Uncovering the proximal proteome of CTR1 through TurboID-mediated proximity labeling.

Protein-protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene-Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID-based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 in Nicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using the N. benthamiana transient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.

Ethylene-triggered subcellular trafficking of CTR1 enhances the response to ethylene gas.

The phytohormone ethylene controls plant growth and stress responses. Ethylene-exposed dark-grown Arabidopsis seedlings exhibit dramatic growth reduction, yet the seedlings rapidly return to the basal growth rate when ethylene gas is removed. However, the underlying mechanism governing this acclimation of dark-grown seedlings to ethylene remains enigmatic. Here, we report that ethylene triggers the translocation of the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling, from the endoplasmic reticulum to the nucleus. Nuclear-localized CTR1 stabilizes the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor by interacting with and inhibiting EIN3-BINDING F-box (EBF) proteins, thus enhancing the ethylene response and delaying growth recovery. Furthermore, Arabidopsis plants with enhanced nuclear-localized CTR1 exhibited improved tolerance to drought and salinity stress. These findings uncover a mechanism of the ethylene signaling pathway that links the spatiotemporal dynamics of cellular signaling components to physiological responses.

In vitro Auto- and Substrate-Ubiquitination Assays.

The precise regulation of the homeostasis of the cellular proteome is critical for the appropriate growth and development of plants. It also allows the plants to respond to various environmental stresses, by modulating their biochemical and physiological aspects in a timely manner. Ubiquitination of cellular proteins is one of the major protein degradation routes for maintaining cellular protein homeostasis, and ubiquitin E3 ligases, components of ubiquitin ligase complexes, play an important role in the selective degradation of target proteins via substrate-specific interactions. Thus, understanding the role of E3 ligases and their substrate regulation uncovers their specific cellular and physiological functions. Here, we provide protocols for auto- and substrate-ubiquitination analyses that utilize the combination of in vitro purified E3 ubiquitin ligase proteins and immunoprecipitation.

Biochemical and Molecular Characterization of the Rice Chalcone Isomerase Family.

Chalcone isomerase (CHI) is a key enzyme in flavonoid biosynthesis. In plants, CHIs occur in multigene families, and they are divided into four types, types I-IV. Type I and II CHIs are bona fide CHIs with CHI activity, and type III and IV CHIs are non-catalytic members with different functions. Rice contains seven CHI family genes (OsCHIs). Molecular analysis suggested that OsCHI3 is a type I CHI, and the other OsCHIs were classified into types III and IV. To elucidate their biochemical functions, OsCHI1, OsCHI3, OsCHI6, and OsCHI7 were expressed in Escherichia coli, and the recombinant OsCHI proteins were purified. An activity assay of recombinant OsCHIs showed that OsCHI3 catalyzed the isomerization of naringenin chalcone and isoliquiritigenin, whereas the other recombinant OsCHIs had no CHI activity. OsCHI3 also exhibited a strong preference to naringenin chalcone compared to isoliquiritigenin, which agrees well with the catalytic properties of type I CHIs. These results ascertain OsCHI3 to be a bona fide CHI in rice. OsCHI3 and the other OsCHIs were expressed constitutively throughout the rice growth period and different tissues. OsCHI3 expression was induced immediately in response to ultra-violet (UV) stress, suggesting its involvement in the biosynthesis of sakuranetin, a flavonoid phytoalexin in rice.

Reciprocal antagonistic regulation of E3 ligases controls ACC synthase stability and responses to stress.

Ethylene influences plant growth, development, and stress responses via crosstalk with other phytohormones; however, the underlying molecular mechanisms are still unclear. Here, we describe a mechanistic link between the brassinosteroid (BR) and ethylene biosynthesis, which regulates cellular protein homeostasis and stress responses. We demonstrate that as a scaffold, 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), a rate-limiting enzyme in ethylene biosynthesis, promote the interaction between Seven-in-Absentia of Arabidopsis (SINAT), a RING-domain containing E3 ligase involved in stress response, and ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-like (EOL) proteins, the E3 ligase adaptors that target a subset of ACS isoforms. Each E3 ligase promotes the degradation of the other, and this reciprocally antagonistic interaction affects the protein stability of ACS. Furthermore, 14-3-3, a phosphoprotein-binding protein, interacts with SINAT in a BR-dependent manner, thus activating reciprocal degradation. Disrupted reciprocal degradation between the E3 ligases compromises the survival of plants in carbon-deficient conditions. Our study reveals a mechanism by which plants respond to stress by modulating the homeostasis of ACS and its cognate E3 ligases.

