Functional kaurene-synthase-like diterpene synthases lacking a gamma domain are widely present in Oryza and related species.

Various diterpene synthases have been functionally identified in cultivated rice (Oryza sativa). These are the homologs of ent-copalyl diphosphate (ent-CDP) synthase and ent-kaurene synthase (KS) that are responsible for the biosynthesis of gibberellins, diterpenoid phytohormones. We isolated a cDNA encoding full-length OsKSL12, a previously uncharacterized KS like (KSL) enzyme that consists of a ß-domain and an α-domain with an active center, but lacks an N-terminal γ-domain. Functional analysis using a bacterial expression system showed that recombinant OsKSL12 converted ent-CDP into ent-manool or ent-13-epi-manool. Comparative genomics revealed that functional OsKSL12 homologs exist in diverse wild species in the Oryzeae-Oryza nivara (Oryza rufipogon), Oryza coarctata, Oryza granulata, Leersia perrieri, and Leersia tisseranti. KSL12 homologs in O. granulata, L. perrieri, and L. tisseranti preferentially reacted with geranylgeranyl diphosphate rather than ent-CDP, resulting in geranyllinalool rather than ent-manool or ent-13-epi-manool as the main product, meaning that KSL12 functionally diversified during evolution in the Oryzeae.

Identification and characterization of (+)-α-bisabolol and 7-epi-silphiperfol-5-ene synthases from Artemisia abrotanum.

Triquinane is a type of sesquiterpenoid with a unique structure that contains a fused tricyclopentane ring and exhibits a wide range of bioactivities. Like other sesquiterpenoids, the first committed step in triquinane-type sesquiterpenoid biosynthesis is the cyclization of farnesyl pyrophosphate (FPP), a common precursor of all sesquiterpenoids, catalyzed by sesquiterpene synthase. Artemisia abrotanum L. (Asteraceae), a common plant used in the culinary and cosmetics industries, has been reported to accumulate high levels of triquinane silphiperfol-5-en-3-one A. This compound is potentially biosynthesized from the cyclization of FPP into 7-epi-silphiperfol-5-ene followed by a multi-step oxidation to silphiperfol-5-en-3-one A. In this study, we aimed to identify the sesquiterpene synthase responsible for the synthesis of 7-epi-silphiperfol-5-ene. We performed RNA sequencing of A. abrotanum leaves and gene candidates were mined by homology searches using the triquinane α-isocomene synthase of chamomile (MrTPS2) as query. After gene cloning, we obtained five variants of putative sesquiterpene synthase showing greater than 85% amino acid identity to MrTPS2 and greater than 95% amino acid identity to each other. Heterologous expression of these variants in a FPP-high-producing yeast strain revealed the first four variants to be (+)-α-bisabolol synthases (AabrBOS1-4). However, the fifth candidate cyclized FPP into 7-epi-silphiperfol-5-ene and can therefore be defined as a 7-epi-silphiperfol-5-ene synthase (AabrSPS). These findings revealed the first committed enzyme involved in silphiperfol-5-en-3-one A and (+)-α-bisabolol biosyntheses in A. abrotanum. Furthermore, the results of this study will be useful for enhancing the production of these compounds for further applications.

Identification of Chimeric αßγ Diterpene Synthases Possessing both Type†II Terpene Cyclase and Prenyltransferase Activities.

Two unusual diterpene synthases composed of three domains (α, ß, and γ) were identified from fungal Penicillium species. They are the first enzymes found to possess both type II terpene cyclase (TC) and prenyltransferase (PT) activities. These enzymes were characterized by heterologous expression in Aspergillus oryzae and in vitro experiments with wild-type, mutated, and truncated enzymes. The results revealed that the α domain in the C-terminal region of these enzymes was responsible for the PT activity, whereas the ßγ domains in the N-terminal region composed the type II TC, and formed copalyl diphosphate (2). Additionally, between the α and ßγ domains, there is a characteristic linker region, in which minimal secondary structure is predicted. This linker does not exist in the characterized three-domain (αßγ) terpene synthases known as monofunctional type I or type II TCs, or bifunctional type I and type II TC enzymes. Therefore, both the catalytic activities and protein architecture substantially differentiate these new enzymes from the previously characterized terpene synthases.

Specifically and wash-free labeling of SNAP-tag fused proteins with a hybrid sensor to monitor local micro-viscosity.

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.

GOT1/AST1 expression status as a prognostic biomarker in pancreatic ductal adenocarcinoma.

