RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.
Animal ; Brain/metabolism ; Calcium/*metabolism ; Calmodulin/*metabolism ; GTP Phosphohydrolases/*metabolism ; Guanosine Diphosphate/metabolism ; Guanosine Triphosphate/*metabolism ; Rats ; Support, Non-U.S. Gov't ; Synaptosomes/metabolism

The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.
Amino Acid Sequence ; Binding Sites ; Catalytic Domain ; Cloning, Molecular ; Crystallography, X-Ray ; Escherichia coli ; Guanosine Diphosphate ; metabolism ; Guanosine Triphosphate ; metabolism ; Humans ; Models, Molecular ; Molecular Conformation ; Molecular Sequence Data ; Plasmids ; metabolism ; Protein Binding ; Protein Structure, Tertiary ; genetics ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; ral GTP-Binding Proteins ; chemistry ; genetics ; metabolism ; ral Guanine Nucleotide Exchange Factor ; chemistry ; genetics ; metabolism

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.
Citrate (si)-Synthase ; chemistry ; Cloning, Molecular ; Conserved Sequence ; Escherichia coli ; cytology ; enzymology ; GTP Phosphohydrolases ; chemistry ; genetics ; isolation & purification ; metabolism ; Guanosine Diphosphate ; pharmacology ; Guanosine Triphosphate ; analogs & derivatives ; pharmacology ; Molecular Chaperones ; chemistry ; genetics ; isolation & purification ; metabolism ; Protein Denaturation ; drug effects ; Protein Renaturation ; drug effects ; Ribosomes ; metabolism ; alpha-Glucosidases ; chemistry

Lack of exercise may be one of the main causes of obesity in the recent modernized society. With the rapid increase of obese population worldwide, the economic costs of overweight have become a great burden in many countries. The costs of obesity may be divided into the direct costs to the health system and the indirect or social costs to the individuals and the community (for example, sick days and individuals' expenditure on weight loss). The direct costs depend on the main part of the diseases caused by obesity and the cost of these diseases. Some diseases thathave been included in the calculation are Type 2 diabetes, heart disease, hypertension, endometrial cancer, arthritis and colorectal cancer. One of the problems in this assessment is the relative risk of the diseases in different communities and ethnic groups. Several methods have been used to calculate these costs and produceda wide range of results from different countries. One of the latest estimates ofcosts of obesity is from the United States and was 10.2% of the total health care cost. Little data is yet available in Asian countries; however, the Korean Obesity Society reported a total cost of obesity estimated at about 0.11 % of GDP or about 2.13% of the total health care cost. It is now necessary to develop a strategy for life style modifications at the nationwide level.
Arthritis ; Asian Continental Ancestry Group ; Colorectal Neoplasms ; Endometrial Neoplasms ; Energy Metabolism ; Ethnic Groups ; Female ; Guanosine Diphosphate ; Health Care Costs ; Health Expenditures ; Heart Diseases ; Humans ; Hypertension ; Life Style ; Obesity ; Overweight ; Sick Leave ; United States

A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹⁰-1 × 10⁻⁵ mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻⁶ mol/L). Also it could be removed by intracellular dialysis of either GDP-β-S (5 × 10⁻⁴mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻⁶ mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.
Animals ; Arginine Vasopressin ; pharmacology ; Ganglia, Spinal ; cytology ; Guanosine Diphosphate ; analogs & derivatives ; pharmacology ; Indoles ; Maleimides ; Membrane Potentials ; Neurons ; drug effects ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; metabolism ; Signal Transduction ; Thionucleotides ; pharmacology ; gamma-Aminobutyric Acid ; pharmacology

Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a class of atlastin (ATL) GTPases. Purified Drosophila ATL is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, we determined the preliminary crystal structure of the cytosolic segment of Drosophila ATL in a GDP-bound state. The structure reveals a GTPase domain dimer with the subsequent three-helix bundles associating with their own GTPase domains and pointing in opposite directions. This conformation is similar to that of human ATL1, to which GDP and high concentrations of inorganic phosphate, but not GDP only, were included. Drosophila ATL restored ER morphology defects in mammalian cells lacking ATLs, and measurements of nucleotide-dependent dimerization and GTPase activity were comparable for Drosophila ATL and human ATL1. However, purified and reconstituted human ATL1 exhibited no in vitro fusion activity. When the cytosolic segment of human ATL1 was connected to the transmembrane (TM) region and C-terminal tail (CT) of Drosophila ATL, the chimera still exhibited no fusion activity, though its GTPase activity was normal. These results suggest that GDP-bound ATLs may adopt multiple conformations and the in vitro fusion activity of ATL cannot be achieved by a simple collection of functional domains.
Animals ; Dimerization ; Drosophila ; Drosophila Proteins ; chemistry ; genetics ; Endoplasmic Reticulum ; chemistry ; GTP Phosphohydrolases ; chemistry ; genetics ; GTP-Binding Proteins ; chemistry ; genetics ; Guanosine Diphosphate ; chemistry ; metabolism ; Humans ; Membrane Proteins ; chemistry ; genetics ; Mutation ; Protein Conformation ; Protein Structure, Secondary

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m⁷GDP and m²²⁷GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²(+). Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m⁷GDP cap from mRNAs in the presence of Mg²(+) or Mn²(+). We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.
Amino Acid Motifs ; Biocatalysis ; Cell Nucleus ; enzymology ; Consensus Sequence ; Cytoplasm ; enzymology ; metabolism ; Guanosine Diphosphate ; metabolism ; Histidine ; metabolism ; Humans ; Hydrolysis ; Luciferases ; genetics ; Magnesium ; metabolism ; Manganese ; metabolism ; Mutagenesis ; Mutation ; Pyrophosphatases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; RNA Caps ; chemistry ; metabolism ; pharmacology ; RNA, Small Nucleolar ; chemistry ; metabolism ; pharmacology

To investigate the regulatory role of purine nucleotide on uncoupling proteins (UCPs), the activity of UCPs and the expressions of UCP4 and UCP5 in mitochondria of rat brain tissues incubated with GDP were observed in vitro. The cerebral hemispheres of adult male Sprague-Dawley rats were removed and clipped into 8-10 mm3 tissue mass which incubated with 1 mmol/L GDP (GDP group), or only incubation medium (control group), for 30 min in vitro. The mitochondria from incubated tissue mass of rat brain were isolated by centrifugation. The activity of UCPs was detected by the method of [3H]-GTP binding with UCPs specifically. The maximal binding content (Bmax) and the dissociation constant (Kd) were determined from Scatchard plot. The mRNA and protein expressions of UCP4 and UCP5 were measured by RT-PCR and Western blot, respectively. The results showed that Bmax was increased and Kd was decreased in rat brain mitochondria in GDP group compared with that in control group. But the mRNA and protein expressions of UCP4 and UCP5 exhibited no statistically significant changes. It is thus suggested that GDP inhibits the activity of mitochondrial UCPs in rat brain in vitro, but exhibits no effect on the expressions of UCP4 and UCP5.
Animals ; Brain ; drug effects ; metabolism ; Guanosine Diphosphate ; pharmacology ; In Vitro Techniques ; Ion Channels ; metabolism ; Male ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Proteins ; metabolism ; Mitochondrial Uncoupling Proteins ; Nerve Tissue Proteins ; metabolism ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction