Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4.

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1–5, each the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4, and confirmed the interaction using glutathione-S-transferase-fusion-pulldown. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca2+-dependent manner to a motif present in the central cytosolic loop of NCKX4, and was abolished by the double mutant I328D/F334D. When co-transfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ~2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, co-expression of wild type calmodulin, but not a Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ binding to calmodulin pre-positioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.

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Physiological concentrations of insulin and T3 stimulate 3T3-L1 adipocyte acyl-CoA synthetase gene transcription

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.270.5.e873 ◽

1996 ◽

Vol 270 (5) ◽

pp. E873-E881 ◽

Cited By ~ 3

Author(s):

M. S. Kansara ◽

A. K. Mehra ◽

J. Von Hagen ◽

E. Kabotyansky ◽

P. J. Smith

Keyword(s):

Gene Transcription ◽

Fold Increase ◽

Mrna Levels ◽

Dependent Manner ◽

Long Chain Fatty Acids ◽

Reciprocal Regulation ◽

Stearoyl Coa Desaturase ◽

Dose Dependent ◽

Long Chain ◽

Stimulation Of

Acyl-CoAsynthetase (ACS) is a key gene for cellular utilization of long-chain fatty acids. We characterized its regulation by physiological concentrations of insulin that acutely regulate metabolism. Our results demonstrate that subnanomolar insulin rapidly and maximally stimulates ACS gene transcription in the absence of protein synthesis; 0.5 nM insulin produced a 2.3 +/- 0.1-fold increase in ACS mRNA levels and induced ACS gene transcription 2.4 +/- 0.3-fold. The insulin sensitivity of ACS was compared with lipoprotein lipase (LPL) and stearoyl-CoA desaturase-1 (SCD-1), which were both less sensitive to insulin. Physiological triiodothyronine (10 nm) also induced ACS mRNA 2.4 +/- 0.1-fold and gene transcription 2.8 +/- 0.3-fold and coordinately induced LPL and SCD-1 mRNA and gene transcription. Because insulin and adenosine 3',5'-cyclic monophosphate often regulate genes involved in lipid and carbohydrate metabolism in a reciprocal manner, we evaluated effects of 1-methyl-3-isobutylxanthine (MIX).ACS mRNA levels were strongly downregulated by MIX in a dose-dependent manner, and ACS gene transcription inhibited in a coordinate manner with LPL and SCD-1. These data demonstrate a uniquely sensitive pattern of stimulation of ACS gene transcription by insulin with reciprocal regulation by MIX, and they suggest a significant role for ACS as a tightly regulated “gatekeeper” gene participating in the control of adipocyte metabolism.

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Tivantinib, A c-Met Inhibitor in Clinical Trials, Is Susceptible to ABCG2-Mediated Drug Resistance

Cancers ◽

10.3390/cancers12010186 ◽

2020 ◽

Vol 12 (1) ◽

pp. 186 ◽

Cited By ~ 5

Author(s):

Zhuo-Xun Wu ◽

Yuqi Yang ◽

Qiu-Xu Teng ◽

Jing-Quan Wang ◽

Zi-Ning Lei ◽

...

Keyword(s):