Prognostication in pancreatic ductal adenocarcinoma (PDAC) remains a challenge. Recently, a link between mutated KRAS and glutamic-oxaloacetic transaminase (GOT1/AST1) has been described as part of the metabolic reprogramming in PDAC. The clinical relevance of this novel metabolic KRAS-GOT1 link has not been determined in primary human patient samples. Here we studied the GOT1 expression status as a prognostic biomarker in PDAC. We employed three independent PDAC cohorts with clinicopathological- and follow-up data: a) ICGC, comprising 57 patients with whole-exome sequencing and genome-wide expression profiling; b) ULM, composed of 122 surgically-treated patients with tissue-samples and KRAS status; c) a validation cohort of 140 primary diagnostic biopsy samples. GOT1 expression was assessed by RNA level (ICGC) or immunolabeling (ULM/validation cohort). GOT1 expression varied (ICGC) and correlation with the KRAS mutation- and expression status was imperfect (P = 0.2, ICGC; P = 0.8, ULM). Clinicopathological characteristics did not differ when patients were separated based on GOT1 high vs. low (P = 0.08-1.0); however, overall survival was longer in patients with GOT1-expressing tumors (P = 0.093, ICGC; P = 0.049, ULM). Multivariate analysis confirmed GOT1 as an independent prognostic marker (P = 0.009). Assessment in univariate (P = 0.002) and multivariate models in the validation cohort (P = 0.019), containing 66% stage IV patients, confirmed the independency of GOT1. We propose the GOT1 expression status as a simple and reliable prognostic biomarker in pancreatic ductal adenocarcinoma.

Identification of reference genes for tissue-specific gene expression in Panax notoginseng using quantitative real-time PCR.

Validated internal controls are prerequisites to accurately normalize gene expression levels. Here, 14 candidate reference genes in Panax notoginseng were characterized. Primer specificity and amplification efficiency were evaluated for each gene. Candidates were subjected to transcript quantification in the root, fibrous root, rhizome, leaf, receptacle, pedicel, and fruit tissues. Expression stability (M value) and normalization factor variation (Vn/Vn+1) were determined by geNorm. 26S-2 and ACT-2 exhibited the highest expression stability among the tissues. Gene expression of dammarenediol synthase was accurately detected after normalization to 26S-2 and ACT-2 was performed. Results were consistent when each or both of 26S-2 and ACT-2 were applied as internal control. Hence, this study provides useful information to normalize gene expression accurately in the tissue-specific transcripts of P. notoginseng.

Identification of proteomic biomarkers predicting prostate cancer aggressiveness and lethality despite biopsy-sampling error.

BACKGROUND: Key challenges of biopsy-based determination of prostate cancer aggressiveness include tumour heterogeneity, biopsy-sampling error, and variations in biopsy interpretation. The resulting uncertainty in risk assessment leads to significant overtreatment, with associated costs and morbidity. We developed a performance-based strategy to identify protein biomarkers predictive of prostate cancer aggressiveness and lethality regardless of biopsy-sampling variation. METHODS: Prostatectomy samples from a large patient cohort with long follow-up were blindly assessed by expert pathologists who identified the tissue regions with the highest and lowest Gleason grade from each patient. To simulate biopsy-sampling error, a core from a high- and a low-Gleason area from each patient sample was used to generate a 'high' and a 'low' tumour microarray, respectively. RESULTS: Using a quantitative proteomics approach, we identified from 160 candidates 12 biomarkers that predicted prostate cancer aggressiveness (surgical Gleason and TNM stage) and lethal outcome robustly in both high- and low-Gleason areas. Conversely, a previously reported lethal outcome-predictive marker signature for prostatectomy tissue was unable to perform under circumstances of maximal sampling error. CONCLUSIONS: Our results have important implications for cancer biomarker discovery in general and development of a sampling error-resistant clinical biopsy test for prediction of prostate cancer aggressiveness.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic ß cells.

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet ß-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and ß-subunits of RGGT in clonal INS 832/13 ß-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or ß-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Trypanosoma brucei solanesyl-diphosphate synthase localizes to the mitochondrion.

Polyprenyl-diphosphate synthase is a key enzyme in the biosynthesis of ubiquinone, a molecule considered essential for a typical eukaryotic cell. Its orthologue in the American stercorarian flagellate Trypanosoma cruzi, solanesyl diphosphate synthase, has been previously localized into the glycosomes. We wondered whether this unique cellular localization is shared by other trypanosome species. Using digitonin permeabilization, immunofluorescence and in situ tagging techniques, we show that in Trypanosoma brucei, the African salivarian flagellate, the enzyme localizes to the mitochondrion.

A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits.

