Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Loss of miR-31-5p drives hematopoietic stem cell malignant transformation and restoration eliminates leukemia stem cells in mice.

Leukemia stem cells (LSCs) propagate leukemia and are responsible for the high frequency of relapse of treated patients. The ability to target LSCs remains elusive, indicating a need to understand the underlying mechanism of LSC formation. Here, we report that miR-31-5p is reduced or undetectable in human LSCs compared to hematopoietic stem progenitor cells (HSPCs). Inhibition of miR-31-5p in HSPCs promotes the expression of its target gene FIH, encoding FIH [factor inhibiting hypoxia-inducing factor 1α (HIF-1α)], to suppress HIF-1α signaling. Increased FIH resulted in a switch from glycolysis to oxidative phosphorylation (OXPHOS) as the predominant mode of energy metabolism and increased the abundance of the oncometabolite fumarate. Increased fumarate promoted the conversion of HSPCs to LSCs and initiated myeloid leukemia-like disease in NOD-Prkdcscid IL2rgtm1/Bcgen (B-NDG) mice. We further demonstrated that miR-31-5p inhibited long- and short-term hematopoietic stem cells with a high frequency of LSCs. In combination with the chemotherapeutic agent Ara-C (cytosine arabinoside), restoration of miR-31-5p using G7 poly (amidoamine) nanosized dendriplex encapsulating miR-31-5p eliminated LSCs and inhibited acute myeloid leukemia (AML) progression in patient-derived xenograft mouse models. These results demonstrated a mechanism of HSC malignant transformation through altered energy metabolism and provided a potential therapeutic strategy to treat patients with AML.

Molecular and Biochemical Characterization of Jasmonic Acid Carboxyl Methyltransferase Involved in Aroma Compound Production of Methyl Jasmonate during Black Tea Processing.

Methyl jasmonate (MeJA), a volatile organic compound, is a principal flowery aromatic compound in tea. During the processing of black tea, MeJA is produced by jasmonic acid carboxyl methyltransferase (JMT) of the jasmonic acid (JA) substrate, forming a specific floral fragrance. CsJMT was cloned from tea leaves; the three-dimensional structure of CsJMT was predicted. Enzyme activity was identified, and protein purification was investigated. Site-directed deletions revealed that N-10, S-22, and Q-25 residues in the beginning amino acids played a key functional role in enzyme activity. The expression patterns of CsJMT in tea organs differed; the highest expression of CsJMT was observed in the fermentation process of black tea. These results aid in further understanding the synthesis of MeJA during black tea processing, which is crucial for improving black tea quality using specific fragrances and could be applied to the aromatic compound regulation and tea breeding improvement in further studies.

Discovering High Potent Hsp90 Inhibitors as Antinasopharyngeal Carcinoma Agents through Fragment Assembling Approach.

Hsp90 is a new promising target for cancer treatment. Many inhibitors have been discovered as therapeutic agents, and some have passed Phase I and II. However, no one is approved by FDA yet. Novel and druggable Hsp90 inhibitors are still demanding. Here, we report a new way to discover high potent Hsp90 inhibitors as antinasopharyngeal carcinoma agents through assembling fragments. With chemotyping analysis, we extract seven chemotypes from 3482 known Hsp90 inhibitors, screen 500 fragments that are compatible with the chemotypes, and confirm 15 anti-Hsp90 fragments. Click chemistry is employed to construct 172 molecules and synthesize 21 compounds among them. The best inhibitor 3d was further optimized and resulted in more potent 4f (IC50 = 0.16 µM). In vitro and in vivo experiments confirmed that 4f is a promising agent against nasopharyngeal carcinoma. This study may provide a strategy in discovering new ligands against targets without well-understood structures.

OSBP-Related Protein 5L Maintains Intracellular IP3/Ca2+ Signaling and Proliferation in T Cells by Facilitating PIP2 Hydrolysis.

