Genome mining of cryptic bisabolenes that were biosynthesized by intramembrane terpene synthases from Antrodia cinnamomea.

Terpenoids represent the largest structural family of natural products (NPs) and have various applications in the pharmaceutical, food and fragrance industries. Their diverse scaffolds are generated via a multi-step cyclization cascade of linear isoprene substrates catalysed by terpene synthases (TPSs). Bisabolene NPs, which are sesquiterpenes (C15), have wide applications in medicines and biofuels and serve as bioactive substances in ecology. Despite the discovery of some canonical class I TPSs that synthesize bisabolenes from plants, bacteria and insects, it remained unknown whether any bisabolene synthases from fungi could produce bisabolenes as a main product. Antrodia cinnamomea, a Basidiomycota fungus, is a medicinal mushroom indigenous to Taiwan and a known prolific producer of bioactive terpenoids, but little is known regarding the enzymes involved in the biosynthetic pathways. Here, we applied a genome mining approach against A. cinnamomea and discovered two non-canonical UbiA-type TPSs that both synthesize (+)-(S,Z)-α-bisabolene (1). It was determined that two tailoring enzymes, a P450 monooxygenase and a methyltransferase, install a C14-methyl ester on the bisabolene scaffold. In addition, four new bisabolene derivatives, 2 and 4-6, were characterized from heterologous reconstitution in Saccharomyces cerevisiae. Our study uncovered enzymatic tools to generate structurally diverse bisabolene NPs. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Enzyme-Catalyzed Azepinoindole Formation in Clavine Alkaloid Biosynthesis.

(-)-Aurantioclavine (1), which contains a characteristic seven-membered ring fused to an indole ring, belongs to the azepinoindole class of fungal clavine alkaloids. Here we show that starting from a 4-dimethylallyl-l-tryptophan precursor, a flavin adenine dinucleotide (FAD)-binding oxidase and a catalase-like heme-containing protein are involved in the biosynthesis of 1. The function of these two enzymes was characterized by heterologous expression, in vitro characterization, and deuterium labeling experiments.

The Biosynthesis of Norsesquiterpene Aculenes Requires Three Cytochrome P450 Enzymes to Catalyze a Stepwise Demethylation Process.

Aculenes are a unique class of norsequiterpenes (C14 ) that are produced by Aspergillus aculeatus. The nordaucane skeleton in aculenes A-D may be derived from an ent-daucane precursor through demethylation, however, the enzymes involved remain unexplored. We identified the biosynthetic gene cluster and characterized the biosynthetic pathway based on gene inactivation, feeding experiments, and heterologous reconstitution in Saccharomyces cerevisiae and Aspergillus oryzae. We discovered that three cytochrome P450 monoxygenases are required to catalyze the stepwise demethylation process. AneF converts the 12-methyl group into a carboxylic acid and AneD installs the 10-hydroxy group for later tautomerization and stabilization. Finally, AneG installs an electron-withdrawing carbonyl group at the C-2 position, which triggers C-12 decarboxylation to yield the nordaucane skeleton. Additionally, a terpene cyclase (AneC) was found that forms a new product (dauca-4,7-diene).

Development of upconversion nanoparticle-conjugated indium phosphide quantum dot for matrix metalloproteinase-2 cancer transformation sensing.

Aim: Matrix metalloproteinase-2 (MMP2) plays an important role in extracellular matrix remodeling, that is, it increases significantly during cancer progression. In this regard, MMP2 monitoring is important. Experiment: A well-designed MMP2-sensitive polypeptide chain was used to link indium phosphide quantum dots (InP QDs) with upconversion nanoparticles (UCNPs) to form a nanocomposite that was utilized as biosensor. Results: We produced a biosensor that can be recognized by MMP2 and determined the presence or absence of MMP2 in cells by identifying difference in fluorescence wavelength. The InP QDs modified the arginylglycylaspartic acid molecules as targeting ligand based on chitosan. Conclusion: The MMP2-based biosensor, named UCNP-p@InP-cRGD, is sensitive and can be applied for biosensing probes.

Biosynthesis of Complex Indole Alkaloids: Elucidation of the Concise Pathway of Okaramines.

