Protein-protein interactions between jasmonate-related master regulator MYC and transcriptional mediator MED25 depend on a short binding domain.

A network of protein-protein interactions (PPI) is involved in the activation of (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), a plant hormone that regulates plant defense responses as well as plant growth and development. In the absence of JA-Ile, inhibitory protein jasmonate-ZIM-domain (JAZ) represses JA-related transcription factors, including a master regulator, MYC. In contrast, when JA-Ile accumulates in response to environmental stresses, PPI occurs between JAZ and the F-box protein COI1, which triggers JAZ degradation, resulting in derepressed MYC that can interact with the transcriptional mediator MED25 and upregulate JA-Ile-related gene expression. Activated JA signaling is eventually suppressed through the catabolism of JA-Ile and feedback suppression by JAZ splice variants containing a cryptic MYC-interacting domain (CMID). However, the detailed structural basis of some PPIs involved in JA-Ile signaling remains unclear. Herein, we analyzed PPI between MYC3 and MED25, focusing on the key interactions that activate the JA-Ile signaling pathway. Biochemical assays revealed that a short binding domain of MED25 (CMIDM) is responsible for the interaction with MYC, and that a bipartite interaction is critical for the formation of a stable complex. We also show the mode of interaction between MED25 and MYC is closely related to that of CMID and MYC. In addition, quantitative analyses on the binding of MYC3-JAZs and MYC3-MED25 revealed the order of binding affinity as JAZJas < MED25CMIDM < JAZCMID, suggesting a mechanism for how the transcriptional machinery causes activation and negative feedback regulation during jasmonate signaling. These results further illuminate the transcriptional machinery responsible for JA-Ile signaling.

Genome Editing Reveals Both the Crucial Role of OsCOI2 in Jasmonate Signaling and the Functional Diversity of COI1 Homologs in Rice.

Jasmonic acid (JA) regulates plant growth, development and stress responses. Coronatine insensitive 1 (COI1) and jasmonate zinc-finger inflorescence meristem-domain (JAZ) proteins form a receptor complex for jasmonoyl-l-isoleucine, a biologically active form of JA. Three COIs (OsCOI1a, OsCOI1b and OsCOI2) are encoded in the rice genome. In the present study, we generated mutants for each rice COI gene using genome editing to reveal the physiological functions of the three rice COIs. The oscoi2 mutants, but not the oscoi1a and oscoi1b mutants, exhibited severely low fertility, indicating the crucial role of OsCOI2 in rice fertility. Transcriptomic analysis revealed that the transcriptional changes after methyl jasmonate (MeJA) treatment were moderate in the leaves of oscoi2 mutants compared to those in the wild type or oscoi1a and oscoi1b mutants. MeJA-induced chlorophyll degradation and accumulation of antimicrobial secondary metabolites were suppressed in oscoi2 mutants. These results indicate that OsCOI2 plays a central role in JA response in rice leaves. In contrast, the assessment of growth inhibition upon exogenous application of JA to seedlings of each mutant revealed that rice COIs are redundantly involved in shoot growth, whereas OsCOI2 plays a primary role in root growth. In addition, a co-immunoprecipitation assay showed that OsJAZ2 and OsJAZ5 containing divergent Jas motifs physically interacted only with OsCOI2, whereas OsJAZ4 with a canonical Jas motif interacts with all three rice COIs. The present study demonstrated the functional diversity of rice COIs, thereby providing clues to the mechanisms regulating the various physiological functions of JA.

Ligand-receptor interactions in plant hormone signaling.

Small-molecule plant hormones principally control plant growth, development, differentiation, and environmental responses. Nine types of plant hormones are ubiquitous in angiosperms, and the molecular mechanisms of their hormone actions have been elucidated during the last two decades by genomic decoding of model plants with genetic mutants. In particular, the discovery of hormone receptors has greatly contributed to the understanding of signal transduction systems. The three-dimensional structure of the ligand-receptor complex has been determined for eight of the nine hormones by X-ray crystal structure analysis, and ligand perception mechanisms have been revealed at the atomic level. Collective research has revealed the molecular function of plant hormones that act as either molecular glue or an allosteric regulator for activation of receptors. In this review, we present an overview of the respective hormone signal transduction and describe the structural bases of ligand-receptor interactions.

Coordinately regulated transcription factors EIN3/EIL1 and MYCs in ethylene and jasmonate signaling interact with the same domain of MED25.