Phospholipase C (PLC) isoforms play central roles in signaling cascades by cleaving PIP2 into the second messengers IP3 and DAG. In this study, to our knowledge, we uncover that ORP5L interacts physically with PLCγ1 in T cells, extracts PIP2 from the plasma membrane via its ORD domain (OSBP-related domain), presents it to PLCγ1 (enabling IP3 generation), and eventually maintains intracellular Ca2+ homeostasis. Through this mechanism, ORP5L promotes T cell proliferation in a Ca2+-activated NFAT2-dependent manner. To our knowledge, our study uncovers a new key function of ORP5L as a critical cofactor for PLCγ1 catalysis and its crucial role in human T cell proliferation.

Enantiomeric Trimethylallantoin Monomers, Dimers, and Trimethyltriuret: Evidence for an Alternative Catabolic Pathway of Caffeine in Tea Plant.

Racemic trimethylallantoin monomer (1), mesomeric and racemic trimethylallantoin dimers (2 and 3), were isolated from tea. Two pairs of optically pure enantiomers (1a, 1b and 3a, 3b) were separated by chiral column from the two racemes (1 and 3). Their structures were elucidated by a combination of extensive spectroscopic techniques, single-crystal X-ray diffraction, and experimental and calculated electronic circular dichroism. A novel caffeine catabolic pathway was proposed based on the caffeine stable isotopic tracer experiments.

Caffeine Content and Related Gene Expression: Novel Insight into Caffeine Metabolism in Camellia Plants Containing Low, Normal, and High Caffeine Concentrations.

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

ORP4L Extracts and Presents PIP2 from Plasma Membrane for PLCß3 Catalysis: Targeting It Eradicates Leukemia Stem Cells.

Leukemia stem cells (LSCs) are a rare subpopulation of abnormal hematopoietic stem cells (HSCs) that propagates leukemia and are responsible for the high frequency of relapse in therapies. Detailed insights into LSCs' survival will facilitate the identification of targets for therapeutic approaches. Here, we develop an inhibitor, LYZ-81, which targets ORP4L with high affinity and specificity and selectively eradicates LCSs in vitro and in vivo. ORP4L is expressed in LSCs but not in normal HSCs and is essential for LSC bioenergetics and survival. It extracts PIP2 from the plasma membrane and presents it to PLCß3, enabling IP3 generation and subsequent Ca2+-dependent bioenergetics. LYZ-81 binds ORP4L competitively with PIP2 and blocks PIP2 hydrolysis, resulting in defective Ca2+ signaling. The results provide evidence that LSCs can be eradicated through the inhibition of ORP4L by LYZ-81, which may serve as a starting point of drug development for the elimination of LSCs to eventually cure leukemia.

MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

The enhanced expression of miR-31 has been observed in many human malignancies including lung cancer, and this microRNA regulates several aspects of oncogenesis. However, the role of miR-31-5p in energy metabolism remains elusive. Here, we confirm that H1299 and A549 cells, 2 lung cancer cell lines, relay on aerobic glycolysis as main source of ATP. Inhibition of miR-31-5p leads to decreased glycolysis and ATP production, while miR-31-5p overexpression increases them. Hypoxia inducible factor 1 (HIF-1) up-regulates the expression of glycolytic enzymes, and the HIF-1α inhibitor (FIH) inhibits HIF-1 activity. Because FIH is a direct target of miR-31-5p, inhibition of miR-31-5p results in enhanced FIH expression and suppression of HIF-1 signaling, while overexpression of miR-31-5p has the opposite effects. Via this mechanism, miR-31-5p up-regulates aerobic glycolytic genes and maintains energy homeostasis. To further validate the mechanism of miR-31-5p in glycolysis regulation, we show that overexpression or knockdown of FIH rescued the effects of miR-31-5p or miR-31-5p inhibitor on HIF activation and its target gene expression, respectively. Finally, by means of an A549 cell xenograft mouse model, we demonstrate that the miR-31-5p promotes cell proliferation via enhancing glycolysis. In summary, this study reveals that miR-31-5p promotes the Warburg effect via direct targeting of FIH.-Zhu, B., Cao, X., Zhang, W., Pan, G., Yi, Q., Zhong, W., Yan, D. MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

Significantly increased amino acid accumulation in a novel albino branch of the tea plant (Camellia sinensis).

