bluuebunny/crossref_metadata_2025 huggingface dataset:
Reciprocal positive regulation of serine‐threonine kinase receptor‐associated protein (STRAP) and B‐myb by direct interaction
Authors: Ha Hyunjung , Seong Hyun‐A
Published: June 2021

Serine‐threonine kinase receptor‐associated protein (STRAP) is a TGF‐β receptor interacting protein and negatively regulates TGF‐β signaling by stabilizing the physical interaction between TGF‐β receptor and Smad7. In this study we report that STRAP physically interacts with B‐myb in vivo and in vitro, and this binding is mediated by an amino‐terminal DNA‐binding domain of B‐myb and three carboxyl‐terminal WD40 repeats of STRAP. Analysis of the activities of the interacting proteins shows that STRAP enhances B‐myb transcriptional activity in a dose‐dependent manner by stimulating the nuclear translocation of B‐myb. Moreover, B‐myb exhibits an enhancement of STRAP activities, including an inhibition of TGF‐β signaling and a stimulation of p53 signaling, in a dose‐dependent manner. These results suggest a reciprocal activating interaction between STRAP and B‐myb in vivo.
Regulation of glycogen synthase kinase‐3beta by cell confluence
Authors: Chang Shiao‐Chi , Hsu Li‐Jin
Published: June 2021

Contact inhibition of cell growth is a fundamental property of normal cells to maintain tissue homeostasis. Loss of contact inhibition is associated with cellular transformation and enables cancer cells to grow in an uncontrolled fashion. E‐cadherin, a major component of the adherens junction, recruits beta‐catenin to the cell membrane and has been suggested to be responsible for the regulation of contact inhibition. E‐cadherin binding prevents beta‐catenin nuclear translocation and transactivation of genes that promote the cell cycle. Phosphorylation of beta‐catenin by casein kinase 1 and glycogen synthase kinase‐3beta (GSK‐3beta) leads to its ubiquitination and degradation. GSK‐3beta has been implicated in diverse cellular functions including cell division and apoptosis. However, it is unclear whether cell‐cell junctions regulate GSK‐3beta activity. Our data showed that the percentages of cells in S and G2/M phases of the cell cycle were increased in a variety of cells when cultured at low densities. Downregulation of GSK‐3beta activity by phosphorylation at serine 9 and dephosphorylation of its downstream target beta‐catenin were detected in non‐confluent cells, as compared to the cells in high density culture. Moreover, nuclear accumulation of GSK‐3beta was observed in non‐confluent cells. Taken together, our results suggest a role of cell‐cell junctions in regulating GSK‐3beta activity.
INVESTIGATING THE ROLE OF RORγT IN HUMAN TH17 CELLS
Authors: John Tamika Leola , Manel Nicolas , Littman Dan
Published: June 2021

CD4+ Helper T‐cells aid in coordinating the immune response by activating and directing other immune cells. For the past two decades, it has been thought that there are only two major subsets of differentiated helper T‐cells: Th1 and Th2 cells. However, a new subset of helper T‐cells was discovered recently. This new subset has been termed Th17 cells because its hallmark secreted cytokine is interleukin‐17. Previous research has shown a positive correlation between high levels of Th17 cells and incidence of auto‐immune diseases. Given Th17 cells' involvement in autoimmunity, the requirements for Th17 differentiation have come under investigation. It has been reported that the protein RORγT, a transcription factor, is required for the differentiation of Th17 cells in mice. The aim of this project was to assess whether RORγT is necessary for the differentiation of Th17 cells in humans. Short‐hairpin RNAs were used to attempt the silencing of RORγT in human T‐cells to determine its role in the production of interleukin‐17. Results suggest that RORγT is involved in human Th17 cell differentiation.

