9 research outputs found

    A STRUCTURAL AND BIOCHEMICAL STUDY ON RIC-8A, AN INTRACELLULAR GUANINE NUCLEOTIDE EXCHANGE FACTOR AND FOLDING CHAPERONE FOR THE INHIBITORY G-PROTEIN ALPHA SUBUNIT-1

    Get PDF
    Heterotrimeric G-proteins (Gαβγ) regulate many cellular processes in the G-protein signaling pathways. The α-subunit (Gα) in the heterotrimer is activated by G-protein-coupled receptor (GPCR) as the guanine-nucleotide exchange factor (GEF), which catalyzes the GDP-release and GTP-binding reactions at Gα nucleotide-binding site, at the cell membrane. Intracellular GEFs for Gα subunits have been identified; among them, the mammalian isoform A of resistance to inhibitors of cholinesterase-8 (Ric-8A) catalyzes nucleotide exchange and functions as a folding chaperone for inhibitory Gα (Gαi1). In a nucleotide-free complex with Gαi1, Ric-8A likely assumes the GEF and chaperone roles by inducing a molten globule-like state. Tall et al. recently discovered that Casein Kinase II phosphorylates Ric-8A at two conserved sites (S435 and T440), which upon phosphorylation, elevate both the GEF and chaperone activities. To understand the molecular mechanism under which Ric-8A interacts with Gαi1, we conducted hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) and identified a putative protein-protein interactive site (residues 454-470) on Ric-8A. Site-directed mutagenesis generated single alanine mutants of Ric-8A along the putative Gαi1-binding sequence and tryptophan fluorescence GEF assays identified five residues (V455, T456, R458, P466, and G469) as binding “hotspot”. We also solved a 2.2Å resolution, X-ray crystal structure of a 452-residue long fragment (R452) of the full-length Ric-8A. The crystal structure depicts a phosphorylated Ric-8A 1-452 molecule (pR452). Mapping sequence conservation scores and HDX protection profile on the pR452 crystal structure provides insights about the Ric-8A, Gαi1 interaction. Low-resolution, solution structures of both R452 and pR452 were also determined using Small Angle X-ray Scattering (SAXS). Phosphorylation of R452 at S435 and T440 likely induces subtle conformational changes on the molecule. Steady-State GTPase assay results indicated that not only does R452 retain measurable GEF activity towards Gαi1, phosphorylation of R452 also elevates the GEF activity at high Ric-8A concentrations. With information from the biochemical assessments and Ric-8A protein structures, we conclude that (A) Gαi1 likely binds to Ric-8A residues 454 to 470 and other under-characterized sites on Ric-8A because (B) R452 retains important structural elements for the GEF activity towards Gαi1 and (C) phosphorylation of Ric-8A induces elevated Ric-8A GEF activity which is accompanied by conformational changes

    Switch II Region in Gαi1: Specificity for Ric-8A

    Get PDF
    On the cell surface are G protein coupled receptors that bind to agonists, causing activation of intracellular G proteins, by catalyzing exchange of GTP for GDP at the G protein alpha subunit (Ga). G proteins are also activated by guanine nucleotide exchange factors (GEF) inside of the cell; these include Ric-8A and Ric-8B. GTP-bound Ga can stimulate the activity of intracellular enzymes. For example, Gas activates adenylyl cyclase, while Gai1 inhibits the activity of this enzyme. Biochemical studies have shown that Ric-8A is a GEF towards Gai1 whereas its isoform Ric-8B acts on Gas. Previous studies in our laboratory and others have shown that a region in Gαi1 called switch II binds to Ric-8A. In this study, we test the hypothesis that differences in amino acid sequence between Gai1 and Gas in switch II are responsible for the ability of these G proteins to discriminate between Ric-8A and Ric-8B. The switch regions of Gai1 and Gas differ in only three amino acids. We predict that, by mutating these amino acids in Gai1 to their corresponding residues in Gas, affinity for Ric-8A will be impaired. Single mutation primers (S206D, K209R and H213Q) were made, transformed and amplified through a polymerase chain reaction (PCR). Once mutant plasmid was expressed in E. coli cells, it is purified, and a tryptophan fluorescence assay is performed. This assay technique detects changes in the fluorescence of tryptophan 211, a side chain in switch II that is sensitive to the exchange of GTP for GDP. Our research sheds light on how mutants in Gai1 in the switch II region plays important role in specificity of binding for Ric-8A

    Application of sulfur SAD to small crystals with a large asymmetric unit and anomalous substructure