Biochemical Characterization of a Flavonoid O-methyltransferase from Perilla Leaves and Its Application in 7-Methoxyflavonoid Production.

Methylation is a common structural modification that can alter and improve the biological activities of natural compounds. O-Methyltransferases (OMTs) catalyze the methylation of a wide array of secondary metabolites, including flavonoids, and are potentially useful tools for the biotechnological production of valuable natural products. An OMT gene (PfOMT3) was isolated from perilla leaves as a putative flavonoid OMT (FOMT). Phylogenetic analysis and sequence comparisons showed that PfOMT3 is a class II OMT. Recombinant PfOMT3 catalyzed the methylation of flavonoid substrates, whereas no methylated product was detected in PfOMT3 reactions with phenylpropanoid substrates. Structural analyses of the methylation products revealed that PfOMT3 regiospecifically transfers a methyl group to the 7-OH of flavonoids. These results indicate that PfOMT3 is an FOMT that catalyzes the 7-O-methylation of flavonoids. PfOMT3 methylated diverse flavonoids regardless of their backbone structure. Chrysin, naringenin and apigenin were found to be the preferred substrates of PfOMT3. Recombinant PfOMT3 showed moderate OMT activity toward eriodictyol, luteolin and kaempferol. To assess the biotechnological potential of PfOMT3, the biotransformation of flavonoids was performed using PfOMT3-transformed Escherichia coli. Naringenin and kaempferol were successfully bioconverted to the 7-methylated products sakuranetin and rhamnocitrin, respectively, by E. coli harboring PfOMT3.

Two Chalcone Synthase Isozymes Participate Redundantly in UV-Induced Sakuranetin Synthesis in Rice.

: Chalcone synthase (CHS) is a key enzyme in the flavonoid pathway, participating in the production of phenolic phytoalexins. The rice genome contains 31 CHS family genes (OsCHSs). The molecular characterization of OsCHSs suggests that OsCHS8 and OsCHS24 belong in the bona fide CHSs, while the other members are categorized in the non-CHS group of type III polyketide synthases (PKSs). Biochemical analyses of recombinant OsCHSs also showed that OsCHS24 and OsCHS8 catalyze the formation of naringenin chalcone from p-coumaroyl-CoA and malonyl-CoA, while the other OsCHSs had no detectable CHS activity. OsCHS24 is kinetically more efficient than OsCHS8. Of the OsCHSs, OsCHS24 also showed the highest expression levels in different tissues and developmental stages, suggesting that it is the major CHS isoform in rice. In oschs24 mutant leaves, sakuranetin content decreased to 64.6% and 80.2% of those in wild-type leaves at 2 and 4 days after UV irradiation, respectively, even though OsCHS24 expression was mostly suppressed. Instead, the OsCHS8 expression was markedly increased in the oschs24 mutant under UV stress conditions compared to that in the wild-type, which likely supports the UV-induced production of sakuranetin in oschs24. These results suggest that OsCHS24 acts as the main CHS isozyme and OsCHS8 redundantly contributes to the UV-induced production of sakuranetin in rice leaves.

Comparisons of the Mucosal Healing Process between Flat and Protruded Type after Endoscopic Submucosal Dissection for Gastric Neoplasms.

BACKGROUND/AIMS: The mucosal healing process after endoscopic submucosal dissection (ESD) is mostly scarring change (flat type), but a protruded lesion is occasionally found. We investigated the factors influencing the mucosal healing process, such as the flat and protruded types. METHODS: A total of 2,096 ESD cases were performed from February 2005 to December 2013, and 1,757 underwent follow-up endoscopy after 3 months to check the healing type of the ulceration. We retrospectively reviewed the medical charts to analyze demographic, endoscopic, and pathological findings between the 2 groups. RESULTS: Forty-eight cases were of the protruded type and 1,709 were of the flat type. In univariate analysis, the protruded type was found more in the antrum, anterior wall, and greater curvature (p < 0.001). In protruded types, the Helicobacter pylori (H. pylori) infection rate was lower (p < 0.017), the mean length of ESD specimen was shorter (p < 0.012), the fibrosis rate was lower (p < 0.033), and the mean number of hot biopsy and clips during ESD were less (p < 0.008 and p < 0.001 respectively). CONCLUSIONS: The healing type of mucosal ulceration after ESD seemed to be influenced by location, specimen size, and the presence of an H. pylori infection.