MAIN CONCLUSION: A normal tea plant with one albino branch was discovered. RNA sequencing, albinism phenotype and ultrastructural observations provided a valuable understanding of the albino mechanism in tea plants. Tea plants with a specific color (white or yellow) have been studied extensively. A normal tea plant (Camellia sinensis cv. quntizhong) with one albino branch was discovered in a local tea plantation in Huangshan, Anhui, China. The pure albino leaves on this special branch had accumulated a fairly high content of amino acids, especially theanine (45.31 mg/g DW), and had a low concentration of polyphenols and an extremely low chlorophyll (Chl) content compared with control leaves. Ultrastructural observation of an albino leaf revealed no chloroplasts, whereas it was viable in the control leaf. RNA sequencing and differentially expressed gene (DEG) analysis were performed on the albino leaves and on control leaves from a normal green branch. The related genes involved in theanine and polyphenol biosynthesis were also investigated in this study. DEG expression patterns in Chl biosynthesis or degradation, carotenoid biosynthesis or degradation, chloroplast development, and biosynthesis were influenced in the albino leaves. Chloroplast deletion in albino leaves had probably destroyed the balance of carbon and nitrogen metabolism, leading to a high accumulation of free amino acids and a low concentration of polyphenols in the albino leaves. The obtained results can provide insight into the mechanism underlying this special albino branch phenotype, and are a valuable contribution toward understanding the albino mechanism in tea plants.

ORP4L Facilitates Macrophage Survival via G-Protein-Coupled Signaling: ORP4L-/- Mice Display a Reduction of Atherosclerosis.

RATIONALE: Macrophage survival within the arterial wall is a central factor contributing to atherogenesis. Oxysterols, major components of oxidized low-density lipoprotein, exert cytotoxic effects on macrophages. OBJECTIVE: To determine whether oxysterol-binding protein-related protein 4 L (ORP4L), an oxysterol-binding protein, affects macrophage survival and the pathogenesis of atherosclerosis. METHODS AND RESULTS: By hiring cell biological approaches and ORP4L-/- mice, we show that ORP4L coexpresses with and forms a complex with Gαq/11 and phospholipase C (PLC)-ß3 in macrophages. ORP4L facilitates G-protein-coupled ligand-induced PLCß3 activation, IP3 production, and Ca2+ release from the endoplasmic reticulum. Through this mechanism, ORP4L sustains antiapoptotic Bcl-XL expression through Ca2+-mediated c-AMP responsive element binding protein transcriptional regulation and thus protects macrophages from apoptosis. Excessive stimulation with the oxysterol 25-hydroxycholesterol disassembles the ORP4L/Gαq/11/PLCß3 complexes, resulting in reduced PLCß3 activity, IP3 production, and Ca2+ release, as well as decreased Bcl-XL expression and increased apoptosis. Overexpression of ORP4L counteracts these oxysterol-induced defects. Mice lacking ORP4L exhibit increased apoptosis of macrophages in atherosclerotic lesions and a reduced lesion size. CONCLUSIONS: ORP4L is crucial for macrophage survival. It counteracts the cytotoxicity of oxysterols/oxidized low-density lipoprotein to protect macrophage from apoptosis, thus playing an important role in the development of atherosclerosis.

Oxysterol-binding protein-related protein 4L promotes cell proliferation by sustaining intracellular Ca2+ homeostasis in cervical carcinoma cell lines.