The okaramines are a class of complex indole alkaloids isolated from Penicillium and Aspergillus species. Their potent insecticidal activity arises from selectively activating glutamate-gated chloride channels (GluCls) in invertebrates, not affecting human ligand-gated anion channels. Okaramines B (1) and D (2) contain a polycyclic skeleton, including an azocine ring and an unprecedented 2-dimethyl-3-methyl-azetidine ring. Owing to their complex scaffold, okaramines have inspired many total synthesis efforts, but the enzymology of the okaramine biosynthetic pathway remains unexplored. Here, we identified and characterized the biosynthetic gene cluster (oka) of 1 and 2, then elucidated the pathway with target gene inactivation, heterologous reconstitution, and biochemical characterization. Notably, we characterized an α-ketoglutarate-dependent non-heme FeII dioxygenase that forged the azetidine ring on the okaramine skeleton.

High frequency of additional gene mutations in acute myeloid leukemia with MLL partial tandem duplication: DNMT3A mutation is associated with poor prognosis.

The mutational profiles of acute myeloid leukemia (AML) with partial tandem duplication of mixed-lineage leukemia gene (MLL-PTD) have not been comprehensively studied. We studied 19 gene mutations for 98 patients with MLL-PTD AML to determine the mutation frequency and clinical correlations. MLL-PTD was screened by reverse-transcriptase PCR and confirmed by real-time quantitative PCR. The mutational analyses were performed with PCR-based assays followed by direct sequencing. Gene mutations of signaling pathways occurred in 63.3% of patients, with FLT3-ITD (44.9%) and FLT3-TKD (13.3%) being the most frequent. 66% of patients had gene mutations involving epigenetic regulation, and DNMT3A (32.7%), IDH2 (18.4%), TET2 (18.4%), and IDH1 (10.2%) mutations were most common. Genes of transcription pathways and tumor suppressors accounted for 23.5% and 10.2% of patients. RUNX1 mutation occurred in 23.5% of patients, while none had NPM1 or double CEBPA mutation. 90.8% of MLL-PTD AML patients had at least one additional gene mutation. Of 55 MLL-PTD AML patients who received standard chemotherapy, age older than 50 years and DNMT3A mutation were associated with inferior outcome. In conclusion, gene mutations involving DNA methylation and activated signaling pathway were common co-existed gene mutations. DNMT3A mutation was a poor prognostic factor in MLL-PTD AML.

Functional interaction between Smad3 and S100A4 (metastatin-1) for TGF-beta-mediated cancer cell invasiveness.

TGF-beta (transforming growth factor-beta) induces a cytostatic response in most normal cell types. In cancer cells, however, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we show that S100A4, a metastasis-associated protein, also called metastatin-1, can physically and functionally interact with Smad3, an important mediator of TGF-beta signalling. In agreement with its known property, S100A4 binds to Smad3 in a Ca2+-dependent manner. The S100A4-binding site is located in the N-terminal region of Smad3. S100A4 can potentiate transcriptional activity of Smad3 and the related Smad2. When exogenously expressed in MCF10CA1a.cl1, an MCF10-derived breast cancer cell line, S100A4 increases TGF-beta-induced MMP-9 (matrix metalloproteinase-9) expression. On the other hand, depletion of S100A4 by siRNA (small interfering RNA) from the MDA-MB231 cell line results in attenuation of MMP-9 induction by TGF-beta. Consistent with these observations, S100A4 increases cell invasion ability induced by TGF-beta in MCF10CA1a.cl1 cells, and depletion of the protein in MDA-MB-231 cells inhibits it. Because expression of both S100A4 and TGF-beta is highly elevated in many types of malignant tumours, S100A4 and Smad3 may co-operatively increase metastatic activity of some types of cancer cells.

Pin1 promotes transforming growth factor-beta-induced migration and invasion.

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-beta responses. TGF-beta binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-beta-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-beta, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-beta-induced growth-inhibitory responses and a number of TGF-beta/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-beta-mediated migration and invasion. Accordingly, TGF-beta induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-beta-induced migration and invasion of cancer cells.

Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics.

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.