Clemmensengotfredsen4150

Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-P) with an alkyne moiety to detect and identify specific binding targets of curcumin in HCT116 colon cancer cells. Through click chemistry, a fluorescent tag or a biotin tag is attached to the probe-modified curcumin targets for visualization or affinity purification followed by mass spectrometric identification. A quantitative proteomics approach of isobaric tags for relative and absolute quantification (iTRAQ)™ is applied to distinguish specific curcumin targets from nonspecific binding proteins.Auxin plays important roles in almost all aspects of plant growth and development. Chemical genetics is an effective approach to understand auxin action, especially in nonmodel plant species, in which auxin-related mutants are not yet available. Among auxin-related chemical tools, we present approaches to utilize auxin biosynthesis inhibitors. The inhibitors are effective not only to understand auxin biosynthesis but also to understand auxin action. The effectiveness of the inhibitors can be assessed based on in vitro or in vivo assays. The in vitro assay employs enzyme inhibition assays. The in vivo assay employs UPLC-MS/MS-based analysis of endogenous IAA and its intermediates or metabolites.The gaseous hormone ethylene regulates a diverse range of plant development and stress responses. Ethylene biosynthesis is tightly regulated by the transcriptional and posttranscriptional regulation of ethylene biosynthetic enzymes. ACC synthase (ACS) is the rate-limiting enzyme that controls the speed of ethylene biosynthesis in plant tissues, thus serving as a primary target for biotic and abiotic stresses to modulate ethylene production. Despite the critical role of ACS in ethylene biosynthesis, only a few regulatory components regulating ACS stability or ACS transcript levels have been identified and characterized. Here we show a genetic approach for identifying novel regulatory components in ethylene biosynthesis by screening EMS-mutagenized Arabidopsis seeds.Plant stress tolerance relies on intricate signaling networks that are not fully understood. Several plant hormones are involved in the adaptation to different environmental conditions. Abscisic acid (ABA) has an essential role in stress tolerance, especially in the adaptation to drought. During the last years, chemical genomics has gained attention as an alternative approach to decipher complex traits. Additionally, chemical-based strategies have been very useful to untangle genetic redundancy, which is hard to address by other approaches such as classical genetics. Here, we describe the use of an ABA-inducible luciferase (LUC) reporter line for the high-throughput identification of chemical activators of the ABA signaling pathway. In this assay, seven-day-old pMAPKKK18-LUC+ seedlings are grown on 96-well plates and treated with test compounds. Next, the activity of the LUC reporter is quantified semiautomatically by image analysis. Candidate compounds able to activate the reporter are thus identified and subjected to a secondary screen by analyzing their effect on ABA-related phenotypes (e.g., inhibition of seed germination). This assay is fast, high-throughput, nondestructive, semiquantitative and can be applied to any other luciferase reporter lines, making it ideal for forward chemical genetic screenings.Small molecules that can activate abscisic acid (ABA) receptors represent valuable probes to study ABA perception and signaling. Additionally, these compounds have the potential to be used in the field to counteract the negative effect of drought stress on plant productivity. The PYR/PYL ABA receptors, in their ligand-bound conformation, inactivate protein phosphatases 2C (PP2Cs), triggering physiological responses that are essential for plant adaptation to environmental stresses, including drought. Based on this ligand-induced PP2C inactivation mechanism, we have developed an in vitro assay for the identification of ABA-receptor agonists by high-throughput screening of chemical libraries. The assay allows simultaneous use of different ABA receptors, increasing the chances to find new agonists and eliminates the need for parallel screening. In this chapter, we describe detailed procedures for the identification of ABA agonists using this multiplexed assay in a medium- (96-well plates) or a high-throughput (384-well plates) setup.Ca2+-based second messenger signaling is used by many signal perception mechanisms to modulate specific cellular responses. The well-characterized phytohormone auxin elicits a very rapid Ca2+ signal, but the molecular players involved in auxin-induced Ca2+ signaling are still largely unknown. The complicated and often redundant nature of the plant Ca2+ signaling machinery makes the use of mutants and transgenic lines a painstaking process, which makes a pharmacological approach an attractive alternative to study these processes. Here, we describe the development and utilization of a screening assay that can be used to probe a compound library for inhibitors of auxin-induced Ca2+ entry in plant cell suspensions.Interfering peptides (iPs) have been recognized as valuable substances to specifically target protein-protein interactions (PPIs) in senescence and disease. Although the concept of iPs has been validated for several PPIs in medical and pharmaceutical research, little attention so far has been paid to the enormous potential iPs that may provide to target and control plant growth and developmental processes or plant environmental responses. However, recent research on PPIs in the ethylene signaling pathway has identified the synthetic peptide NOP-1 derived from the nuclear localization signal of ethylene regulator EIN2 as an efficient inhibitor of typical ethylene responses such as ripening, aging, and senescence. Biophysical and biochemical studies on purified recombinant proteins of the ethylene receptor family from various plant species demonstrate that the synthetic peptide binds in the nM-μM range at the plant target. STA-9090 ic50 Here, we describe methods to evaluate and quantify the effect of the NOP-1 peptide on flower senescence as a typical ethylene response in the intact plant system.