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https://www.readbyqxmd.com/read/30107565/e-coli-elongation-factor-tu-bound-to-a-gtp-analogue-displays-an-open-conformation-equivalent-to-the-gdp-bound-form
#1
Jesper S Johansen, Darius Kavaliauskas, Shawn H Pfeil, Mickaël Blaise, Barry S Cooperman, Yale E Goldman, Søren S Thirup, Charlotte R Knudsen
According to the traditional view, GTPases act as molecular switches, which cycle between distinct 'on' and 'off' conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex. GTP hydrolysis is thought to cause the release of EF-Tu from aminoacyl-tRNA and the ribosome due to a dramatic conformational change following Pi release. Here, the crystal structure of Escherichia coli EF-Tu in complex with a non-hydrolysable GTP analogue (GDPNP) has been determined...
August 11, 2018: Nucleic Acids Research
https://www.readbyqxmd.com/read/29761917/cationic-europium-complexes-for-visualizing-fluctuations-in-mitochondrial-atp-levels-in-living-cells
#2
Romain Mailhot, Thomas Traviss-Pollard, Robert Pal, Stephen J Butler
The ability to study cellular metabolism and enzymatic processes involving adenosine triphosphate (ATP) is impeded by the lack of imaging probes capable of signalling the concentration and distribution of intracellular ATP rapidly, with high sensitivity. We report here the first example of a luminescent lanthanide complex capable of visualizing changes in the concentration of ATP in the mitochondria of living cells. Four cationic europium(III) complexes [Eu.1-4]+ have been synthesized and their binding capabilities towards nucleoside polyphosphate anions examined in aqueous solution at physiological pH...
May 15, 2018: Chemistry: a European Journal
https://www.readbyqxmd.com/read/29731302/quantitative-control-of-gpcr-organization-and-signaling-by-endocytosis-in-epithelial-morphogenesis
#3
Ankita Jha, Thomas S van Zanten, Jean-Marc Philippe, Satyajit Mayor, Thomas Lecuit
Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells...
May 21, 2018: Current Biology: CB
https://www.readbyqxmd.com/read/29505244/single-cell-mass-spectrometry-of-subpopulations-selected-by-fluorescence-microscopy
#4
Linwen Zhang, Christopher J Sevinsky, Brian M Davis, Akos Vertes
Specific subpopulations in a heterogeneous collection of cells, for example, cancer stem cells in a tumor, are often associated with biological or medical conditions. Fluorescence microscopy, based on biomarkers labeled with fluorescent probes, is a widely used technique for the visualization and selection of such cells. Phenotypic differences for these subpopulations at the molecular level can be identified by their untargeted analysis by single-cell mass spectrometry (MS). Here, we combine capillary microsampling MS with fluorescence microscopy for the analysis of metabolite and lipid levels in single cells to discern the heterogeneity of subpopulations corresponding to mitotic stages...
April 3, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29460621/differential-protein-dynamics-of-regulators-of-g-protein-signaling-role-in-specificity-of-small-molecule-inhibitors
#5
Vincent S Shaw, Hossein Mohammadiarani, Harish Vashisth, Richard R Neubig
Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins...
March 7, 2018: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/29357897/parkinson-disease-associated-mutations-in-lrrk2-cause-centrosomal-defects-via-rab8a-phosphorylation
#6
Jesús Madero-Pérez, Elena Fdez, Belén Fernández, Antonio J Lara Ordóñez, Marian Blanca Ramírez, Patricia Gómez-Suaga, Dieter Waschbüsch, Evy Lobbestael, Veerle Baekelandt, Angus C Nairn, Javier Ruiz-Martínez, Ana Aiastui, Adolfo López de Munain, Pawel Lis, Thomas Comptdaer, Jean-Marc Taymans, Marie-Christine Chartier-Harlin, Alexandria Beilina, Adriano Gonnelli, Mark R Cookson, Elisa Greggio, Sabine Hilfiker
BACKGROUND: Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. METHODS: Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning...
January 23, 2018: Molecular Neurodegeneration
https://www.readbyqxmd.com/read/29148177/high-concentrations-of-gtp-induce-conformational-changes-in-the-essential-bacterial-gtpase-enga-and-enhance-its-binding-to-the-ribosome
#7
Catarina da Silveira Tomé, Anne-Emmanuelle Foucher, Jean-Michel Jault, Dominique Housset
EngA is a conserved bacterial GTPase involved in ribosome biogenesis. While essential in bacteria, EngA does not have any human orthologue and can thus be an interesting target for new antibacterial compounds. EngA is the only known GTPase bearing two G domains, making unique its catalytic cycle and the induced modulation of its conformation and interaction with the ribosome. We have investigated nucleotide-induced conformational changes in EngA in order to unveil their role in ribosome binding. SAXS and limited proteolysis were used to probe EngA conformational changes, and revealed a change in protein structure and a distinct rate of proteolysis induced by GTP...
