36 research outputs found
Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.
ABSTRACT G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Giā£2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Giā£2 fusion protein for Gā¤ā„, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of Gā£ā¤ā„ disassembly. GPCRs interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptide
Assessing the dynamics and complexity of disease pathogenicity using 4-dimensional immunological data
Investigating disease pathogenesis and personalized prognostics are major biomedical
needs. Because patients sharing the same diagnosis can experience different outcomes,
such as survival or death, physicians need new personalized tools, including those
that rapidly differentiate several inflammatory phases. To address these topics, a
pattern recognition-based method (PRM) that follows an inverse problem approach
was designed to assess, in <10min, eight concepts: synergy, pleiotropy, complexity,
dynamics, ambiguity, circularity, personalized outcomes, and explanatory prognostics
(pathogenesis). By creating thousands of secondary combinations derived from blood
leukocyte data, the PRM measures synergic, pleiotropic, complex and dynamic
data interactions, which provide personalized prognostics while some undesirable
featuresāsuch as false results and the ambiguity associated with data circularity-are
prevented. Here, this method is compared to Principal Component Analysis (PCA) and
evaluated with data collected from hantavirus-infected humans and birds that appeared
to be healthy. When human data were examined, the PRM predicted 96.9 % of all
surviving patients while PCA did not distinguish outcomes. Demonstrating applications
in personalized prognosis, eight PRM data structures sufficed to identify all but one of
the survivors. Dynamic data patterns also distinguished survivors from non-survivors, as
well as one subset of non-survivors, which exhibited chronic inflammation. When the
PRM explored avian data, it differentiated immune profiles consistent with no, early, or
late inflammation. Yet, PCA did not recognize patterns in avian data. Findings support the
notion that immune responses, while variable, are rather deterministic: a low number of
complex and dynamic data combinations may be enough to, rapidly, unmask conditions
that are neither directly observable nor reliably forecasted.Conacyt of Mexico (Consejo
Nacional de Ciencia y TecnologĆahttp://www.frontiersin.org/Immunologyam2020Veterinary Tropical Disease
Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.This work was supported by National Science Foundation (NSF) Grant MCB0956027 and National Institutes of Health Grant R03 MH081231-01 from the Molecular Libraries Program (to A. W. N.); University of New Mexico Center for Molecular Discovery Molecular Libraries Probe Production Centers (UNMCMD MLPCN) National Institutes of Health Grants U54MH084690 and R01HL081062 (to L. A. S.); UNM National Center for Research Resources (NCRR) Grant 5P20RR016480 (to L. G. H.); National Institutes of Health Grant R21 CA170375-01 through the NCI (to A. W. N., L. G. H., and J. E. G.); National Institutes of Health Grants NS066429 and AI092130 (to T. B.); and University of Kansas Specialized Chemistry Center (KUSCC) MLPCN National Institutes of Health Grant U54HG005031 (to J. A.)
FRET Detection of Lymphocyte Function-Associated Antigen-1 Conformational Extension
Lymphocyte function-associated antigen 1 (LFA-1, CD11a/CD18, Ī±LĪ²2-integrin) and its ligands are essential for adhesion between T-cells and antigen-presenting cells, formation of the immunological synapse, and other immune cell interactions. LFA-1 function is regulated through conformational changes that include the modulation of ligand binding affinity and molecular extension. However, the relationship between molecular conformation and function is unclear. Here fluorescence resonance energy transfer (FRET) with new LFA-1-specific fluorescent probes showed that triggering of the pathway used for T-cell activation induced rapid unquenching of the FRET signal consistent with extension of the molecule. Analysis of the FRET quenching at rest revealed an unexpected result that can be interpreted as a previously unknown LFA-1 conformation
Singlet-State Electron Transfer between a Porphyrin and Ubiquinone:Ā A Transient Resonance Raman and Quantum Chemical Study
Singlet-State Electron Transfer between a Porphyrin and Ubiquinone:Ā A Transient Resonance Raman and Quantum Chemical Study 1
Photoinduced Electron Transfer in Porphyrin Quinone Donor/Acceptor Pairs:Ā pH-Dependent Free Ion Yield
Photoinduced Electron Transfer in Porphyrin Quinone Donor/Acceptor Pairs:Ā pH-Dependent Free Ion Yield
FRET Detection of Cellular Ī±(4)-Integrin Conformational Activation
Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing Ī±(4)-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the Ī±(4)-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was ā¼50 Ć
between resting and Mn(2+) activated receptors and ā¼25 Ć
after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of Ī±(4)-integrin and its affinity provides a mechanism for Dembo's catch-bond concept