scholarly journals Architecture of human Rag GTPase heterodimers and their complex with mTORC1

Science ◽  
2019 ◽  
Vol 366 (6462) ◽  
pp. 203-210 ◽  
Author(s):  
Madhanagopal Anandapadamanaban ◽  
Glenn R. Masson ◽  
Olga Perisic ◽  
Alex Berndt ◽  
Jonathan Kaufman ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
The Other ◽  
Allosteric Activation ◽  
Amino Acid Availability ◽  
Cryo Electron Microscopy ◽  
Gtp Binding ◽  
Regulate Cell Growth ◽  
Hotspot Mutations ◽  
Rag Gtpase ◽  
Cell Growth And Proliferation

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.

Peroxidases from Tulip Bulbs (Tulipa fosteriana, L.) Oxidize Xenobiotics NNitrosodimethylamine and N-Nitroso-N-methylaniline in vitro

1997 ◽  
Vol 62 (11) ◽  
pp. 1804-1814 ◽  
Author(s):  
Marie Stiborová ◽  
Hana Hansíková
Keyword(s):  
Cytochromes P450 ◽  
Kinetic Studies ◽  
The Other ◽  
Maximal Velocity ◽  
Michaelis Constant ◽  
Other Hand ◽  
Plant Peroxidases ◽  
Tulip Bulbs

Tulip bulbs (Tulipa fosteriana, L.) contain peroxidases catalyzing the oxidation of the xenobiotics N-nitrosodimethylamine (NDMA) and N-nitroso-N-methylaniline (NMA). Three anionic (A1, A2, A3) and four cationic (B, C, D, E) peroxidases were purified from this tissue, partially characterized and used for kinetic studies. Demethylation of NDMA and NMA producing formaldehyde is catalyzed by one anionic (A1) and three cationic (C, D, E) peroxidases. The oxidation of NDMA by tulip peroxidases exhibits the Michaelis-Menten kinetics. The apparent Michaelis constant and the maximal velocity values for this substrate were determined. On the other hand, non-Michaelian kinetics for the NMA oxidation were observed with tulip peroxidases. The most abundant cationic peroxidase (peroxidase C) was used for detailed enzymatic studies. In addition to formation of formaldehyde, methylaniline, aniline, 4-aminophenol and phenol were found to be metabolites formed from NMA. Phenol was formed presumably by N-demethylation via a benzenediazonium ion, while methylaniline, aniline and 4-aminophenol were products of denitrosation of the substrate. The efficiencies of plant peroxidases to oxidize NDMA and NMA in vitro are compared with those of cytochromes P450 and discussed.

scholarly journals Receptor-operated Ca2+ channels in gastric parietal cells: gastrin and carbachol induce Ca2+ influx in depleting intracellular Ca2+ stores

1993 ◽  
Vol 289 (1) ◽  
pp. 117-124 ◽  
Author(s):  
S Roche ◽  
J P Bali ◽  
R Magous
Keyword(s):  
Steady State ◽  
Receptor Activation ◽  
Parietal Cells ◽  
The Other ◽  
Bivalent Cation ◽  
Gtp Binding ◽  
Gastric Parietal Cells ◽  
Type Receptor ◽  
Ca2 Channels ◽  
Stimulation Of

The mechanism whereby gastrin-type receptor and muscarinic M3-type receptor regulate free intracellular Ca2+ concentration ([Ca2+]i) was studied in rabbit gastric parietal cells stimulated by either gastrin or carbachol. Both agonists induced a biphasic [Ca2+]i response: a transient [Ca2+]i rise, followed by a sustained steady state depending on extracellular Ca2+. Gastrin and carbachol also caused a rapid and transient increase in Mn2+ influx (a tracer for bivalent-cation entry). Pre-stimulation of cells with one agonist drastically decreased both [Ca2+]i increase and Mn2+ influx induced by the other. Neither diltiazem nor pertussistoxin treatment had any effect on agonist-stimulated Mn2+ entry. Thapsigargin, a Ca(2+)-pump inhibitor, induced a biphasic [Ca2+]i increase, and enhanced the rate of Mn2+ entry. Preincubation of cells with thapsigargin inhibits the [Ca2+]i increase as well as Mn2+ entry stimulated by gastrin or by carbachol. Thapsigargin induced a weak but significant increase in Ins(1,4,5)P3 content, but this agent had no effect on the agonist-evoked Ins(1,4,5)P3 response. In permeabilized parietal cells, Ins(1,4,5)P3 and caffeine caused an immediate Ca2+ release from intracellular pools, followed by a reloading of Ca2+ pools which can be prevented in the presence of thapsigargin. We conclude that (i) gastrin and carbachol mobilize common Ca2+ intracellular stores, (ii) Ca2+ permeability secondary to receptor activation involves neither a voltage-sensitive Ca2+ channel nor a GTP-binding protein from the G1 family, and (iii) agonists regulate common Ca2+ channels in depleting intracellular Ca2+ stores.