Ethylene (ET) and jasmonate (JA) are plant hormones that act synergistically to regulate plant development and defense against necrotrophic fungi infections, and antagonistically in response to wounds and apical hook formation. Previous studies revealed that the coordination of these responses is due to dynamic protein-protein interactions (PPI) between their master transcription factors (TFs) EIN3/EIL1 and MYC in ET and JA signaling, respectively. In addition, both TFs are activated via interactions with the same transcriptional mediator MED25, which upregulates downstream gene expression. Herein, we analyzed the PPI between EIN3/EIL1 and MED25, and as with the PPI between MYC3 and MED25, found that the short binding domain of MED25 (CMIDM) is also responsible for the interaction with EIN3/EIL1 - a finding which suggests that both TFs compete for binding with MED25. These results further inform our understanding of the coordination between the ET and JA regulatory systems.

A comprehensive in vitro fluorescence anisotropy assay system for screening ligands of the jasmonate COI1-JAZ co-receptor in plants.

The phytohormone (+)-7-iso-jasmonoyl-l-isoleucine regulates many developmental and stress responses in plants and induces protein-protein interactions between COI1, the F-box component of E3 ubiquitin ligase, and jasmonate ZIM domain (JAZ) repressors. These interactions cause JAZ degradation and activate jasmonate (JA), leading to plant defense responses, growth inhibition, and senescence. Thirteen JAZ subtypes are encoded in the Arabidopsis thaliana genome, but a detailed understanding of the physiological functions of these JAZ subtypes remains unclear, partially because of the genetic redundancy of JAZ genes. One strategy to elucidate the complex JA signaling pathways is to develop a reliable and comprehensive binding assay system of the ligands with all combinations of the co-receptors. Herein, we report the development of a fluorescence anisotropy-based in vitro binding assay system to screen for the ligands of the COI1-JAZ co-receptors. Our assay enabled the first quantitative analysis of the affinity values and JAZ-subtype selectivity of various endogenous JA derivatives, such as coronatine, jasmonic acid, and 12-hydroxyjasmonoyl-l-isoleucine. Because of its high signal-to-noise ratio and convenient mix-and-read assay system, our screening approach can be used in plate reader-based assays of both agonists and antagonists of COI1-JAZ co-receptors.

The alkyne-tag Raman imaging of coronatine, a plant pathogen virulence factor, in Commelina communis and its possible mode of action.

We previously reported that coronatine, a virulence factor of plant bacteria, facilitates bacterial infection through an ER (endoplasmic reticulum)-mediated, non-canonical mechanism in the model dicot plant, Arabidopsis thaliana. Here, we report that this same ER-mechanism is ubiquitous among dicots and monocots, and works by affecting the ethylene signaling pathway widely found in plants. The subcellular localization of coronatine by the alkyne-tag Raman imaging (ATRI) approach provided a convincing clue.

Reactive group-embedded affinity labeling reagent for efficient intracellular protein labeling.

Affinity labeling of a target protein is a powerful method for chemical biology studies. However, it is still difficult to label intracellular proteins efficiently in living cells. We propose the novel design strategy of a reactive group-embedded affinity labeling reagent for efficient protein labeling. With FKBP12 as the model target protein, the ligand binding pocket-oriented labeling reagent could label intracellular protein, whereas protein surface-oriented reagent was ineffective for labeling in living cells, partially because of the intracellular protein fluctuation under the macromolecular crowding effects. These results provide new insight for efficient intracellular protein labeling.

Protein ligand-tethered synthetic calcium indicator for localization control and spatiotemporal calcium imaging in plant cells.

In plant biology, calcium ions are involved in a variety of intriguing biological phenomena as a secondary messenger. However, most conventional calcium indicators are not applicable for plant cells because of the difficulty with their localization control in plant cells. We here introduce a method to monitor spatiotemporal Ca(2+) dynamics in living plant cells based on linking the synthetic calcium indicator Calcium Green-1 to a natural product-based protein ligand. In a proof-of-concept study using cultured BY-2 cells overexpressing the target protein for the ligand, the ligand-tethered probe accumulated in the cytosol and nucleus, and enabled real-time monitoring of the cytosolic and nucleus Ca(2+) dynamics under the physiological condition. The present strategy using ligand-tethered fluorescent sensors may be successfully applied to reveal the spatiotemporal dynamics of calcium ions in living plant cells.

Analysis of Cell-Surface Receptor Dynamics through Covalent Labeling by Catalyst-Tethered Antibody.

A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces.

Intracellular protein-responsive supramolecules: protein sensing and in-cell construction of inhibitor assay system.

