An affinity pull-down approach to identify the plant cyclic nucleotide interactome
MetadataShow full item record
AbstractCyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry - techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants. © Springer Science+Business Media New York 2013.
JournalMethods in Molecular Biology
- The arabidopsis cyclic nucleotide interactome.
- Authors: Donaldson L, Meier S, Gehring C
- Issue date: 2016 May 11
- Cyclic nucleotides.
- Authors: Newton RP, Smith CJ
- Issue date: 2004 Sep
- Large-scale identification of tubulin-binding proteins provides insight on subcellular trafficking, metabolic channeling, and signaling in plant cells.
- Authors: Chuong SD, Good AG, Taylor GJ, Freeman MC, Moorhead GB, Muench DG
- Issue date: 2004 Oct
- Protein complexes characterization in Arabidopsis thaliana by tandem affinity purification coupled to mass spectrometry analysis.
- Authors: Bigeard J, Pflieger D, Colcombet J, Gérard L, Mireau H, Hirt H
- Issue date: 2014
- Detection of reactive oxygen species downstream of cyclic nucleotide signals in plants.
- Authors: Walker RK, Berkowitz GA
- Issue date: 2013
Showing items related by title, author, creator and subject.
Cooperative effect by monopodal silica-supported niobium com-plexes pairs enhancing catalytic cyclic carbonate productionD'Elia, Valerio; Dong, Hailin; Rossini, Aaron J; Widdifield, Cory M.; Vummaleti, Sai V. C.; Minenkov, Yury; Poater, Albert; Abou-Hamad, Edy; Pelletier, Jeremie D. A.; Cavallo, Luigi; Emsley, Lyndon; Basset, Jean-Marie (American Chemical Society (ACS), 2015-05-07)Recent discoveries highlighted the activity and the intriguing mechanistic features of NbCl5 as a molecular catalyst for the cycloaddition of CO2 and epoxides under ambient conditions. This has inspired the preparation of novel silica supported Nb-species by reacting a molecular niobium precursor [NbCl5•OEt2] with silica dehydroxylated at 700 °C (SiO2-700) or at 200 oC (SiO2-200) to generate diverse surface complexes. The product of the reaction between SiO2-700 and [NbCl5•OEt2] was identified as a monopodal supported surface species [≡SiONbCl4•OEt2] (1a). The reactions of SiO2-200 with the niobium precursor, according to two different protocols, generated surface complexes 2a and 3a presenting significant, but different, populations of the monopodal surface complex along with bipodal [(≡SiO)2NbCl3•OEt2]. 93Nb SSNMR spectra of 1a-3a and 31P SSNMR on their PMe3 derivatives (1b-3b) led to the unambiguous assignment of 1a as a single site, monopodal Nb-species while 2a and 3a were found to present two distinct surface-supported components, with 2a being mostly monopodal [≡SiONbCl4•OEt2] and 3a being mostly bipodal [≡S ONbCl3•OEt2]. Double-quantum/single-quantum 31P NMR correlation experiment carried out on 2b supported the existence of vicinal Nb centers on the silica surface for this species. 1a-3a were active heterogeneous catalysts for the synthesis of propylene carbonate from CO2 and propylene oxide under mild catalytic conditions; the performance of 2a was found to significantly surpass that of 1a and 3a. With the support of a systematic DFT study carried out on model silica surfaces, the observed differences in catalytic efficiency were correlated with an unprece-dented cooperative effect between two neighboring Nb centers on the surface of 2a. This is in an excellent agreement with our previous discoveries regarding the mechanism of the NbCl5 catalyzed cycloaddition in the homogeneous phase.
A New Role for CO2: Controlling Agent of the Anionic Ring-Opening Polymerization of Cyclic EstersVarghese, Jobi K.; Goncalves, Theo; Huang, Kuo-Wei; Hadjichristidis, Nikolaos; Gnanou, Yves; Feng, Xiaoshuang (American Chemical Society (ACS), 2017-08-15)Conventional anionic ring-opening of polymerization (AROP) of cyclic esters suffers from the nonselective and concomitant attack of the monomer and of the polymer chains by the growing active species, which results in polyester samples with uncontrolled molar masses and broad polydispersity due to the competition between propagation and transesterification reactions. In this report, we describe a new AROP system mediated by a controlled amount of CO2 which prevents transesterification reactions from occurring. Using lithium monomethyl diethylene glycoxide (MEEOLi) as initiator and 1.5 equiv of CO2, ε-caprolactone could be polymerized under truly “living” conditions in dichloromethane (DCM) at 70 °C, as evidenced by the control of molar masses, the narrow polydispersity indexes (Mn up to ∼40 kg/mol, Đ < 1.16), and also successful chain extension experiments. Lithium carbonate used as initiator in the presence of 0.5 equiv of CO2 afforded similar polymerization results. Experiments carried out with other alkoxide salts and solvents demonstrate that CO2 is indispensable as well as lithium and noncoordinating solvents for the suppression of transesterifications. A similar strategy was applied for the AROP of l-lactide (LLA). At −20 °C, LLA could be polymerized under living conditions with undetectable level of transesterification as demonstrated by MALDI-ToF analysis. To account for the polymerization mechanism occurring in the presence of a slight excess of CO2, we resorted to computational studies. It appears that a fast equilibrium takes place between two tetrameric aggregates, one dormant comprising four carbonates (RCO3Li)4, and an active one involving three carbonates and one alkoxide (RCO3Li)3(ROLi). The latter is shown to selectively ring-open cyclic ester without indulging in transesterifications like (ROLi)4 precursors.
Cyclic hygrothermal aging of aircraft lightning protections: Phenomenological overviewLubineau, Gilles; El Yagoubi, Jalal; Saghir, Shahid; Selvakumaran, Lakshmi; Askari, Abe H. (American Institute of Aeronautics and Astronautics (AIAA), 2012-04-23)The recent transition to full composite fuselages is a major breakthrough in aeronautical design. To ensure satisfactory lightning protection, the electrical properties of the primary composite structure need to be improved and a design solution lies on bonding a metallic fishnet to the fuselage. An important issue is the prediction of the aging behavior of this top layer when exposed to severe aging conditions representative of flight environment. Multiple aging mechanisms affect this top layer made of thermoset resin and metallic fishnet with complex geometry and a first modeling strategy is a necessary starting point to better understand its evolution. This paper describes the overall phenomenology and the different mechanisms that can be expected in such a structure.