scholarly journals Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

2011 ◽  
Vol 286 (27) ◽  
pp. 24417-24425 ◽  
Author(s):  
Chi-Yuan Chou ◽  
Liang Tong
Keyword(s):  
Binding Sites ◽  
Analytical Ultracentrifugation ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
The Other ◽  
Biotin Carboxylase ◽  
Binding Modes ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

scholarly journals Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes

2000 ◽  
Vol 351 (3) ◽  
pp. 789-794 ◽  
Author(s):  
Jose M. LIZCANO ◽  
Keith F. TIPTON ◽  
Mercedes UNZETA
Keyword(s):  
Amine Oxidase ◽  
Reaction Pathway ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
Tryptophan Fluorescence ◽  
The Other ◽  
Time Dependent ◽  
High Substrate ◽  
Before And After ◽  
Lag Period

The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37°C. After an initial lag-period, activation was first-order with time and complete after approx. 20h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37°C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37°C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in Vmax for benzylamine oxidation, with no significant alterations in the Km or the Ksi for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others.

Kinetic Studies on Sorption of Textile Dyes Using Lamina and Petiole Parts of Eichhornia crassipes

2008 ◽  
Vol 3 (2) ◽  
Author(s):  
S Renganathan ◽  
R Venkatakrishnan ◽  
P Gautam ◽  
Lima Rose Miranda ◽  
M Velan
Keyword(s):  
Diffusion Coefficient ◽  
Kinetic Model ◽  
Eichhornia Crassipes ◽  
Kinetic Studies ◽  
Reactive Dye ◽  
The Other ◽  
Order Kinetic ◽  
Uptake Capacity ◽  
Fast Red ◽  
Order Kinetic Model

Sorption capacity of two different parts of Eichhornia crassipes such as Lamina and Petiole on a Basic dye (Basic Aurophine-O), Acidic dye (Acid Fast Red-A) and Reactive dye (Brill Red-5B) was studied in a batch system. The maximum equilibrium uptake capacity of petiole was 25.5 mg/g for Basic Aurophine-O, 23.1 mg/g for Acid Fast Red-A and 0.18 mg/g for Brill Red-5B. The equilibrium uptake capacity of petiole was found to be more in Basic Aurophine-O dye when compared to all other dyes studied. The maximum equilibrium uptake capacity of lamina was 27.0 mg/g for Basic Aurophine-O, 25.2 mg/g for Acid Fast Red-A and 4.2 mg/g for Brill Red-5B. The equilibrium uptake capacity of lamina was found to be more in Basic Aurophine-O when compared to Brill Red-5B and Acid Fast Red-A dyes studied in the present investigation. From the result, it was observed that the uptake capacity of dyes using E. crassipes part Lamina was found to be more when compared to the other E. crassipes part of Petiole used in the present study. Sorption results were found to be fitted very well with the Pseudo-second order kinetic model when compared to Pseudo-first order kinetic model. The intra particle diffusion coefficient (Ki) and diffusion coefficient (Di) obtained for the removal of dyes studied using Lamina was found to be more when compared to the other part of Petiole. The polynomial equations are developed and can be used as a ready reckoner equation to find out the percentage color removal of dyes studied in the present investigation at different period of time intervals.

