'An affinity probe capillary electrophoresis (APCE) assay for guanine-nucleotide-binding proteins (G proteins) was developed using BODIPY FL GTPgammaS (BGTPgammaS), a fluorescently labeled GTP analogue, as the affinity probe. In the assay, BGTPgammaS was incubated with samples containing G proteins and the resulting mixtures of BGTPgammaS-G protein complexes and free BGTPgammaS ... More
Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding.
AuthorsTrapasso F, Krakowiak A, Cesari R, Arkles J, Yendamuri S, Ishii H, Vecchione A, Kuroki T, Bieganowski P, Pace HC, Huebner K, Croce CM, Brenner C
JournalProc Natl Acad Sci U S A
PubMed ID12574506
'The FHIT gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine ... More
Fhit-nucleotide specificity probed with novel fluorescent and fluorogenic substrates.
AuthorsDraganescu A, Hodawadekar SC, Gee KR, Brenner C
JournalJ Biol Chem
PubMed ID10671479
'Fhit, a member of the histidine triad superfamily of nucleotide-binding proteins, binds and cleaves diadenosine polyphosphates and functions as a tumor suppressor in human epithelial cancers. Function of Fhit in tumor suppression does not require diadenosine polyphosphate cleavage but correlates with the ability to form substrate complexes. As diadenosine polyphosphates ... More
Mechanism of regulation of prokaryotic tubulin-like GTPase FtsZ by membrane protein EzrA.
AuthorsChung KM, Hsu HH, Yeh HY, Chang BY
JournalJ Biol Chem
PubMed ID17043359
'At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring ... More
Use of a native affinity ligand for the detection of G proteins by capillary isoelectric focusing with laser-induced fluorescence detection.
AuthorsCunliffe JM, Liu Z, Pawliszyn J, Kennedy RT
JournalElectrophoresis
PubMed ID15274014
'Affinity probe capillary isoelectric focusing (CIEF) with laser-induced fluorescence was explored for detection of Ras-like G proteins. In the assay, a fluorescent BODIPY FL GTP analogue (BGTPgammaS) and G protein were incubated resulting in formation of BGTPgammaS-G protein complex. Excess BGTPgammaS was separated from BGTPgammaS-G protein complex by CIEF using ... More
Nucleobindin 1 is a calcium-regulated guanine nucleotide dissociation inhibitor of G{alpha}i1.
AuthorsKapoor N, Gupta R, Menon ST, Folta-Stogniew E, Raleigh DP, Sakmar TP,
JournalJ Biol Chem
PubMed ID20679342
'Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that ... More
Thermodynamic characterization of the binding of activator of G protein signaling 3 (AGS3) and peptides derived from AGS3 with G alpha i1.
AuthorsAdhikari A, Sprang SR,
JournalJ Biol Chem
PubMed ID14530282
Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) that contains four G protein regulatory (GPR) or GoLoco motifs in its C-terminal domain. The entire C-terminal domain (AGS3-C) as well as certain peptides corresponding to individual GPR motifs of AGS3 bound to G alpha i1 ... More
Autoprocessing of the Vibrio cholerae RTX toxin by the cysteine protease domain.
AuthorsSheahan KL, Cordero CL, Satchell KJ
JournalEMBO J
PubMed ID17464284
Vibrio cholerae RTX is a large multifunctional bacterial toxin that causes actin crosslinking. Due to its size, it was predicted to undergo proteolytic cleavage during translocation into host cells to deliver activity domains to the cytosol. In this study, we identified a domain within the RTX toxin that is conserved ... More
Structural determinants for GoLoco-induced inhibition of nucleotide release by Galpha subunits.
Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind ... More
Low-affinity interactions of BODIPY-FL-GTPgammaS and BODIPY-FL-GppNHp with G(i)- and G(s)-proteins.
AuthorsGille A, Seifert R
JournalNaunyn Schmiedebergs Arch Pharmacol
PubMed ID12920505
BODIPY-FL-guanosine 5'-[gamma-thio]triphosphate (B-GTPgammaS) and BODIPY-FL-guanosine 5'-[beta,gamma-imido]triphosphate (B-GppNHp) induce fluorescence changes upon binding to purified G(s)/G(i)-proteins and were suggested to serve as probes for monitoring receptor-mediated G-protein activation. However, B-GTPgammaS and B-GppNHp bound to receptor-Galpha(s)/Galpha(i) fusion proteins expressed in Sf9 cell membranes with 1100- to 5600-fold- and 17- to 55-fold lower ... More
A direct fluorescence-based assay for RGS domain GTPase accelerating activity.
AuthorsWillard FS, Kimple AJ, Johnston CA, Siderovski DP
JournalAnal Biochem
PubMed ID15840508
Diverse extracellular signals regulate seven transmembrane-spanning receptors to modulate cellular physiology. These receptors signal primarily through activation of heterotrimeric guanine nucleotide binding proteins (G proteins). A major determinant of heterotrimeric G protein signaling in vivo and in vitro is the intrinsic GTPase activity of the Galpha subunit. RGS (regulator of ... More
Fluorescent BODIPY-GTP analogs: real-time measurement of nucleotide binding to G proteins.