This investigation was supported, in part, by UMBC through the NIH NRSA Award GM 08663 to the MARC U*STAR Program at UMBC and by the HHMI Undergraduate Scholars Program at the University of Maryland, Baltimore County (UMBC) and the Howard Hughes Medical Institute.
Roles of DNA Topoisomerases in Transcription in HuT 78 Cells Treated with Interleukin‐2
Authors: Foglesong Paul David , Pham Minh‐Hieu , Kondapalli Navya , Contreras Leonard , Singh Thakur Aditya , Jaiswal Tripti , Kong Suk Bin
Published: June 2021

DNA topoisomerase I (Topo I) breaks and rejoins one strand of DNA, and DNA topoisomerase (Topo II) breaks and rejoins both strands of DNA. Nuclei of human cells contain two type I enzymes, Topo I and Topo IIIα, and two type II enzymes, Topo IIα and Topo IIβ. We are investigating the involvement of these enzymes in transcription in HuT 78 cells, a human T cell leukemia line that expresses CD 25, a high affinity receptor for interleukin‐2 (IL‐2). The specific activities of both Topo I and Topo II increased several fold in nuclear extracts prepared from HuT 78 cells within the first 12 hrs. after treatment with IL‐2 suggesting that both Topo I and Topo II function in transcription in activated human T cells. We are using specific monoclonal antibodies and antitumor drugs to determine which of the four human DNA topoisomerases are activated upon mitogenesis of HuT 78 by IL‐2. Evidence to date suggests that Topo I is the principal swivelase for transcription and Topo IIβ, a component of the chromosomal scaffold, is the main enzyme that breaks and rejoins DNA in order to make a loop of chromatin accessible for transcription. This work was supported by 5G11 HD052388‐02 and CA 74388.
Sequential Action of ATP on the Enhancer Binding AAA+ ATPase NtrC1
Authors: Nixon B. Tracy , Sysoeva Tatyana A. , Chowdhury Saikat , Chen Baoyu , Guo Liang
Published: June 2021

Structural changes in the catalytic cycle of AAA+ ATPases mediate contact between these remodeling proteins and macromolecular targets. These molecular motors fulfill biologically important roles in all kingdoms of life, defects in them being associated with many human diseases. Despite broad interest in these molecular machines, there is still a critical gap in our knowledge of how they perform mechanical work. Our static and time resolved SAS experiments define such changes in σ54‐dependent AAA+ ATPases. These activators remodel σ54 in bacterial RNA polymerase to initiate transcription, as for example, in Borrelia burgdorferi to promote Lyme's disease. New studies of the NtrC1 protein of Aquifex aeolicus show that the interaction between the 7 ATP binding sites of NtrC1 ATPase and ATP is complex. SAXS data titrating the ATP analog ADP‐BeFx and time resolved SAXS data for binding in the presence of saturating analog provide direct evidence for two phases of binding. Initially, binding to a subset of sites stablizes the ring by compacting it (Rg reduces from 47 Åto 42Å); subsequent binding then stabilizes an extended loop form with scattering signatures located at 0.08 to 0.14 Å‐1. We hypothesize that only the latter state is competent to bind σ54.
Nascent peptide‐dependent ribosome stalling in drug‐inducible antibiotic resistance
Authors: Ramu Haripriya , Subramanian Sai Lakshmi , Vazquez‐Laslop Nora , Mankin Alexander S.
Published: June 2021

Ribosome‐nascent peptide interactions are involved in regulation of expression of several bacterial genes. One such example is the induction of an antibiotic resistance gene, ermC (erythromycin resistance methyltransferase). Expression of ermC is controlled by ribosome stalling at a short upstream ORF (uORF) and depends on the sequence of the nascent peptide and the presence of erythromycin. A number of other genes that confer resistance to ribosome‐targeting antibiotics are inducible, but in most cases, the underlying mechanisms are unknown. A comprehensive sequence analysis of the upstream regions of these genes showed the presence of uORFs some of which have not been identified previously. Sequences of the encoded peptides show remarkable diversity. Toe‐printing experiments in a cell‐free transcription‐translation system revealed erythromycin‐dependent ribosome stalling at a variety of these leader ORFs. Alanine scanning mutagenesis is carried out to identify the critical sequences of nascent peptides required for drug‐dependent ribosome stalling. These data are expected to provide significant insight into the molecular mechanisms of ribosome ‐ nascent peptide interactions.
Evidence for a conformational change in thimet oligopeptidase and the machinery behind it
Authors: Scognamillo Lori A , Sigman Jeffrey A. , Glucksman Marc J. , Pabon Amanda , Wolfson Adele J.
Published: June 2021