Renin-angiotensin system in hypothyroid rats: effects of potassium iodide and triiodo-L-thyronine

1984 ◽  
Vol 105 (4) ◽  
pp. 505-510 ◽  
Author(s):  
E. Jiménez ◽  
M. Montiel ◽  
J. A. Narváez ◽  
M. Morell
Keyword(s):  
Plasma Renin Activity ◽  
Potassium Iodide ◽  
Renin Angiotensin System ◽  
Kinetic Studies ◽  
Plasma Renin ◽  
The Other ◽  
Renin Substrate ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Previously Treated

Abstract. Kinetic studies of the renin-angiotensin system (RAS) were carried out by measuring plasma renin activity (PRA), plasma renin concentration (PRC) and plasma renin substrate (PRS). Changes in this system were studied during hypothyroidism, after administration of propylthiouracil (PTU), and in thyroidectomized rats. A significant decrease in PRA and PRC was observed in those animals previously treated with PTU. However, a significant increase in PRC, and a decrease in PRS, were found in T animals, but no changes in PRA were observed. In these animals, after daily administration of potassium iodide for I week (T+KI), no changes in RAS were observed in comparison with T rats. Nevertheless, administration of daily doses of triiodo-ithyronine (T+T3) induced a significant increase in PRA, leaving PRC unaltered. In this case the changes in PRA were related to the increase in PRS after T3 treatment. These results suggest that two different mechanisms were involved in renin release, one activated in T rats and the other in pharmacological hypothyroidism.

scholarly journals Gating mechanism of human N-type voltage-gated calcium channel

2021 ◽  
Author(s):  
Yanli Dong ◽  
Yiwei Gao ◽  
Shuai Xu ◽  
Yuhang Wang ◽  
Zhuoya Yu ◽  
...  
Keyword(s):  
Resting State ◽  
Complex Structure ◽  
The Other ◽  
Nociceptive Transmission ◽  
Voltage Gated ◽  
Gating Mechanisms ◽  
Cryo Electron Microscopy ◽  
Gating Mechanism ◽  
Voltage Sensing Domain ◽  
Structural Insights

N-type voltage-gated calcium (CaV) channels mediate Ca2+ influx at the presynaptic terminals in response to action potential and play vital roles in synaptogenesis, neurotransmitter releasing, and nociceptive transmission. Here we elucidate a cryo-electron microscopy (cryo-EM) structure of the human CaV2.2 complex at resolution of 2.8 Å. This complex structure reveals how the CaV2.2, β1, and α2δ1 subunits are assembled. In our structure, the second voltage-sensing domain (VSD) is stabilized at a resting-state conformation, which is distinct from the other three VSDs of CaV2.2 as well as activated VSDs observed in previous structures of CaV channels. The structure also shows that the intracellular gate formed by S6 helices is closed, and a W-helix from the DII-III linker is determined to act as a blocking-ball that causes closed-state inactivation in CaV2.2. Collectively, our structure provides previously unseen structural insights into fundamental gating mechanisms of CaV channels.

scholarly journals DCTPP1, an Oncogene Regulated by miR-378a-3p, Promotes Proliferation of Breast Cancer via DNA Repair Signaling Pathway

2021 ◽  
Vol 11 ◽  
Author(s):  
Ming Niu ◽  
Ming Shan ◽  
Yang Liu ◽  
Yanni Song ◽  
Ji-guang Han ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Dna Repair ◽  
Survival Rates ◽  
The Other ◽  
Poor Survival ◽  
Induced Apoptosis ◽  
Cell Growth And Proliferation

Breast cancer (BRCA) is one of the most deadly cancers worldwide, with poor survival rates that could be due to its high proliferation. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is implicated in numerous diseases, including cancers. However, its role in BRCA is unclear. In this study, we used bioinformatic analyses of the ONCOMINE, UALCAN, and GEPIA databases to determine the expression pattern of DCTPP1 in BRCA. We found that elevated DCTPP1 levels correlate with poor BRCA prognosis. DCTPP1 silencing inhibited BRCA cell proliferation and induced apoptosis in vitro, as well as in vivo. Our data show that this tumorigenic effect depends on DNA repair signaling. Moreover, we found that DCTPP1 is directly modulated by miR-378a-3p, whose downregulation is linked to BRCA progression. Our results showed down-regulation of miR-378a-3p in BRCA. Upregulation of miR-378a-3p, on the other hand, can inhibit BRCA cell growth and proliferation. This study shows that reduced miR-378a-3p level enhances DCTPP1 expression in BRCA, which promotes proliferation by activating DNA repair signaling in BRCA.

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