AuthorsMcEwen DP, Gee KR, Kang HC, Neubig RR
JournalAnal Biochem
PubMed ID11262163
Three BODIPY GTPgammaS analogs (FL, 515, and TR), BODIPY FL GppNHp and BODIPY FL GTP molecules were synthesized as possible fluorescent probes to study guanine nucleotide binding spectroscopically. Binding to G(alphao) increases baseline analog fluorescence by 6-, 8.5-, 2.8-, 3.5-, and 3.0-fold, respectively. Binding of GTPgammaS and GppNHp analogs to ... More
RGS12 and RGS14 GoLoco motifs are G alpha(i) interaction sites with guanine nucleotide dissociation inhibitor Activity.
AuthorsKimple RJ, De Vries L, Tronchère H, Behe CI, Morris RA, Gist Farquhar M, Siderovski DP
JournalJ Biol Chem
PubMed ID11387333
The regulators of G-protein signaling (RGS) proteins accelerate the intrinsic guanosine triphosphatase activity of heterotrimeric G-protein alpha subunits and are thus recognized as key modulators of G-protein-coupled receptor signaling. RGS12 and RGS14 contain not only the hallmark RGS box responsible for GTPase-accelerating activity but also a single G alpha(i/o)-Loco (GoLoco) ... More
Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactive second GoLoco motif.
GoLoco ('Galpha(i/o)-Loco' interaction) motif proteins have recently been identified as novel GDIs (guanine nucleotide dissociation inhibitors) for heterotrimeric G-protein alpha subunits. G18 is a member of the mammalian GoLoco-motif gene family and was uncovered by analyses of human and mouse genomes for anonymous open-reading frames. The encoded G18 polypeptide is ... More
Fluorescence-based adenylyl cyclase assay adaptable to high throughput screening.
The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these ... More
Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues.
Hydrolysis of fluorescent GTP analogues BODIPY FL guanosine 5 '-O-(thiotriphosphate) (BGTPgammaS) and BODIPY FL GTP (BGTP) by Galpha(i1) and Galpha was characterized using on-line capillary electrophoresis (o) laser-induced fluorescence assays in order that changes in sub-strate, substrate-enzyme complex, and product could be monitored separately. Apparent k values (V /[E]) (max ... More
Spontaneous nucleotide exchange in low molecular weight GTPases by fluorescently labeled gamma-phosphate-linked GTP analogs.
Regulated guanosine nucleotide exchange and hydrolysis constitute the fundamental activities of low molecular weight GTPases. We show that three guanosine 5'-triphosphate analogs with BODIPY fluorophores coupled via the gamma phosphate bind to the GTPases Cdc42, Rac1, RhoA, and Ras and displace guanosine 5'-diphosphate with high intrinsic exchange rates in the ... More
Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers.
AuthorsPace HC, Hodawadekar SC, Draganescu A, Huang J, Bieganowski P, Pekarsky Y, Croce CM, Brenner C
JournalCurr Biol
PubMed ID10959838
BACKGROUND: The nucleotide-binding protein Fhit, among the earliest and most frequently inactivated proteins in lung cancer, suppresses tumor formation by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion protein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression ... More
The Sec14-like phosphatidylinositol transfer proteins Sec14l3/SEC14L2 act as GTPase proteins to mediate Wnt/Ca2+ signaling.
Authors
JournalElife
PubMed ID28463110
Targeting nucleotide exchange to inhibit constitutively active G protein α subunits in cancer cells.
Authors
JournalSci Signal
PubMed ID30181242
m6A enhances the phase separation potential of mRNA.
Authors
JournalNature
PubMed ID31292544
Identification of small molecule inhibitors of the Chikungunya virus nsP1 RNA capping enzyme.
Authors
JournalAntiviral Res
PubMed ID29680670
Identification of a novel antiviral inhibitor of the flavivirus guanylyltransferase enzyme.
Authors
JournalJ Virol
PubMed ID22674988
Analysis of flavivirus NS5 methyltransferase cap binding.
Authors
JournalJ Mol Biol
PubMed ID19101564
A Sensitive and Robust High-Throughput Screening Assay for Inhibitors of the Chikungunya Virus nsP1 Capping Enzyme.
Authors
JournalPLoS One
PubMed ID27427769
A brain somatic RHEB doublet mutation causes focal cortical dysplasia type II.
Authors
JournalExp Mol Med
PubMed ID31337748
Tannins from Syzygium guineense suppress Wnt signaling and proliferation of Wnt-dependent tumors through a direct effect on secreted Wnts.
Authors
JournalCancer Lett
PubMed ID30098400
Structure of an inactive conformation of GTP-bound RhoA GTPase.