Peppers (Capsicum spp.) display a variety of fruit colors that are reflected by the composition and amount of diverse carotenoid pigments accumulated in the pericarp. Three independent loci, c1, c2, and y, are known to determine the mature color of pepper fruits by their allelic combinations. We examined the inheritance of fruit color in recombinant inbred lines (RILs) derived from an interspecific cross between C. annuum cv. TF68 (red) and C. chinense cv. Habanero (orange). The c2 gene encodes phytoene synthase (PSY), a rate-limiting enzyme in the carotenoid biosynthesis pathway. TF68 has a dominant c2+ allele whereas Habanero is homozygous for the recessive c2 allele, which determined RIL fruit color. Here we report that the recessive c2 allele has a point mutation in the PSY gene that occurs at a splice acceptor site of the fifth intron leading to both a frame shift and premature translational termination, suggesting that impaired activity of PSY is responsible for orange fruit color. During ripening, PSY is expressed at a significantly high level in orange colored fruits compared to red ones. Interestingly, the PSY gene of red Habanero has a conserved splice acceptor dinucleotide AG. Further analysis suggests that red Habanero is a wild type revertant of the PSY mutant orange Habanero.

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts.

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.

Synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate and its use in a high-throughput fluorometric assay for Rab geranylgeranyltransferase.

Protein prenylation is one of the most common post-translational modifications affecting hundreds of eukaryotic proteins. Rab geranylgeranyl transferase prenylates exclusively the GTPases of Rab family, and inhibition of this enzyme induces apoptosis in cancer cells, making it an attractive anticancer target. To efficiently test for possible inhibitors of this enzyme, a robust high-throughput assay is required. Here, we present protocols for the synthesis of a fluorescent analogue of geranylgeranyl pyrophosphate NBD-FPP. We utilized this fluorescent probe to design a high-throughput fluorometric assay of Rab prenylation. This continuous fluorometric assay offers the advantage of being sensitive, cost-effective and amendable to miniaturization. The protocol includes the synthesis of the fluorescent substrate, setup of the assay, assay procedure and data analysis. The procedure for the Rab geranylgeranyl transferase (RabGGTase) plate assay depends on the number of compounds in the screen but generally can be performed within a day.

An assay for thiaminase I in complex biological samples.

An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, resulting in a large decrease in absorbance at 411nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples.

Dysfunction of peroxisomes in twitcher mice brain: a possible mechanism of psychosine-induced disease.

Psychosine (galactosylsphingosine) accumulates in the brain of Krabbe disease (KD) patients as well as twitcher mice, a murine model of KD, resulting in loss of oligodendrocytes and myelin. This study documents progressive loss of peroxisomal proteins/functions and induction of expression of inflammatory cytokine TNF-alpha in twitcher brain. The observed decrease in peroxisomal proteins was accompanied by decreased level of peroxisome proliferator-activated receptor-alpha (PPAR-alpha), one of the transcription factors required for expression of peroxisomal protein genes. The role of psychosine in down-regulation of PPAR-alpha activity was further supported by decreased PPAR-alpha mediated PPRE transcriptional activity in cells transfected with PPAR-alpha and PPRE reporters. The psychosine-induced down-regulation of PPAR activity and cell death was attenuated by sPLA2 inhibitor. Therefore, this study provides the first evidence of peroxisomal abnormality in a lysosomal disorder, suggesting that such dysfunction of peroxisomes may play a role in the pathogenesis of Krabbe disease.

Functional characterization of OsPPT1, which encodes p-hydroxybenzoate polyprenyltransferase involved in ubiquinone biosynthesis in Oryza sativa.

Prenylation of the aromatic intermediate p-hydroxybenzoate (PHB) is a critical step in ubiquinone (UQ) biosynthesis. The enzyme that catalyzes this prenylation reaction is p-hydroxybenzoate polyprenyltransferase (PPT), which substitutes an aromatic proton at the m-position of PHB with a prenyl chain provided by polyprenyl diphosphate synthase. The rice genome contains three PPT candidates that share significant similarity with the yeast PPT (COQ2 gene), and the rice gene showing the highest similarity to COQ2 was isolated by reverse transcription-PCR and designated OsPPT1a. The deduced amino acid sequence of OsPPT1a contained a putative mitochondrial sorting signal at the N-terminus and conserved domains for putative substrate-binding sites typical of PPT protein family members. The subcellular localization of OsPPT1a protein was shown to be mainly in mitochondria based on studies using a green fluorescent protein-PPT fusion. A yeast complementation study revealed that OsPPT1a expression successfully recovered the growth defect of the coq2 mutant. A prenyltransferase assay using recombinant protein showed that OsPPT1a accepted prenyl diphosphates of various chain lengths as prenyl donors, whereas it showed strict substrate specificity for the aromatic substrate PHB as a prenyl acceptor. The apparent K (m) values for geranyl diphosphate and PHB were 59.7 and 6.04 microM, respectively. The requirement by OsPPT1a and COQ2 for divalent cations was also studied, with Mg2+ found to produce the highest enzyme activity. Northern analysis showed that OsPPT1a mRNA was accumulated in all tissues of O. sativa. These results suggest that OsPPT1a is a functional PPT involved in UQ biosynthesis in O. sativa.