    Get PDF
    The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al. (2019), Structure, 27, 1137-1141]. Here, alternative approaches to determining the 40-atom sulfur substructure of the 103 kDa Ric-8A dimer that composes the asymmetric unit are explored. At the data-collection wavelength of 1.77 Å measured at the Frontier micro-focusing Macromolecular Crystallography (FMX) beamline at National Synchrotron Light Source II, the sulfur anomalous signal strength, |Δ|/σΔ (d\u27\u27/sig), approaches 1.4 at 3.4 Å resolution. The highly redundant, 11 000 000-reflection data set measured from 18 crystals was segmented into isomorphous clusters using BLEND in the CCP4 program suite. Data sets within clusters or sets of clusters were scaled and merged using AIMLESS from CCP4 or, alternatively, the phenix.scale_and_merge tool from the Phenix suite. The latter proved to be the more effective in extracting anomalous signals. The HySS tool in Phenix, SHELXC/D and PRASA as implemented in the CRANK2 program suite were each employed to determine the sulfur substructure. All of these approaches were effective, although HySS, as a component of the phenix.autosol tool, required data from all crystals to find the positions of the sulfur atoms. Critical contributors in this case study to successful phase determination by SAD included (i) the high-flux FMX beamline, featuring helical-mode data collection and a helium-filled beam path, (ii) as recognized by many authors, a very highly redundant, multiple-crystal data set and (iii) the inclusion within that data set of data from crystals that were scanned over large ω ranges, yielding highly isomorphous and highly redundant intensity measurements

    Ric-8A, a G protein chaperone with nucleotide exchange activity induces long-range secondary structure changes in Gα

    Get PDF
    Cytosolic Ric-8A has guanine nucleotide exchange factor (GEF) activity and is a chaperone for several classes of heterotrimeric G protein α subunits in vertebrates. Using Hydrogen-Deuterium Exchange-Mass Spectrometry (HDX-MS) we show that Ric-8A disrupts the secondary structure of the Gα Ras-like domain that girds the guanine nucleotide-binding site, and destabilizes the interface between the Gαi1 Ras and helical domains, allowing domain separation and nucleotide release. These changes are largely reversed upon binding GTP and dissociation of Ric-8A. HDX-MS identifies a potential Gα interaction site in Ric-8A. Alanine scanning reveals residues crucial for GEF activity within that sequence. HDX confirms that, like G protein-coupled receptors (GPCRs), Ric-8A binds the C-terminus of Gα. In contrast to GPCRs, Ric-8A interacts with Switches I and II of Gα and possibly at the Gα domain interface. These extensive interactions provide both allosteric and direct catalysis of GDP unbinding and release and GTP binding

    Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A

    Get PDF
    Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1-4, 5-6, and 7-9. 2D H-N-TROSY spectra of [H,N]-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding

    BDNF VAL66MET Polymorphism Elevates the Risk of Bladder Cancer via MiRNA-146b in Micro-Vehicles

    No full text
    Background/Aims: Emerging studies on brain-derived neurotrophic factor (BDNF) have shown that might be novel biomarkers and therapeutic targets for cancer. We explore the role of BDNF in the tumorigenesis of bladder cancer and the underlying molecular mechanism. Methods: 368 patients with diagnosed bladder cancer and 352 healthy controls were enrolled to evaluate the association of BDNF and the miR-146b. Bioinformatics algorithm analysis and luciferase assay were performed to identify the target genes of miR-146b. Real-time PCR and western-blot were carried out to validate the relationship between miR-146b and CRK. MTT assay and FACS were used to evaluated the proliferation and apoptosis of cancer cells. MVs were isolated and transfect into the culture cells to confirm the above observation. Results: The clinical study shows that BDNF Met/Met was significantly associated with the risk of bladder cancer. In addition, comparing with Val/Val and Val/Met, Met/Met has lower miR-146b level. Luciferase assay shows that BDNF Val/Val is apparently enhanced miR-146b promoter-luciferase, but not BDNF Met/Met. Based on luciferase assay, CRK is a direct target gene of miR-146b. MiR-146b mimics significantly inhibited the expression of CRK and activation of AKT level. The expression of CRK and the activation of AKT (p-AKT) were significantly inhibited by MV-BDNF Val/Val-miR-146b or MV-BDNF Val/Met-miR-146b, but not MV-BDNF Met/Met-miR-146b. MV-BDNF Val/Val-miR-146b or Val/Met-miR-146b obviously inhibited cell proliferation, which eliminated by CRK. Meanwhile, with MV-BDNF Met/Met-miR-146b or Met/Met-miR-146b+CRK did not affect the proliferation. MV-BDNF Val/Val-miR-146b or Val/Met-miR-146b enhanced cell apoptosis, which could be eliminated by CRK. Meanwhile, MV-BDNF Met/Met-miR-146b or Met/Met-miR-146b+CRK did not promote apoptosis. Conclusion: BDNF VAL66MET polymorphism is associated with miR-146b and its target gene CRK. MiR-146b and CRK mediated BDNF VAL66MET polymorphism associated proliferation and apoptosis via activation of AKT. Thus, BDNF Val66Met is associated with the risk of bladder cancer, and the BDNF variant could be used a biomarker for the diagnosis of bladder cancer
    corecore