The metalloenzyme thimet oligopeptidase (TOP) has been shown to cleave numerous structurally distinct physiological peptides all under 18 amino acids in length. Our previous data shows that conformational changes in the enzyme influences its substrate specificity. The data, supported by homology modeling of TOP to a substrate‐bound form of DcP carboxypeptidase, indicates that a hinge‐bend movement in TOP may be crucial to enzymatic activity and could provide a mechanism for TOP's size restriction. Using the homology model, we have identified several charged residues on the enzyme surface that line the entrance to the substrate‐binding crevice. We propose that these residues stabilize the closed form of the enzyme via ionic interactions. Quenched fluorescence‐based activity assays performed with the single and double mutants in which the charge on opposite sides of the cleft are reversed showed that these amino acids were crucial in maintaining enzymatic activity. Furthermore, chemical denaturation and fluorescence anisotropy reveal that the ability of the enzyme to form a closed structure may have been compromised. The data lends further support to the importance of hinge‐bend movements in TOP and supports a role for surface charges in stabilizing the closed form of the enzyme.
Peroxiredoxin VI in Complex With a Transition Inhibitor
Authors: Wilson Ryan Scott , Khanal Akhil , Bahnson Brian
Published: June 2021

Peroxiredoxin VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs. Its antioxidant properties are due to its ability to reduce hydroperoxides found in lung surfactants and thus prevent the toxicity associated with hyperoxia. Prdx6 is a bifunctional protein that contains two distinct active sites. One active site catalyzes a phospholipase A2 (PLA2) type hydrolysis of phospholipids, and the second active site catalyzes the reduction of lipid hydroperoxides typical of 1‐cys peroxiredoxins. The structure of Prdx6 has been solved; however, there is no structural evidence to elucidate its catalytic mechanism. This study is designed to solve the structure of Prdx6 in complex with MJ33, a PLA2 transition‐state inhibitor, in an effort to understand the mechanism and conformational change Prdx6 undergoes during its catalytic cycle. Here we report that Prdx6 has been successfully purified to homogeneity. Our crystallization screens have produced protein crystals that have diffracted to a resolution of 2.8Å. This research is supported by the Chemistry Alumni Scholars.
Metabolomics of Genotype by Diet Interactions underlying Metabolic Syndrome in Drosophila
Authors: Reed Laura Katie , Gibson Greg
Published: June 2021

The underlying genetic architecture and environmental contribution to Metabolic Syndrome (MetS) is just beginning to be described, so we are using genomic approaches to dissect natural genetic variation for a similar condition in Drosophila. Larvae from 150 near isogenic lines were raised on high fat and high sugar diets and the traits of weight gain, blood sugar concentration, lipid storage, and survival were measured. We find that genotype explains 30% of the phenotypic variation, while diet only explains less than 8% percent of the variation, and the interaction between diet and genotype is responsible for at much as 24% of the total variation for these metabolic traits. We also find greater variation in the physiological traits in flies raised on the high fat diet, indicating that perturbation of an evolutionarily stable trait may be contributing to the prevalence of MetS. Metabolomic profiling using GC‐MS on the most extreme lines allowed us to characterize over 70 metabolites that show significant variation with genotype, diet, or their interaction. PCA of the metabolomic profiles indicates strong correlations between metabolite levels, diet, and weight, as well as phenotypic perturbation on a high fat diet. We argue that dietary and lifestyle shifts may lead to disease by destabilizing normal buffering of physiological processes.
Spontaneously Hypertensive Rat (SHR) Display Alterations in Carbohydrate Metabolism And Function At An Early Age
Authors: Lauzier Benjamin , Labarthe Francois , Bouchard Bertrand , McDuff Janie , Gélinas Roselle , Foisy Sylvain , Charron Guy , Des Rosiers Christine
Published: June 2021

