Metabolome analysis of biosynthetic mutants reveals a diversity of metabolic changes and allows identification of a large number of new compounds in Arabidopsis

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.     

Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.     

Parthenolide attenuates LPS-induced fever, circulating cytokines and markers of brain inflammation in rats

Parthenolide, a sesquiterpene lactone, has been reported to exhibit a variety of anti-inflammatory and immunomodulatory effects. To test the effect of parthenolide on brain inflammatory responses, brain oxidative stress and fever, we treated rats with parthenolide (1 mg/kg), simultaneously or 1 h prior to a systemic (i.p.) challenge with a moderate dose (100 μg/kg) of lipopolysaccharide (LPS). The initial hypothermia was exaggerated; the second phase of the biphasic LPS-induced fever and circulating interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) were significantly attenuated only in parthenolide-pretreated animals. In the hypothalamus, markers of NFκB/NF-IL6 pathway activation (inhibitor κBα, NF-IL6 and the serin/threonin kinase-like protein mRNA expression) and markers of oxidative stress (including nuclear respiratory factor 1) and NFκB immunoreactivity were significantly reduced while NF-IL6 immunoreactivity and suppressor of cytokine signaling 3 mRNA expression remained unaltered, 8 h after LPS-stimulation with parthenolide-pretreatment. Importantly, this response was accompanied by decreased mRNA expression of the rate limiting enzyme in prostaglandin synthesis, cyclooxygenase 2 (COX2), known for its critical role in fever induction pathways. A direct action of parthenolide on brain cells was also confirmed in a primary neuro-glial cell culture of the vascular organ of the lamina terminalis a pivotal brain structure for fever manifestation with a leaky blood-brain barrier. In summary, pretreatment with parthenolide attenuates the febrile response during LPS-induced systemic inflammation by reducing circulating IL-6 and TNFα and decreasing hypothalamic NFκB/NF-IL6 activation, oxidative stress and expression of COX2. Thus parthenolide appears to have the potential to reduce brain inflammation.     

A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Fast identification of folded human protein domains expressed in <Emphasis Type="Italic">E. coli</Emphasis> suitable for structural analysis

Background  

High-throughput protein structure analysis of individual protein domains requires analysis of large numbers of expression clones to identify suitable constructs for structure determination. For this purpose, methods need to be implemented for fast and reliable screening of the expressed proteins as early as possible in the overall process from cloning to structure determination.     

Mechanistic studies on N-acetylmuramic acid 6-phosphate hydrolase (MurQ): an etherase involved in peptidoglycan recycling

Peptidoglycan recycling is a process in which bacteria import cell wall degradation products and incorporate them back into either peptidoglycan biosynthesis or basic metabolic pathways. The enzyme MurQ is an N-acetylmuramic acid 6-phosphate (MurNAc 6-phosphate) hydrolase (or etherase) that hydrolyzes the lactyl side chain from MurNAc 6-phosphate and generates GlcNAc 6-phosphate. This study supports a mechanism involving the syn elimination of lactate to give an alpha,beta-unsaturated aldehyde with (E)-stereochemistry, followed by the syn addition of water to give product. The observation of both a kinetic isotope effect slowing the reaction of [2-(2)H]MurNAc 6-phosphate and the incorporation of solvent-derived deuterium into C2 of the product indicates that the C2-H bond is cleaved during catalysis. The observation that the solvent-derived (18)O isotope is incorporated into the C3 position of the product, but not the C1 position, provides evidence of the cleavage of the C3-O bond and argues against imine formation. The finding that 3-chloro-3-deoxy-GlcNAc 6-phosphate serves as an alternate substrate is also consistent with an elimination-addition mechanism. Upon extended incubations of MurQ with GlcNAc 6-phosphate, the alpha,beta-unsaturated aldehydic intermediate accumulates in solution, and (1)H NMR analysis indicates it exists as the ring-closed form of the (E)-alkene. A structural model is developed for the Escherichia coli MurQ and is compared to that of the structural homologue glucosamine-6-phosphate synthase. Putative active site acid/base residues are probed by mutagenesis, and Glu83 and Glu114 are found to be crucial for catalysis. The Glu83Ala mutant is essentially inactive as an etherase yet is capable of exchanging the C2 proton of substrate with solvent-derived deuterium. This suggests that Glu83 may function as the acidic residue that protonates the departing lactate.