Supramolecular nanomaterials responsive to specific intracellular proteins should be greatly promising for protein sensing and imaging, controlled drug release or dynamic regulation of cellular processes. However, valid design strategies to create useful probes are poorly developed, particularly for proteins inside living cells as targets. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent probes consisting of a protein ligand and a fluorophore on the live cell surface, as well as in test tube settings. Herein, we discovered that our self-assembled supramolecular probes having a rhodamine derivative (tetramethylrhodamine or rhodamine-green) can incorporate and stay as less-fluorescent aggregates inside the living cells, so as to sense the protein activity in a reversible manner. Using the overexpressed model protein (dihydrofolate reductase), we demonstrated that this turn-on/off mode is controlled by selective ligand-protein recognition inside the live cells. Not only such a model protein, but also endogenous human carbonic anhydrase and heat shock protein 90 were specifically visualized in living mammalian cells, by use of the similar ligand-tethered supramolecular probes. Furthermore, such reversibility allowed us to intracellularly construct a unique system to evaluate the inhibitors affinity toward specific endogenous proteins in live cells, highlighting the potential of dynamic supramolecules as novel intelligent biomaterials.

Acidic-pH-activatable fluorescence probes for visualizing exocytosis dynamics.

Live imaging of exocytosis dynamics is crucial for a precise spatiotemporal understanding of secretion phenomena, but current approaches have serious limitations. We designed and synthesized small-molecular fluorescent probes that were chemically optimized for sensing acidic intravesicular pH values, and established that they can be used to sensitively and reliably visualize vesicular dynamics following stimulation. This straightforward technique for the visualization of exocytosis as well as endocytosis/reacidification processes with high spatiotemporal precision is expected to be a powerful tool for investigating dynamic cellular phenomena involving changes in the pH value.

Two distinct modes of action of molecular glues in the plant hormone co-receptor COI1-JAZ system.

The plant hormone (3R, 7S)-jasmonoyl-L-isoleucine ((3R, 7S)-JA-Ile) is perceived by the COI1-JAZ co-receptor in Arabidopsis thaliana, leading to the activation of gene expression for plant defense responses, growth, development, and other processes. Therefore, understanding the interaction between the COI1-JAZ co-receptor and its ligands is essential for the development of COI1-JAZ agonists and antagonists as potent chemical tools for regulating (3R, 7S)-JA-Ile signaling. This study demonstrated that COI1-JAZ has two independent modes of ligand perception using a differential scanning fluorimetry (DSF) assay. (3R, 7S)-JA-Ile is perceived through a one-step model in which (3R, 7S)-JA-Ile causes protein-protein interaction between COI1 and JAZ. In contrast, coronatine (COR), a mimic of (3R, 7S)-JA-Ile, is perceived through a two-step model in which COR is first perceived by COI1 and then recruits JAZ to form the COI1-COR-JAZ complex. Our results demonstrate two distinct modes of action of molecular glues causing protein-protein interactions.

Heme binding mechanism of structurally similar iron-regulated surface determinant near transporter domains of Staphylococcus aureus exhibiting different affinities for heme.

Near transporter (NEAT) domains of the iron-regulated surface determinant (Isd) proteins are essential for the import of nutritional heme from host animals to Gram-positive pathogens such as Staphylococcus aureus. The order of transfer of heme between NEAT domains occurs from IsdH to IsdA to IsdC, without any energy input despite the similarity of their three-dimensional structures. We measured the free energy of binding of heme and various metalloporphyrins to each NEAT domain and found that the affinity of heme and non-iron porphyrins for NEAT domains increased gradually in the same order as that for heme transfer. To gain insight into the atomistic mechanism for the differential affinities, we performed in silico molecular dynamics simulation and in vitro site-directed mutagenesis. The simulations revealed that the negatively charged residues that are abundant in the loop between strand ß1b and the 310 helix of IsdH-NEAT3 destabilize the interaction with the propionate group of heme. The higher affinity of IsdC was in part attributed to the formation of a salt bridge between its unique residue, Glu88, and the conserved Arg100 upon binding to heme. In addition, we found that Phe130 of IsdC makes the ß7-ß8 hairpin less flexible in the ligand-free form, which serves to reduce the magnitude of the entropy loss on binding to heme. We confirmed that substitution of these key residues of IsdC decreased its affinity for heme. Furthermore, IsdC mutants, whose affinities for heme were lower than those of IsdA, transferred heme back to IsdA. Thus, NEAT domains have evolved the characteristic residues on the common structural scaffold such that they exhibit different affinities for heme, thus promoting the efficient transfer of heme.

Semisynthetic lectin-4-dimethylaminopyridine conjugates for labeling and profiling glycoproteins on live cell surfaces.

