The synthesis and application of a diazirine-modified uridine analogue for investigating RNA-protein interactions

The roles played by many ncRNAs remain largely unknown. Similarly, relatively little is known about the RNA binding proteins involved in processing ncRNA. Identification of new RNA/RNA binding protein (RBP) interactions may pave the way to gain a better understanding of the complex events occurring within cells during gene expression and ncRNA biogenesis. The development of chemical tools for the isolation of RBPs is of paramount importance. In this context, we report on the synthesis of the uridine phosphoramidite U Dz that bears a diazirine moiety on the nucleobase. RNA probes containing U Dz units were irradiated in the presence of single-stranded DNA binding protein (SSB), which is also known to bind ssRNAs, and shown to efficiently (15% yield) and selectively cross-link to the protein. The corresponding diazirine-modified uridine triphosphate U DzTP was synthesized and its capacity to act as a substrate for the T7 RNA polymerase was tested in transcription assays. U DzTP was accepted with a maximum yield of 38% for a 26mer RNA containing a single incorporation and 28% yield for triple consecutive incorporations. Thus, this uridine analogue represents a convenient biochemical tool for the identification of RNA binding proteins and unraveling the role and function played by ncRNAs.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

RNA editing in kinetoplastids

RNA editing in kinetoplastid protozoa is a post-transcriptional process of uridine insertion or deletion in mitochondrial mRNAs. The process involves two RNA species, the pre-edited mRNA and in most cases a trans-acting guide RNA (gRNA). Sequences within gRNAs define the position and extend of mRNA editing. Both mRNAs and gRNAs are encoded by mitochondrial genes in the kinetoplast DNA (kDNA), which consists of thousands of small circular DNA molecules, called minicircles, encoding thousands of gRNAs, catenated together and with a few mRNA encoding larger circles, the maxicircles, to form a huge DNA network. Editing has been shown to result in translatable mRNAs of bona fide mitochondrial genes as well as novel alternatively edited transcripts that are involved in the maintenance of the kDNA itself. RNA editing occurs within large protein-RNA complexes, editosomes, containing gRNA, preedited and partially edited mRNAs and also structural and catalytically active proteins. Editosomes are diverse in both RNA and protein composition and undergoe structural remodeling during the maturation. The compositional and structural diversity of editosomes further underscores the complexity of the RNA editing process.

Hit proteins, mitochondria and cancer

The histidine triad (HIT) superfamily comprises proteins that share the histidine triad motif, His-ϕ-His-ϕ-His-ϕ-ϕ, where ϕ is a hydrophobic amino acid. HIT proteins are ubiquitous in prokaryotes and eukaryotes. HIT proteins bind nucleotides and exert dinucleotidyl hydrolase, nucleotidylyl transferase or phosphoramidate hydrolase enzymatic activity. In humans, 5 families of HIT proteins are recognized. The accumulated epidemiological and experimental evidence indicates that two branches of the superfamily, the HINT (Histidine Triad Nucleotide Binding) members and FHIT (Fragile Histidine Triad), have tumor suppressor properties but a conclusive physiological role can still not be assigned to these proteins. Aprataxin forms another discrete branch of the HIT superfamily, is implicated in DNA repair mechanisms and unlike the HINT and FHIT members, a defective protein can be conclusively linked to a disease, ataxia with oculomotor apraxia type 1. The scavenger mRNA decapping enzyme, DcpS, forms a fourth branch of the HIT superfamily. Finally, the GalT enzymes, which exert specific nucleoside monophosphate transferase activity, form a fifth branch that is not implicated in tumorigenesis. The molecular mechanisms by which the HINT and FHIT proteins participate in bioenergetics of cancer are just beginning to be unraveled. Their purported actions as tumor suppressors are highlighted in this review.