The SHR, a genetic model of hypertension with a CD36 gene defect, develops left ventricular hypertrophy (LVH) between 9 and 12 weeks of age. We reported that 15‐wk‐old SHR display a reduced exogenous fatty acid (FA) oxidation, while that of carbohydrate (CHO) was unchanged despite an enhanced glycolysis. This study aimed at clarifying whether these alterations are characteristic of this rat strain or result from metabolic gene remodeling linked to LVH. We assessed the metabolic and functional profile of working hearts perfused with 13C‐labeled substrates at 7 weeks (SHR vs Wistar), and cardiac metabolic gene expression by qPCR at 7 and 15 weeks. As young Wistar hearts were not able to maintain stable cardiac output at 80 mmHg, they were perfused at 70 mmHg afterload to match functions with SHR. At the metabolic level, SHR hearts displayed a 40% lower FA β‐oxidation, while CHO oxidation was increased by 50% (p<0.05). qPCR data revealed that SHR hearts showed marginal differences in mRNA levels for genes involved in FA and CHO metabolism at 7 weeks, but a significant increase in all these gene transcripts at 15 weeks. Compared to their 15‐week‐old counterpart, young SHR hearts displayed a similar lower FA oxidation, but an increase in CHO oxidation. The latter difference appears to be explained by transcriptional changes in PDK4 linked to LVH, and may confer to young SHR hearts the capacity to withstand a higher workload.

(Supported by the Fondation Bettencourt Schueller and CIHR).
Muscle Adaptation in Response to Voluntary Exercise in Phosphorylase Kinase Deficient Mice
Authors: Mefford Ashley M , Rice Nancy A.
Published: June 2021

Phosphorylase kinase (PhK), the key regulator of glycogenolysis, is responsible for maintaining blood glucose levels thus providing energy to sustain muscle contraction. A deficiency of PhK is the cause of glycogen storage disease (GSD) type IX in humans. This study investigates the physiological and genetic adaptations that occur in a mouse model of GSD (I/LnJ) in response to voluntary exercise. Juvenile (4‐6 weeks old) I/LnJ and wild‐type C57/Bl6 mice exercised voluntarily for 1, 2 or 5 weeks. Exercise data was calculated as mean daily running time, daily running distance, and average speed. At week 5, I/LnJ mice ran approximately 7.1 km/day, whereas C57/Bl6 mice ran twice as long, 14.9 km/day. The average running speed for I/LnJ mice, 32.1 m/min, was significantly lower than C57/Bl6 mice whose average speed was 38.4 m/min. While a significant training effect was observed in both mouse strains during the 5 week exercise period, no significant difference was observed in heart/body weight ratios in exercised mice compared to non‐exercised controls. Changes in gene expression of glucose transporter 4 (GLUT4), pyruvate dehydrogenase (PDHA1), and phosphofructokinase (PFKM) as a result of exercise have been determined by quantitative RT‐PCR in both I/LnJ and C57/Bl6 mice and indicate genetic factors correlate with the observed physiological changes. It is our long‐term goal to determine dietary and exercise therapies that can be translated to humans patients with GSD. This work was supported by the National Institute of Health grant 2PORR16481 (NAR) and a WKU Faculty Scholarship (NAR).
Characterization of essential fatty acid metabolism using the stable isotope tracer technique and gas chromatography/ mass spectrometric assay
Authors: Lin Yu Hong , Song Byoung‐Joon , Llanos Adolfo R , Lefkowitz William , Uauy Ricardo , Salem Norman
Published: June 2021