Drug Resistance ◽

Critical Role ◽

Hek293 Cells ◽

Atp Binding ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Lung Cancers ◽

Transporter Family ◽

Met Inhibitor ◽

Atp Binding Cassette

Tivantinib, also known as ARQ-197, is a potent non-ATP competitive selective c-Met inhibitor currently under phase 3 clinical trial evaluation for liver and lung cancers. In this study, we explored factors that may lead to tivantinib resistance, especially in regards to its interaction with ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is one of the most important members of the ATP-binding cassette (ABC) transporter family, a group of membrane proteins that play a critical role in mediating multidrug resistance (MDR) in a variety of cancers, including those of the liver and lung. Tivantinib received a high score in docking analysis, indicating a strong interaction between tivantinib and ABCG2, and an ATPase assay indicated that tivantinib stimulated ABCG2 ATPase activity in a concentration-dependent manner. An MTT assay showed that ABCG2 overexpression significantly desensitized both the cancer cells and ABCG2 transfected-HEK293 cells to tivantinib and that this drug resistance can be reversed by ABCG2 inhibitors. Furthermore, tivantinib upregulated the protein expression of ABCG2 without altering the cell surface localization of ABCG2, leading to increased resistance to substrate drugs, such as mitoxantrone. Altogether, these data demonstrate that tivantinib is a substrate of ABCG2, and, therefore, ABCG2 overexpression may decrease its therapeutic effect. Our study provides evidence that the overexpression of ABCG2 should be monitored in clinical settings as an important risk factor for tivantinib drug resistance.

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Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613873 ◽

2000 ◽

Vol 83 (04) ◽

pp. 617-621 ◽

Cited By ~ 3

Author(s):

Masaru Ido ◽

Tatsuya Hayashi ◽

Esteban Gabazza ◽

Koji Suzuki

Keyword(s):

Protein Kinase ◽

Receptor Agonist ◽

Thromboxane A2 ◽

Cytoplasmic Tail ◽

Fold Increase ◽

Human Platelets ◽

Thrombin Receptor ◽

Glutathione S Transferase ◽

Protease Activated Receptor 1 ◽

Stimulation Of

SummaryStimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PAR1 (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A2 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.

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Stimulation of mouse liver glutathione S-transferase activity in propylthiouracil-treated mice in vivo by tri-iodothyronine

Biochemical Journal ◽

10.1042/bj2330595 ◽

1986 ◽

Vol 233 (2) ◽

pp. 595-598 ◽

Cited By ~ 10

Author(s):

M T Williams ◽

H Carrington ◽

A Herrera

Keyword(s):

Dehydrogenase Activity ◽

Actinomycin D ◽

Fold Increase ◽

Transferase Activity ◽

Glutathione S Transferase ◽

Glycerol 3 Phosphate Dehydrogenase ◽

Dose Dependent Increase ◽

Dose Dependent ◽

Stimulation Of

Female C57Bl/6J mice were given drinking water containing 0.05% propylthiouracil to induce a hypothyroid condition. Mitochondrial glycerol-3-phosphate dehydrogenase activity, used as an index of hypothyroidism, was 57.1 +/- 4.5 and 29.4 +/- 3.8 nmol/min per mg of protein for control and propylthiouracil-treated animals respectively. Administration of tri-iodothyronine resulted in an approx. 4.5-fold increase in dehydrogenase activity in propylthiouracil-treated animals. A dose-dependent increase in hepatic GSH S-transferase activity in propylthiouracil-treated animals was observed at tri-iodothyronine concentrations ranging from 2 to 200 micrograms/100 g body wt. This increase in transferase activity was seen only when 1,2-epoxy-3-(p-nitrophenoxy)propane was used as substrate for the transferase. Transferase activity with 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrate was decreased by tri-iodothyronine. Administration of actinomycin D (75 micrograms/100 g body wt.) inhibited the tri-iodothyronine induction of transferase activity. Results of these studies strongly suggest that tri-iodothyronine administration markedly affected the activities of GSH S-transferase by inducing a specific isoenzyme of GSH S-transferase and suppressing other isoenzymic activities.

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Phospholemman is not required for the acute stimulation of Na+-K+-ATPase α2-activity during skeletal muscle fatigue

AJP Cell Physiology ◽

10.1152/ajpcell.00205.2015 ◽

2015 ◽

Vol 309 (12) ◽

pp. C813-C822 ◽

Cited By ~ 11

Author(s):

Palanikumar Manoharan ◽

Tatiana L. Radzyukevich ◽

Hesamedin Hakim Javadi ◽

Cory A. Stiner ◽

Julio A. Landero Figueroa ◽

...

