Calphostin C-induced apoptosis is mediated by a tissue transglutaminase-dependent mechanism involving the DLK/JNK signaling pathway

A role for tissue transglutaminase (TG2) and its substrate dual leucine zipper-bearing kinase (DLK), an upstream component of the c-Jun N-terminal kinase (JNK) signaling pathway, has been previously suggested in the apoptotic response induced by calphostin C. In the current study, we directly tested this hypothesis by examining via pharmacological and RNA-interference approaches whether inhibition of expression or activity of TG2, DLK and JNK in mouse NIH 3T3 fibroblasts and human MDA-MB-231 breast cancer epithelial cells affects calphostin C-induced apoptosis. Our experiments with the selective JNK inhibitor SP600125 reveal that calphostin C is capable of causing JNK activation and JNK-dependent apoptosis in both cell lines. Small interfering RNA-mediated depletion of TG2 alone strongly reduces calphostin C action on JNK activity and apoptosis. Consistent with an active role for DLK in this cascade of event, cells deficient in DLK demonstrate a substantial delay of JNK activation and poly-ADP-ribose polymerase (PARP) cleavage in response to calphostin C, whereas overexpression of a recombinant DLK resistant to silencing, but sensitive to TG2-mediated oligomerization, reverses this effect. Importantly, combined depletion of TG2 and DLK further alters calphostin C effects on JNK activity, Bax translocation, caspase-3 activation, PARP cleavage and cell viability, demonstrating an obligatory role for TG2 and DLK in calphostin C-induced apoptosis.     

Regulatory network analyses reveal genome-wide potentiation of LIF signaling by glucocorticoids and define an innate cell defense response

While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.     

Variations in Body Composition and Plasma Lipids in Response to a High‐Carbohydrate Diet

Objective: To examine the extent to which variations in body composition modulate changes in the lipid profile in response to the ad libitum consumption of a diet rich in carbohydrates (CHOs) (high‐CHO diet: 58% of energy as CHOs) or high in fat and in monounsaturated fatty acids (MUFAs) (high‐MUFA diet: 40% of energy as fat, 23% as MUFAs). Research Methods and Procedures: Sixty‐three men were randomly assigned to one of the two diets that they consumed for 6 to 7 weeks. Body composition and fasting plasma lipid levels were measured at the beginning and the end of the dietary intervention. Results: The high‐CHO and high‐MUFA diets induced significant and comparable reductions in body weight and waist circumference. These changes were accompanied by significant and comparable (p < 0.01) reductions in total plasma cholesterol and low‐density lipoprotein cholesterol levels. However, the high‐MUFA diet had more beneficial effects on plasma triglyceride concentrations (p < 0.01) and on plasma high‐density lipoprotein cholesterol levels (p = 0.02) compared with the high‐CHO diet. Diet‐induced changes in waist circumference were significantly associated with changes in low‐density lipoprotein cholesterol levels in the high‐CHO group (r = 0.39, p = 0.03) but not in the high‐MUFA group (r = 0.16, p = 0.38). Discussion: Improvements in plasma lipids induced by the ad libitum consumption of a high‐CHO diet seem to be partly mediated by changes in body weight, whereas lipid changes induced by the high‐MUFA diet seem to be independent of changes in body weight.     

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.     

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells

Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂^●-) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂^●- level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂^●- level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂^●-production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂^●- levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases.     

Randomized Controlled Ferret Study to Assess the Direct Impact of 2008–09 Trivalent Inactivated Influenza Vaccine on A(H1N1)pdm09 Disease Risk

During spring-summer 2009, several observational studies from Canada showed increased risk of medically-attended, laboratory-confirmed A(H1N1)pdm09 illness among prior recipients of 2008–09 trivalent inactivated influenza vaccine (TIV). Explanatory hypotheses included direct and indirect vaccine effects. In a randomized placebo-controlled ferret study, we tested whether prior receipt of 2008–09 TIV may have directly influenced A(H1N1)pdm09 illness. Thirty-two ferrets (16/group) received 0.5 mL intra-muscular injections of the Canadian-manufactured, commercially-available, non-adjuvanted, split 2008–09 Fluviral or PBS placebo on days 0 and 28. On day 49 all animals were challenged (Ch0) with A(H1N1)pdm09. Four ferrets per group were randomly selected for sacrifice at day 5 post-challenge (Ch+5) and the rest followed until Ch+14. Sera were tested for antibody to vaccine antigens and A(H1N1)pdm09 by hemagglutination inhibition (HI), microneutralization (MN), nucleoprotein-based ELISA and HA1-based microarray assays. Clinical characteristics and nasal virus titers were recorded pre-challenge then post-challenge until sacrifice when lung virus titers, cytokines and inflammatory scores were determined. Baseline characteristics were similar between the two groups of influenza-naïve animals. Antibody rise to vaccine antigens was evident by ELISA and HA1-based microarray but not by HI or MN assays; virus challenge raised antibody to A(H1N1)pdm09 by all assays in both groups. Beginning at Ch+2, vaccinated animals experienced greater loss of appetite and weight than placebo animals, reaching the greatest between-group difference in weight loss relative to baseline at Ch+5 (7.4% vs. 5.2%; p = 0.01). At Ch+5 vaccinated animals had higher lung virus titers (log-mean 4.96 vs. 4.23pfu/mL, respectively; p = 0.01), lung inflammatory scores (5.8 vs. 2.1, respectively; p = 0.051) and cytokine levels (p>0.05). At Ch+14, both groups had recovered. Findings in influenza-naïve, systematically-infected ferrets may not replicate the human experience. While they cannot be considered conclusive to explain human observations, these ferret findings are consistent with direct, adverse effect of prior 2008–09 TIV receipt on A(H1N1)pdm09 illness. As such, they warrant further in-depth investigation and search for possible mechanistic explanations.     

The snoGloBe interaction predictor reveals a broad spectrum of C/D snoRNA RNA targets

Box C/D small nucleolar RNAs (snoRNAs) are a conserved class of RNA known for their role in guiding ribosomal RNA 2′-O-ribose methylation. Recently, C/D snoRNAs were also implicated in regulating the expression of non-ribosomal genes through different modes of binding. Large scale RNA–RNA interaction datasets detect many snoRNAs binding messenger RNA, but are limited by specific experimental conditions. To enable a more comprehensive study of C/D snoRNA interactions, we created snoGloBe, a human C/D snoRNA interaction predictor based on a gradient boosting classifier. SnoGloBe considers the target type, position and sequence of the interactions, enabling it to outperform existing predictors. Interestingly, for specific snoRNAs, snoGloBe identifies strong enrichment of interactions near gene expression regulatory elements including splice sites. Abundance and splicing of predicted targets were altered upon the knockdown of their associated snoRNA. Strikingly, the predicted snoRNA interactions often overlap with the binding sites of functionally related RNA binding proteins, reinforcing their role in gene expression regulation. SnoGloBe is also an excellent tool for discovering viral RNA targets, as shown by its capacity to identify snoRNAs targeting the heavily methylated SARS-CoV-2 RNA. Overall, snoGloBe is capable of identifying experimentally validated binding sites and predicting novel sites with shared regulatory function.