Biochemical Characterization of the Rice Cinnamyl Alcohol Dehydrogenase Gene Family.

Cinnamyl alcohol dehydrogenase (CAD) is involved in the final step of the phenylpropanod pathway, catalyzing the NADPH-dependent reduction of hydroxy-cinnamaldehydes into the corresponding alcohols. The rice genome contains twelve CAD and CAD-like genes, collectively called OsCADs. To elucidate the biochemical function of the OsCADs, OsCAD1, 2, 6, and 7, which are highly expressed in rice, were cloned from rice tissues. The cloned OsCADs were heterologously expressed in Escherichia coli as His-tag fusion proteins. The activity assay of the recombinant OsCADs showed that OsCAD2, 6, and 7 have CAD activity toward hydroxycinnamaldehydes, but OsCAD1 has no detectable catalytic activity. The kinetic parameters of the enzyme reactions demonstrated that OsCAD2 has the highest catalytic activity among the examined enzymes. This result agrees well with the finding that the Zn binding and NADPH binding motifs and the residues constituting the substrate binding pocket in bona fide plant CADs were fully conserved in OsCAD2. Although they have large variations in the residue for the substrate binding pocket, OsCAD6 and 7 catalyzed the reduction of hydroxycinnamaldehydes with a similar efficiency. Alignment of amino acid sequences showed that OsCAD1 lacks the GxxxxP motif for NADPH binding and has mismatches in residues important in the reduction process, which could be responsible for the loss of catalytic activity. OsCAD2 belongs to CAD Class I with bona fide CADs from other plant species and is constitutively expressed throughout the developmental stages of rice, with preferential expression in actively lignifying tissues such as the root, stem, and panicle, suggesting that it is mainly involved in developmental lignification in rice. The expression of OsCAD2 was also induced by biotic and abiotic stresses such as Xanthomonas oryzae pv. oryzae (Xoo) infection and UV-irradiation, suggesting that it plays a role in the defense response of rice, in addition to a bona fide role in developmental lignification. OsCAD6 and 7 belong in CAD Class II. Their expression is relatively lower than that of OsCAD2 and is confined to certain tissues, such as the leaf sheath, stem, and panicle. The expression of OsCAD6 was stimulated by Xoo infection and UV-irradiation. Thus OsCAD6 appears to be an inducible OsCAD that is likely involved in the defense response of rice against biotic and abiotic stresses.

Determination of a radotinib dosage regimen based on dose-response relationships for the treatment of newly diagnosed patients with chronic myeloid leukemia.

Radotinib is a second-generation BCR-ABL1 tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukemia in chronic phase (CP-CML). Here, using the data from a Phase 3 study conducted in patients with newly diagnosed CP-CML, the dose-efficacy as well as dose-safety relationship analyses were performed to determine a safe and effective initial dosage regimen of radotinib. A significant positive association was detected between the starting dose of radotinib adjusted for body weight (Dose/BW) and the probability of dose-limiting toxicity (≥grade 3 hematologic and nonhematologic toxicity) (P = 0.003). In contrast, a significant inverse association was discovered between Dose/BW and the probability of major molecular response (BCR-ABL1/ABL1 ≤ 0.1%) when controlled for sex (P = 0.033). Moreover, frequent dose interruptions and reductions secondary to radotinib toxicities occurred in the Phase 3 study, resulting in nearly half (44%) of patients receiving a reduced dose at a 12-month follow-up. In conclusion, the results of this study demonstrate the need for initial radotinib dose attenuation to improve the long-term efficacy and safety of radotinib. Hence, the authors suggest a new upfront radotinib dose of 400 mg once daily be tested in patients with newly diagnosed CP-CML.

Biotechnological Production of Dimethoxyflavonoids Using a Fusion Flavonoid O-Methyltransferase Possessing Both 3'- and 7-O-Methyltransferase Activities.