January 2018: FEBS Journal
https://www.readbyqxmd.com/read/29115013/a-t7-rna-polymerase-mutant-enhances-the-yield-of-5-thienoguanosine-initiated-rnas
#8
Seth Lyon, Venkat Gopalan
Spectroscopic methods, which are used to establish RNA structure-function relationships, require strategies for post-synthetic, site-specific incorporation of chemical probes into target RNAs. For RNAs larger than 50 nt, the enzymatic incorporation of a nucleoside or nucleotide monophosphate guanosine analogue (G analogue) at their 5'-end is routinely achieved by T7 RNA polymerase (T7RNAP)-mediated in vitro transcription (IVT) of the appropriate DNA template containing a GTP-initiating class III Φ6.5 promoter...
January 18, 2018: Chembiochem: a European Journal of Chemical Biology
https://www.readbyqxmd.com/read/29111616/daclatasvir-and-asunaprevir-treatment-in-patients-infected-by-genotype-1b-of-hepatitis-c-virus-with-no-or-subtle-resistant-associated-substitutions-ras-in-ns5a-y93
#9
Masatoshi Ishigami, Kazuhiko Hayashi, Takashi Honda, Teiji Kuzuya, Yoji Ishizu, Tetsuya Ishikawa, Isao Nakano, Fumihiro Urano, Takashi Kumada, Kentaro Yoshioka, Yoshiki Hirooka, Hidemi Goto
In this study, we investigated the real-world data of the first approved interferon-free regimen in Japan, daclatasvir and asunaprevir (DCV+ASV), in chronic hepatitis C patients infected HCV genotype 1b with no or subtle amount of baseline resistant associated substitutions (RAS). Among 924 patients registered in our multicenter study, 750 patients who were proven not to be infected with NS5A-Y93H RAS by direct sequencing and to have no or subtle amount (less than 20%) of NS5A-Y93H RAS by probe assays (Cycleave or PCR invader assay) were included in this study...
April 2018: Journal of Medical Virology
https://www.readbyqxmd.com/read/29033317/ras-binder-induces-a-modified-switch-ii-pocket-in-gtp-and-gdp-states
#10
Daniel R Gentile, Manoj K Rathinaswamy, Meredith L Jenkins, Steven M Moss, Braden D Siempelkamp, Adam R Renslo, John E Burke, Kevan M Shokat
Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state...
December 21, 2017: Cell Chemical Biology
https://www.readbyqxmd.com/read/28943312/ubiquitin-linkage-specific-affimers-reveal-insights-into-k6-linked-ubiquitin-signaling
#11
Martin A Michel, Kirby N Swatek, Manuela K Hospenthal, David Komander
Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific "affimer" reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer...
October 5, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28870802/%C3%AE-arrestin-biased-dopamine-d2-receptor-partial-agonists-synthesis-and-pharmacological-evaluation
#12
Barbara Männel, Harald Hübner, Dorothée Möller, Peter Gmeiner
β-Arrestin biased G protein-coupled receptor ligands represent important molecular probes and may increase favorable drug action and safety as novel therapeutics. Starting from recently discovered hydroxy-substituted heterocyclic piperazine scaffolds, we have developed a series of dopamine D2 receptor ligands with a pyrazolo[1,5-a]pyridine as secondary pharmacophore that is functionalized in position 3 by a formyl or hydroxyiminomethyl substituent. The ligands, especially the benzoxazinone 9d, were found to stimulate substantial β-arrestin-2 recruitment, while being nearly devoid of activity in a GTPγS binding assay...
October 15, 2017: Bioorganic & Medicinal Chemistry
https://www.readbyqxmd.com/read/28687663/a-module-for-rac-temporal-signal-integration-revealed-with-optogenetics
#13
Brian R Graziano, Delquin Gong, Karen E Anderson, Anne Pipathsouk, Anna R Goldberg, Orion D Weiner
Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation...
August 7, 2017: Journal of Cell Biology
https://www.readbyqxmd.com/read/28482099/rsga-couples-the-maturation-state-of-the-30s-ribosomal-decoding-center-to-activation-of-its-gtpase-pocket
#14
Jorge Pedro López-Alonso, Tatsuya Kaminishi, Takeshi Kikuchi, Yuya Hirata, Idoia Iturrioz, Neha Dhimole, Andreas Schedlbauer, Yoichi Hase, Simon Goto, Daisuke Kurita, Akira Muto, Shu Zhou, Chieko Naoe, Deryck J Mills, David Gil-Carton, Chie Takemoto, Hyouta Himeno, Paola Fucini, Sean R Connell
During 30S ribosomal subunit biogenesis, assembly factors are believed to prevent accumulation of misfolded intermediate states of low free energy that slowly convert into mature 30S subunits, namely, kinetically trapped particles. Among the assembly factors, the circularly permuted GTPase, RsgA, plays a crucial role in the maturation of the 30S decoding center. Here, directed hydroxyl radical probing and single particle cryo-EM are employed to elucidate RsgA΄s mechanism of action. Our results show that RsgA destabilizes the 30S structure, including late binding r-proteins, providing a structural basis for avoiding kinetically trapped assembly intermediates...