Glycoproteins on cell surfaces play important roles in biological processes, including cell-cell interaction/signaling, immune response, and cell differentiation. Given the diversity of the structure of glycans, labeling and imaging of selected glycoproteins are challenging, although several promising strategies have been developed recently. Here, we design and construct semisynthetic reactive lectins (sugar-binding proteins) that are able to selectively label glycoproteins. Congerin II, an animal galectin, and wheat germ agglutinin are conjugated with 4-dimethylaminopyridine (DMAP), a well-known acyl transfer catalyst by our affinity-guided DMAP method and Cu(I)-assisted click chemistry. Selective labeling of glycoproteins is facilitated by the DMAP-tethered lectin catalysts both in vitro and on living cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis enabled us to isolate labeled glycoproteins that are uniquely exposed on distinct cell lines. Furthermore, the combination of immunoprecipitation with mass spectrometry (MS)-fingerprinting techniques allowed us to characterize 48 glycoproteins endogenously expressed on HeLa cells, and some low-abundant glycoproteins, such as epidermal growth factor receptor (EGFR) and neuropilin-1, were successfully identified. Our results demonstrate that semisynthetic DMAP-tethered lectins provide a new tool for labeling and profiling glycoproteins on living cells.

Specific detection and imaging of enzyme activity by signal-amplifiable self-assembling (19)F MRI probes.

Specific turn-on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal-amplifiable self-assembling (19) F NMR/MRI probe for turn-on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are "silent" when aggregated, but exhibit a disassembly driven turn-on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these (19) F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by (19) F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by (19) F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn-on (19) F probes may serve as a screening platform for the activity of MMPs.

Protein organic chemistry and applications for labeling and engineering in live-cell systems.

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry-based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test-tube settings; 2) the bioorthogonal reactions of proteins with non-natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag-probe pair for labeling natural amino acids; and 4) ligand-directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2-4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.

Subtype-selective agonists of plant hormone co-receptor COI1-JAZs identified from the stereoisomers of coronatine.

Severe genetic redundancy is particularly clear in gene families encoding plant hormone receptors, each subtype sharing redundant and specific functions. Genetic redundancy of receptor family members represents a major challenge for the functional dissection of each receptor subtype. A paradigmatic example is the perception of the hormone (+)-7-iso-jasmonoyl-L-isoleucine, perceived by several COI1-JAZ complexes; the specific role of each receptor subtype still remains elusive. Subtype-selective agonists of the receptor are valuable tools for analyzing the responses regulated by individual receptor subtypes. We constructed a stereoisomer library consisting of all stereochemical isomers of coronatine (COR), a mimic of the plant hormone (+)-7-iso-jasmonoyl-L-isoleucine, to identify subtype-selective agonists for COI1-JAZ co-receptors in Arabidopsis thaliana and Solanum lycopersicum. An agonist selective for the Arabidopsis COI1-JAZ9 co-receptor efficiently revealed that JAZ9 is not involved in most of the gene downregulation caused by COR, and the degradation of JAZ9-induced defense without inhibiting growth.

Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes.

Visualization of tumor-specific protein biomarkers on cell membranes has the potential to contribute greatly to basic biological research and therapeutic applications. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore in test tube settings. This method is based on recognition-driven disassembly of the nanoprobes, which induces a clear turn-on fluorescent signal. In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-assembling turn-on probes.

Systematic study of protein detection mechanism of self-assembling 19F NMR/MRI nanoprobes toward rational design and improved sensitivity.

(19)F NMR/MRI probe is expected to be a powerful tool for selective sensing of biologically active agents owing to its high sensitivity and no background signals in live bodies. We have recently reported a unique supramolecular strategy for specific protein detection using a protein ligand-tethered self-assembling (19)F probe. This method is based on a recognition-driven disassembly of the nanoprobes, which induced a clear turn-on signal of (19)F NMR/MRI. In the present study, we conducted a systematic investigation of the relationship between structure and properties of the probe to elucidate the mechanism of this turn-on (19)F NMR sensing in detail. Newly synthesized (19)F probes showed three distinct behaviors in response to the target protein: off/on, always-on, and always-off modes. We clearly demonstrated that these differences in protein response could be explained by differences in the stability of the probe aggregates and that "moderate stability" of the aggregates produced an ideal turn-on response in protein detection. We also successfully controlled the aggregate stability by changing the hydrophobicity/hydrophilicity balance of the probes. The detailed understanding of the detection mechanism allowed us to rationally design a turn-on (19)F NMR probe with improved sensitivity, giving a higher image intensity for the target protein in (19)F MRI.

Ligand-directed tosyl chemistry for protein labeling in vivo.

Here we describe a method for the site-selective attachment of synthetic molecules into specific 'endogenous' proteins in vivo using ligand-directed tosyl (LDT) chemistry. This approach was applied not only for chemically labeling proteins in living cells, tissues and mice but also for constructing a biosensor directly inside cells without genetic engineering. These data establish LDT chemistry as a new tool for the study and manipulation of biological systems.