Although they are less abundant in nature, methoxyflavonoids have distinct physicochemical and pharmacological properties compared to common nonmethylated flavonoids. Thus, enzymatic conversion and biotransformation using genetically engineered microorganisms of flavonoids have been attempted for the efficient production of methoxyflavonoids. Because of their regiospecificity, more than two flavonoid O-methyltransferases (FOMTs) and enzyme reactions are required to biosynthesize di(or poly)-methoxyflavonoids. For the one-step biotechnological production of bioactive di-O-methylflavonoids, we generated a multifunctional FOMT fusing a 3'-OMT (SlOMT3) and a 7-OMT (OsNOMT). The SlOMT3/OsNOMT fusion enzyme possessed both 3'- and 7-OMT activities to diverse flavonoid substrates, which were comparable to those of individual SlOMT3 and OsNOMT. The SlOMT3/OsNOMT enzyme also showed 3'- and 7-OMT activity for 7- or 3'-O-methylflavonoids, respectively, suggesting that the fusion enzyme can sequentially methylate flavonoids into di-O-methylflavonoids. The biotransformation of the flavonoids quercetin, luteolin, eriodictyol, and taxifolin using SlOMT3/OsNOMT-transformed Escherichia coli generated corresponding di-O-methylflavonoids, rhamnazin, velutin, 3',7-di-O-methyleriodictyol, and 3',7-di-O-methyltaxifolin, respectively. These results indicate that dimethoxyflavonoids may be efficiently produced from nonmethylated flavonoid precursors through a one-step biotransformation using the engineered E. coli harboring the SlOMT3/OsNOMT fusion gene.

Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family.

Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as CCR and CCR-like genes, collectively called OsCCRs. To elucidate the functions of OsCCRs, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 OsCCRs, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in Escherichia coli and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that OsCCR20 was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that OsCCR20 is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as Magnaporthe grisea and Xanthomonas oryzae pv. oryzae (Xoo) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.

Optimization of radotinib doses for the treatment of Asian patients with chronic myelogenous leukemia based on dose-response relationship analyses.

A fixed dose regimen for tyrosine kinase inhibitors (TKIs) is postulated to be responsible for variable safety outcomes in the treatment of chronic myelogenous leukemia (CML). The objective of this study was to explore an optimal dosing regimen for a TKI, radotinib, to improve its safety profile. Clinical data were obtained from a Phase 2 study of fixed-dose radotinib in 77 Asian patients with CML. The magnitude of radotinib dose adjusted for patient's body weight (Dose/BW) and the probability of dose-limiting toxicity (DLT) demonstrated a positive association (Logit[P] = 0.86*[Dose/BW]-4.45, p = 0.001). There was a significant difference in the Kaplan-Meier curves for time to first DLT between the patient subgroups of Dose/BW <6 and ≥6 mg/kg (259 versus 83 days). Consequently, a two-tier weight-based dosing regimen may improve the safety of radotinib: 300 mg or 400 mg twice daily for patients weighing ≤65 or >65 kg, respectively.

Antimicrobial activity of UV-induced phenylamides from rice leaves.

Rice produces a wide array of phytoalexins in response to pathogen attacks and UV-irradiation. Except for the flavonoid sakuranetin, most phytoalexins identified in rice are diterpenoid compounds. Analysis of phenolic-enriched fractions from UV-treated rice leaves showed that several phenolic compounds in addition to sakuranetin accumulated remarkably in rice leaves. We isolated two compounds from UV-treated rice leaves using silica gel column chromatography and preparative HPLC. The isolated phenolic compounds were identified as phenylamide compounds: N-trans-cinnamoyltryptamine and N-p-coumaroylserotonin. Expression analysis of biosynthetic genes demonstrated that genes for arylamine biosynthesis were upregulated by UV irradiation. This result suggested that phenylamide biosynthetic pathways are activated in rice leaves by UV treatment. To unravel the role of UV-induced phenylamides as phytoalexins, we examined their antimicrobial activity against rice fungal and bacterial pathogens. N-trans-Cinnamoyltryptamine inhibited the growth of rice brown spot fungus (Bipolaris oryzae). In addition to the known antifungal activity to the blast fungus, sakuranetin had antimicrobial activity toward B. oryzae and Rhizoctonia solani (rice sheath blight fungus). UV-induced phenylamides and sakuranetin also had antimicrobial activity against rice bacterial pathogens for grain rot (Burkholderia glumae), blight (Xanthomonas oryzae pv. oryzae) and leaf streak (X. oryzae pv. oryzicola) diseases. These findings suggested that the UV-induced phenylamides in rice are phytoalexins against a diverse array of pathogens.