June 20, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28472054/a-mathematical-model-coupling-polarity-signaling-to-cell-adhesion-explains-diverse-cell-migration-patterns
#15
William R Holmes, JinSeok Park, Andre Levchenko, Leah Edelstein-Keshet
Protrusion and retraction of lamellipodia are common features of eukaryotic cell motility. As a cell migrates through its extracellular matrix (ECM), lamellipod growth increases cell-ECM contact area and enhances engagement of integrin receptors, locally amplifying ECM input to internal signaling cascades. In contrast, contraction of lamellipodia results in reduced integrin engagement that dampens the level of ECM-induced signaling. These changes in cell shape are both influenced by, and feed back onto ECM signaling...
May 2017: PLoS Computational Biology
https://www.readbyqxmd.com/read/28437082/high-efficiency-1-and-2-ribosomal-frameshiftings-revealed-by-force-spectroscopy
#16
Te-Wei Tsai, Haopeng Yang, Heng Yin, Shoujun Xu, Yuhong Wang
Ribosomal frameshifting is a rare but ubiquitous process that is being studied extensively. Meanwhile, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. We report unambiguous observation of highly efficient "-1" and "-2" frameshiftings on a GA7 G slippery mRNA without the downstream secondary structure, using force-induced remnant magnetization spectroscopy combined with unique probing schemes. The result represents the first experimental evidence of multiple frameshifting steps...
June 16, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28374844/signatures-of-a-macroscopic-switching-transition-for-a-dynamic-microtubule
#17
J S Aparna, Ranjith Padinhateeri, Dibyendu Das
Characterising complex kinetics of non-equilibrium self-assembly of bio-filaments is of general interest. Dynamic instability in microtubules, consisting of successive catastrophes and rescues, is observed to occur as a result of the non-equilibrium conversion of GTP-tubulin to GDP-tubulin. We study this phenomenon using a model for microtubule kinetics with GTP/GDP state-dependent polymerisation, depolymerisation and hydrolysis of subunits. Our results reveal a sharp switch-like transition in the mean velocity of the filaments, from a growth phase to a shrinkage phase, with an associated co-existence of the two phases...
April 4, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28336063/probing-cdc42-polarization-dynamics-in-budding-yeast-using-a-biosensor
#18
Satoshi Okada, Mid Eum Lee, Erfei Bi, Hay-Oak Park
Cdc42 is a small guanosine triphosphatase (GTPase) that plays a central role in polarity development in diverse cell types. Since the activity of Cdc42 is dynamically controlled in time and space, it is required to develop a biosensor to monitor its activation in vivo. In this chapter, we describe the construction and usage of a simple and robust biosensor for monitoring active Cdc42 in budding yeast. This affinity-based biosensor uses a red fluorescent protein fused to a Cdc42- and Rac-interactive binding motif from one of the Cdc42 effector proteins...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28315413/akr1-attenuates-methylmercury-toxicity-through-the-palmitoylation-of-meh1-as-a-subunit-of-the-yeast-ego-complex
#19
Zhi-Ting Zhang, Yousuke Ogiwara, Yuichi Ito, Akinori Hikida, Nobuhiko Miura, Shusuke Kuge, Akira Naganuma, Gi-Wook Hwang
BACKGROUND: We previously reported that palmitoyltransferase activity of Akr1 is required for alleviation of methylmercury toxicity in yeast. In this study, we identified a factor that alleviates methylmercury toxicity among the substrate proteins palmitoylated by Akr1, and investigated the role of this factor in methylmercury toxicity. METHODS: Gene disruption and site-directed mutagenesis were used to examine the relationship of methylmercury toxicity and vacuole function...
July 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28277837/an-overview-of-ca-2-mobilization-assays-in-gpcr-drug-discovery
#20
REVIEW
Qiang Ma, Lingyan Ye, Hongxia Liu, Ying Shi, Naiming Zhou
Calcium ions (Ca(2+)) serve as a second messenger or universal signal transducer implicated in the regulation of a wide range of physiological processes. A change in the concentration of intracellular Ca(2+) is an important step in intracellular signal transduction. G protein-coupled receptors (GPCRs), the largest and most versatile group of cell surface receptors, transduce extracellular signals into intracellular responses via their coupling to heterotrimeric G proteins. Since Ca(2+) plays a crucial role in GPCR-induced signaling, measurement of intracellular Ca(2+) has attracted more and more attention in GPCR-targeted drug discovery...
May 2017: Expert Opinion on Drug Discovery
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