Radiation metabolomics. 4. UPLC-ESI-QTOFMS-Based metabolomics for urinary biomarker discovery in gamma-irradiated rats

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2'-deoxyuridine, 2'deoxyxanthosine, N(1)-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2'-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2'Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma

BACKGROUND ; AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

Analysis of lorazepam and its 30-glucuronide in human urine by capillary electrophoresis: evidence for the formation of two distinct diastereoisomeric glucuronides

Lorazepam (LOR) is a 3-hydroxy-1,4-benzodiazepine that is chiral and undergoes enantiomerization at room temperature. In humans, about 75% of the administered dose of LOR is excreted in the urine as its 30-glucuronide. CE-MS with negative ESI was used to confirm the presence of LOR-30-glucuronide in urines that stemmed from a healthy individual who ingested 1 or 2 mg LOR, whereas free LOR could be detected in extracts prepared from enzymatically hydrolyzed urines. As the 30-glucuronidation reaction occurs at the chiral center of the molecule, two diastereoisomers can theoretically be formed, molecules that can no longer interconvert. The stereoselective formation of LOR glucuronides in humans and in vitro was investigated. MEKC analysis of extracts of the nonhydrolyzed urines suggested the presence of the two different LOR glucuronides in the urine. The formation of the same two diastereoisomers was also observed in vitro employing incubations of LOR with human liver microsomes in the presence of uridine 5'-diphospho-glucuronic acid as coenzyme. The absence of other coenzymes excluded the formation of phase I or other phase II metabolites of LOR. Both results revealed a stereoselectivity, one diastereoisomer being formed in a higher amount than the other. After enzymatic hydrolysis using beta-glucuronidase, these peaks could not be detected any more. Instead, LOR was monitored. Analysis of the extracts prepared from enzymatically hydrolyzed urines by MEKC in the presence of 2-hydroxypropyl-beta-CD revealed the enantiomerization process of LOR (observation of two peaks of equal magnitude connected with a plateau zone). The data presented provide for the first time the evidence of the stereoselectivity of the LOR glucuronidation in humans.

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A)

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.

UPLC-ESI-TOFMS-based metabolomics and gene expression dynamics inspector self-organizing metabolomic maps as tools for understanding the cellular response to ionizing radiation

Global transcriptomic and proteomic profiling platforms have yielded important insights into the complex response to ionizing radiation (IR). Nonetheless, little is known about the ways in which small cellular metabolite concentrations change in response to IR. Here, a metabolomics approach using ultraperformance liquid chromatography coupled with electrospray time-of-flight mass spectrometry was used to profile, over time, the hydrophilic metabolome of TK6 cells exposed to IR doses ranging from 0.5 to 8.0 Gy. Multivariate data analysis of the positive ions revealed dose- and time-dependent clustering of the irradiated cells and identified certain constituents of the water-soluble metabolome as being significantly depleted as early as 1 h after IR. Tandem mass spectrometry was used to confirm metabolite identity. Many of the depleted metabolites are associated with oxidative stress and DNA repair pathways. Included are reduced glutathione, adenosine monophosphate, nicotinamide adenine dinucleotide, and spermine. Similar measurements were performed with a transformed fibroblast cell line, BJ, and it was found that a subset of the identified TK6 metabolites were effective in IR dose discrimination. The GEDI (Gene Expression Dynamics Inspector) algorithm, which is based on self-organizing maps, was used to visualize dynamic global changes in the TK6 metabolome that resulted from IR. It revealed dose-dependent clustering of ions sharing the same trends in concentration change across radiation doses. "Radiation metabolomics," the application of metabolomic analysis to the field of radiobiology, promises to increase our understanding of cellular responses to stressors such as radiation.

Pharmacogenetics-based population pharmacokinetic analysis of etravirine in HIV-1 infected individuals