Efficacy and safety of radotinib in chronic phase chronic myeloid leukemia patients with resistance or intolerance to BCR-ABL1 tyrosine kinase inhibitors.

Radotinib (IY5511HCL), a novel and selective BCR-ABL1 tyrosine kinase inhibitor, has shown pre-clinical and phase I activity and safety in chronic myeloid leukemia. This phase II study investigated the efficacy and safety of radotinib in Philadelphia chromosome-positive chronic phase-chronic myeloid leukemia patients with resistance and/or intolerance to BCR-ABL1 tyrosine kinase inhibitors. Patients received radotinib 400 mg twice daily for 12 cycles based on results from the phase I trial. The primary end point was rate of major cytogenetic response by 12 months. A total of 77 patients were enrolled. Major cytogenetic response was achieved in 50 (65%; cumulative 75%) patients, including 36 (47%) patients with complete cytogenetic response by 12 months. Median time to major cytogenetic response and complete cytogenetic response were 85 days and 256 days, respectively. Major cytogenetic response and complete cytogenetic response rates were similar between imatinib-resistant and imatinib-intolerant patients, but were higher in patients without BCR-ABL1 mutations. Overall and progression-free survival rates at 12 months were 96.1% and 86.3%, respectively. All newly-occurring or worsening grade 3/4 hematologic abnormalities included thrombocytopenia (24.7%) and anemia (5.2%); grade 3/4 drug-related non-hematologic adverse events included fatigue (3.9%), asthenia (3.9%), and nausea (2.6%). The most common biochemistry abnormality was hyperbilirubinemia (grade 3/4 23.4%), and 12 of 18 cases were managed with dose modification. Study findings suggest radotinib is effective and well tolerated in chronic phase-chronic myeloid leukemia patients with resistance and/or intolerance to BCR-ABL1 tyrosine kinase inhibitors and may represent a promising alternative for these patients. (clinicaltrials.gov identifier: 01602952).

Transcriptomic analysis of UV-treated rice leaves reveals UV-induced phytoalexin biosynthetic pathways and their regulatory networks in rice.

Rice produces diterpenoid and flavonoid phytoalexins for defense against pathogen attack. The production of phytoalexins in rice is also induced by UV-irradiation. To understand the metabolic networks involved in UV-induced phytoalexin biosynthesis and their regulation, phytochemical and transcriptomic analyses of UV-treated rice leaves were performed. In response to UV treatment, the accumulation of flavonoids was observed in rice leaves, which may serve as antioxidants against UV-induced oxidative stress. The phytochemical analysis confirmed sakuranetin accumulation and also demonstrated the induction of phenylamide synthesis in rice leaves by UV-irradiation. Transcriptomic analysis established that aromatic amino acid biosynthetic genes were immediately up-regulated after UV treatment. The genes involved in the phenylpropanoid pathway and flavonoid biosynthesis were also up-regulated. These findings suggest that the aromatic amino acid and flavonoid biosynthetic pathways are coordinately activated for the production of flavonoids and phenolic phytoalexins such as sakuranetin and phenylamides. An in silico analysis of UV-induced O-methyltransferase and acyltransferase genes suggested that these genes may be implicated in sakuranetin and phenylamide synthesis, respectively. The transcriptomic analysis also showed up-regulation of both methylerythritol phosphate pathway and the diterpenoid phytoalexin biosynthetic genes in response to UV treatment. A functional gene network analysis of phytoalexin biosynthetic and UV-induced genes for signaling components and transcription factors using RiceNet suggested that regulatory networks comprising signal perceiving receptor kinases, G-proteins, signal transducing mitogen-activated protein kinases and calcium signaling components, and transcription factors control flavonoid and phytoalexin biosynthesis in rice leaves under UV-C stress conditions.