Keyword(s):

Skeletal Muscle ◽

Exercise Capacity ◽

Skeletal Muscles ◽

Contractile Activity ◽

Critical Role ◽

Regulatory Subunit ◽

Dependent Manner ◽

Evoked Contractions ◽

Stimulation Of

The Na+-K+-ATPase α2-isoform in skeletal muscle is rapidly stimulated during muscle use and plays a critical role in fatigue resistance. The acute mechanisms that stimulate α2-activity are not completely known. This study examines whether phosphorylation of phospholemman (PLM/FXYD1), a regulatory subunit of Na+-K+-ATPase, plays a role in the acute stimulation of α2 in working muscles. Mice lacking PLM (PLM KO) have a normal content of the α2-subunit and show normal exercise capacity, in contrast to the greatly reduced exercise capacity of mice that lack α2 in the skeletal muscles. Nerve-evoked contractions in vivo did not induce a change in total PLM or PLM phosphorylated at Ser63 or Ser68, in either WT or PLM KO. Isolated muscles of PLM KO mice maintain contraction and resist fatigue as well as wild type (WT). Rb+ transport by the α2-Na+-K+-ATPase is stimulated to the same extent in contracting WT and contracting PLM KO muscles. Phosphorylation of sarcolemmal membranes prepared from WT but not PLM KO skeletal muscles stimulates the activity of both α1 and α2 in a PLM-dependent manner. The stimulation occurs by an increase in Na+ affinity without significant change in Vmax and is more effective for α1 than α2. These results demonstrate that phosphorylation of PLM is capable of stimulating the activity of both isozymes in skeletal muscle; however, contractile activity alone is not sufficient to induce PLM phosphorylation. Importantly, acute stimulation of α2, sufficient to support exercise and oppose fatigue, does not require PLM or its phosphorylation.

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Curcumin Acetylsalicylate Extends the Lifespan of Caenorhabditis elegans

Molecules ◽

10.3390/molecules26216609 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6609

Author(s):

Lei Zhou ◽

Jin Liu ◽

Lan-Lan Bu ◽

Duan-Fang Liao ◽

Hai-Jun Tu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Heat Shock ◽

Nuclear Translocation ◽

Aging Effect ◽

Dependent Manner ◽

Biological Models ◽

Glutathione S Transferase ◽

C Elegans ◽

Dose Dependent ◽

Stimulation Of

Aspirin and curcumin have been reported beneficial in anti-aging in a variety of biological models. Here, we synthesized a novel compound, curcumin acetylsalicylate (CA), by combining aspirin and curcumin. We characterized how CA affects the lifespan of Caenorhabditis elegans (C. elegans) worms. Our results demonstrated that CA extended the lifespan of worms in a dose-dependent manner and reached its most important anti-aging effect at the concentration of 20 μM. In addition, CA reduced the deposition of lipofuscin or “age pigment” without affecting the reproductivity of worms. CA also caused a rightward shift of C. elegans lifespan curves in the presence of paraquat-induced (5 mM) oxidative stress or 37 °C acute heat shock. Additionally, CA treatment decreased the reactive oxygen species (ROS) level in C. elegans and increased the expression of downstream genes superoxide dismutase (sod)-3, glutathione S-transferase (gst)-4, heat shock protein (hsp)-16.2, and catalase-1 (ctl-1). Notably, CA treatment resulted in nuclear translocation of the DAF-16 transcription factor, which is known for the stimulation of the expression of SOD-3, GST-4, HSP-16, and CTL-1. CA did not produce a longevity effect in daf-16 mutants. In sum, our data indicate that CA delayed the aging of C. elegans without affecting reproductivity, and this effect may be mediated by its activation of DAF-16 and subsequent expressions of antioxidative genes, such as sod-3 and gst-4. Our study suggests that novel anti-aging drugs may be developed by combining two individual drugs.