Objectives: Etravirine (ETV) is metabolized by cytochrome P450 (CYP) 3A, 2C9, and 2C19. Metabolites are glucuronidated by uridine diphosphate glucuronosyltransferases (UGT). To identify the potential impact of genetic and non-genetic factors involved in ETV metabolism, we carried out a two-step pharmacogenetics-based population pharmacokinetic study in HIV-1 infected individuals. Materials and methods: The study population included 144 individuals contributing 289 ETV plasma concentrations and four individuals contributing 23 ETV plasma concentrations collected in a rich sampling design. Genetic variants [n=125 single-nucleotide polymorphisms (SNPs)] in 34 genes with a predicted role in ETV metabolism were selected. A first step population pharmacokinetic model included non-genetic and known genetic factors (seven SNPs in CYP2C, one SNP in CYP3A5) as covariates. Post-hoc individual ETV clearance (CL) was used in a second (discovery) step, in which the effect of the remaining 98 SNPs in CYP3A, P450 cytochrome oxidoreductase (POR), nuclear receptor genes, and UGTs was investigated. Results: A one-compartment model with zero-order absorption best characterized ETV pharmacokinetics. The average ETV CL was 41 (l/h) (CV 51.1%), the volume of distribution was 1325 l, and the mean absorption time was 1.2 h. The administration of darunavir/ritonavir or tenofovir was the only non-genetic covariate influencing ETV CL significantly, resulting in a 40% [95% confidence interval (CI): 13–69%] and a 42% (95% CI: 17–68%) increase in ETV CL, respectively. Carriers of rs4244285 (CYP2C19*2) had 23% (8–38%) lower ETV CL. Co-administered antiretroviral agents and genetic factors explained 16% of the variance in ETV concentrations. None of the SNPs in the discovery step influenced ETV CL. Conclusion: ETV concentrations are highly variable, and co-administered antiretroviral agents and genetic factors explained only a modest part of the interindividual variability in ETV elimination. Opposing effects of interacting drugs effectively abrogate genetic influences on ETV CL, and vice-versa.

Reactivity of hexanuclear ruthenium metallaprisms towards nucleotides and a DNA decamer

The reactivity of three hexacationic arene ruthenium metallaprisms towards isolated nucleotides and a short DNA strand was investigated using NMR spectroscopy, ESI mass spectrometry, UV/Vis and circular dichroism spectroscopy. The metallaprism built from oxalato-bridging ligands reacts rapidly in the presence of deoxyguanosine monophosphate (dGMP) and deoxyadenosine monophosphate, while the benzoquinonato derivative only reacts with dGMP. On the other hand, the larger metallaprism incorporating naphtoquinonato bridges remains stable in the presence of nucleotides. The reactivity of the three hexacationic metallaprisms with the decameric oligonucleotide d(CGCGATCGCG)2 was also investigated. Analysis of the NMR, MS, UV/Vis and CD data suggests that no adducts are formed between the oligonucleotide and the metallaprisms, but electrostatic interactions, leading to partial unwinding of the double-stranded oligonucleotide, were evidenced

Influence of Gilbert's syndrome on the formation of ethyl glucuronide.

A drinking experiment with participants suffering from Gilbert's syndrome was performed to study the possible influence of this glucuronidation disorder on the formation of ethyl glucuronide (EtG). Gilbert's syndrome is a rather common and, in most cases, asymptomatic congenital metabolic aberration with a prevalence of about 5 %. It is characterized by a reduction of the enzyme activity of the uridine diphosphate glucuronosyltransferase (UGT) isoform 1A1 up to 80 %. One of the glucuronidation products is EtG, which is formed in the organism following exposure to ethanol. EtG is used as a short-term marker for ethyl alcohol consumption to prove abstinence in various settings. After 2 days of abstinence from ethanol and giving a void urine sample, 30 study participants drank 0.1 L of sparkling wine (9 g ethanol). 3, 6, 12, and 24 h after drinking, urine samples were collected. 3 hours after drinking, an additional blood sample was taken, in which liver enzyme activities, ethanol, hematological parameters, and bilirubin were measured. EtG and ethyl sulfate (EtS), another short-term marker of ethanol consumption, were determined in the urine samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS); creatinine was measured photometrically. In all participants, EtG and EtS were detected in concentrations showing a wide range (EtG: 3 h sample 0.5-18.43 mg/L and 6 h sample 0.67-13.8 mg/L; EtS: 3 h sample 0.87-6.87 mg/L and 6 h sample 0.29-4.48 mg/L). No evidence of impaired EtG formation was found. Thus, EtG seems to be a suitable marker for ethanol consumption even in individuals with Gilbert's syndrome.

Caffeine affects the biological responses of human hematopoietic cells of myeloid lineage via downregulation of the mTOR pathway and xanthine oxidase activity.