To better understand essential fatty acid metabolism, several in vivo and in vitro studies have been performed. Diet controlled rats were orally administrated a mixture of two or more labeled fatty acids: d5‐linoleate, ‐linolenate, ‐dihomo‐gamma‐linolenate (DGLA), and/or 13C‐U‐ linoleate or eicosapentaenoate. GC/MS NCI assay was employed to measure the isotopic precursors, their respective metabolites in rat organs over a time‐course from 4 h up to 25 d. Results and conclusions. 1. Only 2.6% and 6% of linoleate and linolenate, respectively, were elongated and desaturated in rats with about 78% being utilized for beta‐oxidation, excretion, or other loss and 16~18% was stored in tissues, mainly adipose, as the precursor itself. 2. Eicosapentaenoate was a more efficient substrate for docosahexaenoate (DHA) than linolenate; similarly, DGLA is a better substrate for arachidonate and n‐6 docosapentanoate than was linoleate. 3. Dietary DHA is the primary source of brain DHA. 4. Preformed DHA markedly decreased the accumulation of de novo biosynthesized DHA and n‐6 docosapentaenoate in liver, plasma, heart and brain; but no significant differences in rat hepatocytes delta‐5 or ‐6 desaturase activities have been observed. Decreased accumulation of newly‐synthesized DHA by dietary DHA may relate to increased DHA catabolism.

This project was funded by the DICBR, NIAAA, NIH.
Effects of high fat diet on multiple indices of oxidative stress in the brain
Authors: Liu Ying , Zhang Le , Fernandez‐Kim Sun OK , Morrison Christopher , Bruce‐Keller Annadora , Keller Jeffrey N.
Published: June 2021

Consumption of a high fat diet is known to have deleterious effects on multiple organ systems, although the effects of high fat diet consumption on the brain remains poorly defined. Oxidative stress is believed to contribute to the aging of multiple tissues, including the brain, although currently it remains unclear whether consumption of a high fat diet is capable of altering age‐related changes in oxidative stress within the brain. In the present study we report on the effects of high fat diet consumption on multiple indices of oxidative stress in the brain including the levels of protein carbonyls, HNE‐modified proteins, nitrosylated proteins, and glycated proteins. In addition we report on the effects of a high fat diet on the expression of nuclear factor erythroid 2‐related factor 2 (Nrf2), which is a critical regulator of antioxidant enzymes in the brain. Additionally, we report on the ability of aging to modulate the effects of high fat diet consumption on the brain. Taken together, these data have important implications for understanding how diet may alter the development of oxidative stress and aging within the brain.

Supported by grants from the Chinese Ministry of Education, the Chinese Scholarship Council, National Institute of Aging, and the Hibernia National Bank/Edward G. Schlieder Chair
A large number of protein expression changes occur early in life and precede phenotype onset in different animal models for Huntington's disease
Authors: Zabel Claus , Mao Lei , Woodman Ben , Klaere Yvonne , Nebrich Grit , Klein Oliver , Hartl Daniela , Klose Joachim , Bates Gillian P.
Published: June 2021

Huntington's disease (HD) is fatal in humans within 15 to 20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large 2‐D gel/mass spectrometry based proteomics approach to investigate HD‐induced protein expression alterations and their kinetics at very early stages and during the course of disease. Different HD animal models were investigated at absence, early, intermediate and late stage HD. Preliminary results showed that in at least two animal models HD‐stage‐specific protein changes (above 70%) as well as a drastic alteration in protein expression already occurred before symptoms appeared. Early changes included in both cases the dys‐regulation of proteins involved in glycolysis/gluconeogenesis and the actin‐cytoskeleton formation. This suggests a period of highly variable protein expression which precedes the onset of an HD phenotype. More animal models will be investigated to further characterize protein alteration kinetics and their role in HD. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.
Hypothermia impedes calcium induced mitochondrial permeability transition pore opening in mitochondria harvested after cold ischemia of isolated hearts
Authors: Haumann Johan , Aldakkak Mohammed , Heisner James S , Stowe David F , Camara Amadou KS
Published: June 2021

