Monitoring the real-time kinetics of the hydrolysis reaction of guanine nucleotide-binding proteins

The conversion of guanosine triphosphate (GTP) to guanosine diphosphate (GDP) and inorganic phosphate (Pi) by guanine nucleotide-binding proteins (GNBPs) is a fundamental enzyme reaction in living cells that acts as an important timer in a variety of biological processes. This reaction is intrinsically slow but can be stimulated by GTPase-activating proteins (GAPs) by several orders of magnitude. In the present study, we synthesized and characterized a new fluorescent nucleotide, 2'(3')-O-(N-ethylcarbamoyl-(5'-carboxytetramethylrhodamine) amide)-GTP, or tamraGTP, which is sensitive towards conformational changes of certain GNBPs induced by GTP hydrolysis. Unlike other fluorescent nucleotides, tamra-GTP allows real-time monitoring of the kinetics of the intrinsic and GAP-catalyzed GTP hydrolysis reactions of small GNBPs from the Rho family.     

Structural insights into the interaction of ROCKI with the switch regions of RhoA

The Rho-ROCK pathway modulates the phosphorylation level of a variety of important signaling proteins and is thereby involved in miscellaneous cellular processes including cell migration, neurite outgrowth, and smooth muscle contraction. The observation of the involvement of the Rho-ROCK pathway in tumor invasion and in diseases such as hypertension and bronchial asthma makes it an interesting target for drug development. We herein present the crystal structure of the complex between active RhoA and the Rho-binding domain of ROCKI. The Rho-binding domain structure forms a parallel alpha-helical coiled-coil dimer and, in contrast to the published Rho-protein kinase N structure, binds exclusively to the switch I and II regions of the guanosine 5'-(beta,gamma-imido)triphosphate-bound RhoA. The switch regions of two different RhoA molecules form a predominantly hydrophobic patch, which is complementarily bound by two identical short helices of 13 residues (amino acids 998-1010). The identified ROCK-binding site of RhoA strikingly supports the assumption of a common consensus-binding site for effector recognition.     

Microarray-based AMASE as a novel approach for mutation detection

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.     

Narrow genetic basis for the Australian dingo confirmed through analysis of paternal ancestry

The dingo (Canis lupus dingo) is an iconic animal in the native culture of Australia, but archaeological and molecular records indicate a relatively recent history on the continent. Studies of mitochondrial DNA (mtDNA) imply that the current dingo population was founded by a small population of already tamed dogs from Southeast Asia. However, the maternal genetic data might give a unilateral picture, and the gene pool has yet to be screened for paternal ancestry. We sequenced 14,437?bp of the Y-chromosome (Y-chr) from two dingoes and one New Guinea Singing Dog (NGSD). This positioned dingo and NGSD within the domestic dog Y-chr phylogeny, and produced one haplotype not detected before. With this data, we characterized 47 male dingoes in 30 Y-chr single-nucleotide polymorphism sites using protease-mediated allele-specific extension technology. Only two haplotypes, H3 and H60, were found among the dingoes, at frequencies of 68.1 and 31.9?%, respectively, compared to 27 haplotypes previously established in the domestic dog. While H3 is common among Southeast Asian dogs, H60 was specifically found in dingoes and the NGSD, but was related to Southeast Asian dog Y-chr haplotypes. H3 and H60 were observed exclusively in the western and eastern parts of Australia, respectively, but had a common range in Southeast. Thus, the Y-chr diversity was very low, similar to previous observations for d-loop mtDNA. Overall genetic evidence suggests a very restricted introduction of the first dingoes into Australia, possibly from New Guinea. This study further confirms the dingo as an isolated feral dog.     

Modeling the impact of common noise inputs on the network activity of retinal ganglion cells

Synchronized spontaneous firing among retinal ganglion cells (RGCs), on timescales faster than visual responses, has been reported in many studies. Two candidate mechanisms of synchronized firing include direct coupling and shared noisy inputs. In neighboring parasol cells of primate retina, which exhibit rapid synchronized firing that has been studied extensively, recent experimental work indicates that direct electrical or synaptic coupling is weak, but shared synaptic input in the absence of modulated stimuli is strong. However, previous modeling efforts have not accounted for this aspect of firing in the parasol cell population. Here we develop a new model that incorporates the effects of common noise, and apply it to analyze the light responses and synchronized firing of a large, densely-sampled network of over 250 simultaneously recorded parasol cells. We use a generalized linear model in which the spike rate in each cell is determined by the linear combination of the spatio-temporally filtered visual input, the temporally filtered prior spikes of that cell, and unobserved sources representing common noise. The model accurately captures the statistical structure of the spike trains and the encoding of the visual stimulus, without the direct coupling assumption present in previous modeling work. Finally, we examined the problem of decoding the visual stimulus from the spike train given the estimated parameters. The common-noise model produces Bayesian decoding performance as accurate as that of a model with direct coupling, but with significantly more robustness to spike timing perturbations.     