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Cellular messengers of stimulants of pepsinogen secretion from isolated frog esophageal mucosa

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1986.250.3.g361 ◽

1986 ◽

Vol 250 (3) ◽

pp. G361-G368 ◽

Cited By ~ 1

Author(s):

T. Shirakawa ◽

B. I. Hirschowitz

Keyword(s):

Critical Role ◽

Phosphodiesterase Inhibitor ◽

Calcium Ionophore ◽

Ionophore A23187 ◽

Esophageal Mucosa ◽

Dependent Manner ◽

Bathing Medium ◽

Camp Content ◽

Pepsinogen Secretion ◽

Stimulation Of

The messenger roles of Ca2+ and cyclic nucleotides in the stimulation of pepsinogen secretion by three classes of stimuli [muscarinic (bethanechol), peptidergic (bombesin), and adrenergic (isoproterenol)] were studied in vitro using the peptic gland-bearing esophageal mucosa from the American bullfrog, Rana catesbeiana. Pepsinogen secretion was stimulated in a dose-dependent manner by the calcium ionophore A23187, by dibutyryl cAMP (DBcAMP), and by isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor. Isoproterenol and bethanechol increased the tissue cAMP content in the presence of IBMX. IBMX, which by itself stimulated secretion, was potentiating in combination with bombesin, additive with bethanechol, and less than additive with isoproterenol. Omission of Ca2+ from the bathing medium did not alter basal pepsinogen secretion nor the response to maximally effective doses of isoproterenol but partly inhibited the secretory responses to bethanechol and bombesin. Ca2+-free medium with 1 mM EGTA reduced pepsinogen secretion under all basal and stimulated (including A23187- but not DBcAMP-stimulated) conditions, indicating a critical role for Ca2+ in the secretion of pepsinogen secretion. A23187 by itself produced only an initial (15-20 min) release of pepsinogen, whereas IBMX and DBcAMP produced a delayed sustained secretion. The combination of A23187 with either IBMX or DBcAMP mimicked the responses to bethanechol or bombesin. These results indicate that both calcium and cAMP may be obligatory and interacting intermediates in the full stimulation of pepsinogen secretion by frog esophageal peptic glands with at least cholinergic and peptidergic stimuli.

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The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620999200818155814 ◽

2020 ◽

Vol 20 ◽

Author(s):

Hongtao Li ◽

Peng Chen ◽

Lei Chen ◽

Xinning Wang

Keyword(s):

Cell Growth ◽

Western Blot ◽

Wilms Tumor ◽

Critical Role ◽

Time Dependent ◽

Luciferase Assay ◽

Dependent Manner ◽

Tlr4 Expression ◽

Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

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HYBID (alias KIAA1199/CEMIP) and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.010457 ◽

2020 ◽

Vol 295 (8) ◽

pp. 2483-2494

Author(s):

Hiroyuki Yoshida ◽

Mika Aoki ◽

Aya Komiya ◽

Yoko Endo ◽

Keigo Kawabata ◽

...

Keyword(s):

Signaling Pathways ◽

Fold Increase ◽

Histamine Receptor ◽

Skin Fibroblasts ◽

Dependent Manner ◽

Skin Damage ◽

Photoaged Skin ◽

Molecular Sizes ◽

Ha Synthase ◽

Hyaluronan Binding

The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of HYBID (hyaluronan-binding protein involved in hyaluronan depolymerization, also called CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine's effects on HA synthase (HAS) expression, the molecular sizes of HA species produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine's effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ or PI3K–Akt signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ∼2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation.

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Arginine Decarboxylase Is Essential for Pneumococcal Stress Responses

Pathogens ◽

10.3390/pathogens10030286 ◽

2021 ◽

Vol 10 (3) ◽

pp. 286

Author(s):

Mary Frances Nakamya ◽

Moses B. Ayoola ◽

Leslie A. Shack ◽

Mirghani Mohamed ◽

Edwin Swiatlo ◽

...

Keyword(s):

Stress Responses ◽

Critical Role ◽

Acid Stress ◽

Arginine Decarboxylase ◽

Arginine Deiminase ◽

Dependent Manner ◽

Cellular Processes ◽

Opportunistic Human Pathogen ◽

Thiol Peroxidase ◽

The Impact

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

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