca2+-independent mechanism.

scholarly journals Structure-guided examination of the mechanogating mechanism of PIEZO2

2019 ◽  
Vol 116 (28) ◽  
pp. 14260-14269 ◽  
Author(s):  
Francisco J. Taberner ◽  
Vincenzo Prato ◽  
Irina Schaefer ◽  
Katrin Schrenk-Siemens ◽  
Paul A. Heppenstall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Single Channel ◽  
Hydrophobic Interactions ◽  
Cell Types ◽  
Site Directed Mutagenesis ◽  
Inactivation Kinetics ◽  
Voltage Sensitivity ◽  
Intrinsically Disordered ◽  
Piezo Channels ◽  
Α Helix

Piezo channels are mechanically activated ion channels that confer mechanosensitivity to a variety of different cell types. Piezos oligomerize as propeller-shaped homotrimers that are thought to locally curve the membrane into spherical domes that project into the cell. While several studies have identified domains and amino acids that control important properties such as ion permeability and selectivity as well as inactivation kinetics and voltage sensitivity, only little is known about intraprotein interactions that govern mechanosensitivity—the most unique feature of PIEZOs. Here we used site-directed mutagenesis and patch-clamp recordings to investigate the mechanogating mechanism of PIEZO2. We demonstrate that charged amino acids at the interface between the beam domain—i.e., a long α-helix that protrudes from the intracellular side of the “propeller” blade toward the inner vestibule of the channel—and the C-terminal domain (CTD) as well as hydrophobic interactions between the highly conserved Y2807 of the CTD and pore-lining helices are required to ensure normal mechanosensitivity of PIEZO2. Moreover, single-channel recordings indicate that a previously unrecognized intrinsically disordered domain located adjacent to the beam acts as a cytosolic plug that limits ion permeation possibly by clogging the inner vestibule of both PIEZO1 and PIEZO2. Thus, we have identified several intraprotein domain interfaces that control the mechanical activation of PIEZO1 and PIEZO2 and which might thus serve as promising targets for drugs that modulate the mechanosensitivity of Piezo channels.

Prevalence of the pit and antipit in the genus Glycine

1987 ◽  
Vol 45 ◽  
pp. 986-987
Author(s):  
R. W. Yaklich ◽  
E. L. Vigil ◽  
W. P. Wergin
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Glycine Max ◽  
Seed Coat ◽  
Cell Types ◽  
Abaxial Surface ◽  
Legume Seed ◽  
Initial Report ◽  
Cone Cells ◽  
Glycine Max L

The legume seed coat is the site of sucrose unloading and the metabolism of imported ureides and synthesis of amino acids for the developing embryo. The cell types directly responsible for these functions in the seed coat are not known. We recently described a convex layer of tissue on the inside surface of the soybean (Glycine max L. Merr.) seed coat that was termed “antipit” because it was in direct opposition to the concave pit on the abaxial surface of the cotyledon. Cone cells of the antipit contained numerous hypertrophied Golgi apparatus and laminated rough endoplasmic reticulum common to actively secreting cells. The initial report by Dzikowski (1936) described the morphology of the pit and antipit in G. max and found these structures in only 68 of the 169 seed accessions examined.

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