Disruption of tubulin polymerization and cell proliferation by 1-naphthylarsonic acid

Arsenical compounds exhibit a differential toxicity to cancer cells. Microtubules are a primary target of a number of anticancer drugs, such as arsenical compounds. The interaction of 1-NAA (1-naphthylarsonic acid) has been investigated on microtubule polymerization under in vitro and cellular conditions. Microtubules were extracted from sheep brain. Transmission electron microscopy was used to show microtubule structure in the presence of 1-NAA. Computational docking method was applied for the discovery of ligand-binding sites on the microtubular proteins. Proliferation of HeLa cells and HF2 (human foreskin fibroblasts) was measured by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay method following their incubation with 1-NAA. Fluorescence microscopic labelling was done with the help of α-tubulin monoclonal antibody and Tunel kit was used to investigate the apoptotic effects of 1-NAA on the HeLa cells. 1-NAA inhibits the tubulin polymerization by the formation of abnormal polymers having high affinity to the inner cell wall.     

Comparative modeling of CCRL1, a key protein in masked immune diseases and virtual screening for finding inhibitor of this protein

Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1 and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1- CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1- CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7 causes immune potentiaiton.     

The role of the conserved switch II glutamate in guanine nucleotide exchange factor-mediated nucleotide exchange of GTP-binding proteins

Guanine nucleotide exchange factors (GEFs) regulate the activity of small G proteins by catalysing the intrinsically slow exchange of GDP for GTP. The mechanism involves the formation of trimeric G protein-nucleotide-GEF complexes, followed by the release of nucleotide to form stable binary G protein-GEF complexes. A number of structural studies of G protein-GEF complexes have shown large structural changes induced in the nucleotide binding site. Together with a recent structure of a trimeric complex, these studies have suggested not only some common principles but also large differences in detail in the GEF-mediated exchange reaction. Several structures suggested that a glutamic acid residue in switch II, which is part of the DxxGQE motif and highly conserved in Ras-like G proteins, might have a decisive mechanistic role in GEF-mediated nucleotide exchange reactions. Here we show that mutation of the switch II glutamate to Ala severely impairs GEF-catalysed nucleotide exchange in most, but not all, Ras family G proteins, explaining its high sequence conservation. The residue determines the initial approach of GEF to the nucleotide-loaded G protein and does not appreciably affect the formation of a binary nucleotide-free complex. Its major effect thus appears to be the removal of the P-loop lysine from its interaction with the nucleotide.     

Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA)

Phospholipases A(2) (PLA(2)s) catalyze hydrolysis of fatty acids from the sn-2 position of phospholipids. Here we report the identification and characterization of a membrane-associated intracellular calcium-dependent, adipose-specific PLA(2) that we named AdPLA (adipose-specific phospholipase A(2)). We found that AdPLA was highly expressed specifically in white adipose tissue and was induced during preadipocyte differentiation into adipocytes. Clearance of AdPLA by immunoprecipitation significantly decreased PLA activity in white adipose tissue lysates but had no effect on liver lysates, where expression was hardly detectable. In characterizing AdPLA, we employed radiochemical assays with TLC analysis of the enzyme activity of lysates from COS-7 cells overexpressing AdPLA. For kinetic studies, we produced purified recombinant AdPLA for use in a lipoxidase-coupled spectrophotometric assay. AdPLA generated free fatty acid and lysophospholipid from phosphatidylcholine with a preference for hydrolysis at the sn-2 position. Although we found low but detectable lysophospholipase activity, AdPLA showed no significant activity against a variety of other lipid substrates. Calcium was found to activate AdPLA but was not essential for activity. Studies with known phospholipase inhibitors, including bromoenolactone, methyl arachidonyl fluorophosphate, AACOCF(3), 7,7-dimethyl-5,8-eicosadienoic acid, and thioetheramide, supported that AdPLA is a phospholipase. Mutational studies showed that His-23 and Cys-113 are critical for activity of AdPLA and suggested that AdPLA is likely a His/Cys PLA(2). Overall, although AdPLA is similar to other histidine phospholipases in pH and calcium dependence, AdPLA showed different characteristics in many regards, including predicted catalytic mechanism. AdPLA may therefore represent the first member of a new group of PLA(2)s, group XVI.