Biosynthetic potential of sesquiterpene synthases: alternative products of tobacco 5-epi-aristolochene synthase.

Nicotiana tabacum (tobacco) 5-epi-aristolochene synthase (TEAS) serves as an useful model for understanding the enzyme mechanisms of sesquiterpene biosynthesis. Despite extensive bio-chemical and structural characterization of TEAS, a more detailed analysis of the reaction product spectrum is lacking. This study reports the discovery and quantification of several alternative sesquiterpene products generated by recombinant TEAS in the single-vial GC-MS assay. The combined use of chiral and non-polar stationary phases for gas chromatography separations proved critical for resolving the numerous sesquiterpene products of TEAS for mass spectral analysis and identification. Co-injection studies with available authentic standards from both synthetic and natural sources further corroborated the assignment of several compounds, resulting in an annotated reaction mechanism accounting for their biosynthesis. Moreover, a previously undocumented farnesyl trans-cis isomerization pathway was observed.

Brushes with sage.

Wherein we learn that when working with plant terpene synthases, sometimes it is not the isotopes that get scrambled.

A single-vial analytical and quantitative gas chromatography-mass spectrometry assay for terpene synthases.

A quantitative assay for the analysis of sesquiterpene synthases, wherein each reaction mixture is formulated in glass gas chromatography vials, overlaid with organic solvent such as ethyl acetate, and subsequently vortexed to extract hydrocarbon reaction products into the organic phase after a suitable incubation period, was developed. The product-enriched organic phase is then sampled in an automated fashion and injected directly into a gas chromatograph-mass spectrometer without further workup for analysis and quantification of hydrocarbon products. Application of the vial assay to the analysis of amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, demonstrated the sensitivity of the assay for detection of major and minor reaction products and most notably for the identification of several sesquiterpene products that had escaped previous detection. A steady-state kinetic analysis of tobacco 5-epi-aristolochene synthase (TEAS), another sesquiterpene synthase, validated the quantitative nature of the assay, providing an alternative means to the established method of using radiolabeled substrate, extraction, and scintillation counting. This simplified assay provides a standardized method to facilitate analysis of terpene synthases and diverse mutant enzyme libraries by supplanting the common practice of using larger scale reactions, multiple extractions, and evaporative concentration of the organic phase prior to gas chromatography-mass spectrometry (GC-MS) analysis.

Enantiospecific (+)- and (-)-germacrene D synthases, cloned from goldenrod, reveal a functionally active variant of the universal isoprenoid-biosynthesis aspartate-rich motif.

The naturally occurring, volatile sesquiterpene hydrocarbon germacrene D has strong effects on insect behaviour and genes encoding enzymes that produce this compound are of interest in the study of plant-insect interactions and in a number of biotechnological approaches to pest control. Goldenrod, Solidago canadensis, is unusual in that it produces both enantiomers of germacrene D. Two new sesquiterpene synthase cDNAs, designated Sc11 and Sc19, have been isolated from goldenrod and functional expression in Escherichia coli identified Sc11 as (+)-germacrene D synthase and Sc19 as (-)-germacrene D synthase. Thus, the enantiomers of germacrene D are the products of separate, but closely related (85% amino-acid identity), enzymes. Unlike other sesquiterpene synthases and the related monoterpene synthases and prenyl transferases, which contain the characteristic amino-acid motif DDXX(D,E), Sc11 is unusual in that this motif occurs as (303)NDTYD. Mutagenesis of this motif to (303)DDTYD gave rise to an enzyme that fully retained (+)-germacrene D synthase activity. The converse mutation in Sc19 (D303N) resulted in a less efficient but functional enzyme. Mutagenesis of position 303 to glutamate in both enzymes resulted in loss of activity. These results indicate that the magnesium ion-binding role of the first aspartate in the DDXXD motif may not be as critical as previously thought. Further amino-acid sequence comparisons and molecular modelling of the enzyme structures revealed that very subtle changes to the active site of this family of enzymes are required to alter the reaction pathway to form, in this case, different enantiomers from the same enzyme-bound carbocationic intermediate.