Correction of human myeloid cell function is crucial for the prevention of inflammatory and allergic reactions as well as leukaemia progression. Caffeine, a naturally occurring food component, is known to display anti-inflammatory effects which have previously been ascribed largely to its inhibitory actions on phosphodiesterase. However, more recent studies suggest an additional role in affecting the activity of the mammalian target of rapamycin (mTOR), a master regulator of myeloid cell translational pathways, although detailed molecular events underlying its mode of action have not been elucidated. Here, we report the cellular uptake of caffeine, without metabolisation, by healthy and malignant hematopoietic myeloid cells including monocytes, basophils and primary acute myeloid leukaemia mononuclear blasts. Unmodified caffeine downregulated mTOR signalling, which affected glycolysis and the release of pro-inflammatory/pro-angiogenic cytokines as well as other inflammatory mediators. In monocytes, the effects of caffeine were potentiated by its ability to inhibit xanthine oxidase, an enzyme which plays a central role in human purine catabolism by generating uric acid. In basophils, caffeine also increased intracellular cyclic adenosine monophosphate (cAMP) levels which further enhanced its inhibitory action on mTOR. These results demonstrate an important mode of pharmacological action of caffeine with potentially wide-ranging therapeutic impact for treating non-infectious disorders of the human immune system, where it could be applied directly to inflammatory cells.

The impact of ABCC11 polymorphisms on the risk of early-onset fluoropyrimidine toxicity

A missense variant (c.1637C>T, T546M) in ABCC11 encoding the MRP8 (multidrug resistance protein 8), a transporter of 5-fluorodeoxyuridine monophosphate, has been associated with an increased risk of 5-fluorouracil-related severe leukopenia. To validate this association, we investigated the impact of the ABCC11 variants c.1637C>T, c.538G>A and c.395+1087C>T on the risk of early-onset fluoropyrimidine-related toxicity in 514 cancer patients. The ABCC11 variant c.1637C>T was strongly associated with severe leukopenia in patients carrying risk variants in DPYD, encoding the key fluoropyrimidine-metabolizing enzyme dihydropyrimidine dehydrogenase (odds ratio (OR): 71.0; 95% confidence interval (CI): 2.5-2004.8; Pc.1637C>T*DPYD=0.013). In contrast, in patients without DPYD risk variants, no association with leukopenia (OR: 0.95; 95% CI: 0.34-2.6) or overall fluoropyrimidine-related toxicity (OR: 1.02; 95% CI: 0.5-2.1) was observed. Our study thus suggests that c.1637C>T affects fluoropyrimidine toxicity to leukocytes particularly in patients with high drug exposure, for example, because of reduced fluoropyrimidine catabolism.

Regulation of the cardiac Na+ channel NaV1.5 by post-translational modifications.

The cardiac voltage-gated Na(+) channel, Na(V)1.5, is responsible for the upstroke of the action potential in cardiomyocytes and for efficient propagation of the electrical impulse in the myocardium. Even subtle alterations of Na(V)1.5 function, as caused by mutations in its gene SCN5A, may lead to many different arrhythmic phenotypes in carrier patients. In addition, acquired malfunctions of Na(V)1.5 that are secondary to cardiac disorders such as heart failure and cardiomyopathies, may also play significant roles in arrhythmogenesis. While it is clear that the regulation of Na(V)1.5 protein expression and function tightly depends on genetic mechanisms, recent studies have demonstrated that Na(V)1.5 is the target of various post-translational modifications that are pivotal not only in physiological conditions, but also in disease. In this review, we examine the recent literature demonstrating glycosylation, phosphorylation by Protein Kinases A and C, Ca(2+)/Calmodulin-dependent protein Kinase II, Phosphatidylinositol 3-Kinase, Serum- and Glucocorticoid-inducible Kinases, Fyn and Adenosine Monophosphate-activated Protein Kinase, methylation, acetylation, redox modifications, and ubiquitylation of Na(V)1.5. Modern and sensitive mass spectrometry approaches, applied directly to channel proteins that were purified from native cardiac tissues, have enabled the determination of the precise location of post-translational modification sites, thus providing essential information for understanding the mechanistic details of these regulations. The current challenge is first, to understand the roles of these modifications on the expression and the function of Na(V)1.5, and second, to further identify other chemical modifications. It is postulated that the diversity of phenotypes observed with Na(V)1.5-dependent disorders may partially arise from the complex post-translational modifications of channel protein components.