Oxsterol binding protein-related protein 4 (ORP4) is essential for cell proliferation, but the underlying mechanism is unclear. ORP4 is expressed as three variants, ORP4L, ORP4M and ORP4S. Here, we reported that silencing of ORP4L with specific small interfering RNA (siRNA) inhibited the proliferation of human cervical cancer cell lines C33A, HeLa and CaSki, the reverse effect being observed in ORP4L overexpressing cells. For molecular insight, we found that ORP4L maintained intracellular Ca2+ homeostasis. Through this mechanism, ORP4L activated nuclear factor of activated T cells (NFAT) activity and thus promoted expression of a gene cluster which supported cell proliferation. Of note, ORP4L sustained inositol-1,4,5-trisphosphate receptor 1 (IP3R1) expression at both mRNA and protein levels via Ca2+-dependent NFAT3 activation, which offered a mechanic explanation for the role of ORP4L intracellular Ca2+ homeostasis. Furthermore, ORP4L knockdown markedly inhibited tumor growth in a C33A cell xenograft mouse model. To conclude, our results reveal that ORP4L promotes cell proliferation through maintaining intracellular Ca2+ homeostasis.

ORP4L is essential for T-cell acute lymphoblastic leukemia cell survival.

Metabolic pathways are reprogrammed in cancer to support cell survival. Here, we report that T-cell acute lymphoblastic leukemia (T-ALL) cells are characterized by increased oxidative phosphorylation and robust ATP production. We demonstrate that ORP4L is expressed in T-ALL but not normal T-cells and its abundance is proportional to cellular ATP. ORP4L acts as an adaptor/scaffold assembling CD3ɛ, Gαq/11 and PLCß3 into a complex that activates PLCß3. PLCß3 catalyzes IP3 production in T-ALL as opposed to PLCγ1 in normal T-cells. Up-regulation of ORP4L thus results in a switch in the enzyme responsible for IP3-induced endoplasmic reticulum Ca(2+) release and oxidative phosphorylation. ORP4L knockdown results in suboptimal bioenergetics, cell death and abrogation of T-ALL engraftment in vivo. In summary, we uncovered a signalling pathway operating specifically in T-ALL cells in which ORP4L mediates G protein-coupled ligand-induced PLCß3 activation, resulting in an increase of mitochondrial respiration for cell survival. Targeting ORP4L might represent a promising approach for T-ALL treatment.

Oxysterol-binding protein-related protein 8 (ORP8) increases sensitivity of hepatocellular carcinoma cells to Fas-mediated apoptosis.

Human hepatoma (HCC) has been reported to be strongly resistant to Fas-mediated apoptosis. However, the underlying mechanisms are poorly understood. In this study the function of oxysterol-binding protein-related protein 8 (ORP8) in human hepatoma cells apoptosis was assessed. We found that ORP8 is down-regulated, whereas miR-143, which controls ORP8 expression, is up-regulated in clinical HCC tissues as compared with liver tissue from healthy subjects. ORP8 overexpression triggered apoptosis in primary HCC cells and cell lines, which coincided with a relocation of cytoplasmic Fas to the cell plasma membrane and FasL up-regulation. Co-culture of HepG2 cells or primary HCC cells with Jurkat T-cells or T-cells, respectively, provided further evidence that ORP8 increases HCC cell sensitivity to Fas-mediated apoptosis. ORP8-induced Fas translocation is p53-dependent, and FasL was induced upon ORP8 overexpression via the endoplasmic reticulum stress response. Moreover, ORP8 overexpression and miR-143 inhibition markedly inhibited tumor growth in a HepG2 cell xenograft model. These results indicate that ORP8 induces HCC cell apoptosis through the Fas/FasL pathway. The role of ORP8 in Fas translocation to the plasma membrane and its down-regulation by miR-143 offer a putative mechanistic explanation for HCC resistance to apoptosis. ORP8 may be a potential target for HCC therapy.