Hypothermia decreases ischemia‐reperfusion (IR) injury. IR injury has been associated with mitochondrial permeability transition pore (PTP) opening, and decreased activity of the electron transport chain. We studied mitochondria from isolated hearts subjected to normothermic and hypothermic ischemia. Guinea pig isolated hearts were perfused at constant pressure with Krebs‐Ringer's solution at 37 ºC and subjected to 30 min of ischemia at 37ºC or 17ºC. After 5 min of reperfusion mitochondria were isolated. Mitochondrial [Ca2+]m, pH, membrane potential (ΔΨm) and NADH were measured spectrophotometrically with indo 1, BCECF, rhodamine 123 and autofluorescence. After energizing with pyruvic acid, [CaCl2] (0‐100 μM) and ADP were added. Adding CaCl2 caused a dose dependent [Ca2+]m increase. Ca2+ induced PTP opening, assessed by collapse of ΔΨm, and prevented by cyclosporine A, occurred after adding 50 μM CaCl2 after 37ºC IR, and adding 100 μM CaCl2 after 17ºC IR. ADP decreased ΔΨm, pH and NADH and increased [Ca2+]m in all mitochondria. These bioenergetic markers recovered after 17ºC IR, but not after 37ºC IR. Our study shows that hypothermia protects mitochondria against IR injury as shown by resistance to Ca2+ induced PTP opening and appropriate responses to ADP. This study emphasizes the importance of mitochondrial function in protecting against IR injury, and gives insight into how hypothermia reduces IR injury.
Changed expression profile of complement regulatory proteins (Cregs) on leukocytes after severe injury in humans
Authors: Amara Umme , Kalbitz Miriam , Acker Barbara , Perl Mario , Weiss Manfred , Huber‐Lang Markus
Published: June 2021

Introduction

Complement as crucial element of the innate immune system becomes uncontrolled and excessively activated after severe injury and sepsis. However, protection against complement‐mediated tissue destruction is provided by a variety of soluble and membrane‐bound complement regulatory proteins (Cregs). So far, the cellular expression profile of the Cregs in patients with the multiple injuries is not clear.

Material and Methods

The expression profiles of the most relevant Cregs on leukocytes (neutrophils, monocytes and lymphocytes) were investigated using flow cytometric analysis obtained from either age‐ and sex‐matched healthy volunteers (n=16) or polytraumatized patients (n=12; injury severity score ISS =18) at admission in the emergency room and 4, 12, 24, 120 and 240 h after trauma.

Results

In current study the expression profiles of Cregs on leukocytes were found to be significantly altered, reflected by specific changes in CD35 (complement receptor 1, CR1), CD46 (membrane co‐factor protein, MCP), CD55 (decay accelerating factor, DAF) and CD59 (MAC inhibitor). Some of them demonstrated a cell type‐dependent (neutrophils, monocytes and lymphocytes) different expression pattern, whereas some of them exhibited a comparable cellular expression profile during the trauma course, indicating different regulation pathways. Furthermore, CD59 and CD46 neutrophil expression correlated with injury severity.

Conclusion

The data suggest that Cregs expression profiles on leukocytes from polytraumatized humans are specifically changed after trauma, indicative of a trauma‐induced "complementopathy".
A 13C isotopomer model for NMR and GC‐MS analysis
Authors: Li Wei , Bian Fang , Chaudhuri Priyanajana , Yu Xin
Published: June 2021

13C NMR spectroscopy and GC‐MS, in combination with stable isotope tracer molecules, offer a unique opportunity in studying metabolic regulation. Accurate quantification of fluxes through different metabolic pathways requires comprehensive analysis of isotopomer distribution. In this study, a mathematical model that describes 13C isotopomer distribution in TCA cycle intermediates was developed and applied to the analysis of NMR and GC‐MS data for quantification of substrate utilization and anaplerosis. Rat hearts were perfused with buffer containing 5.5 mM glucose and 0.6 mM [U‐13C] palmitate. 13C NMR and GC‐MS of tissue extracts were performed for positional isotopomer distribution (PID) of glutamate and mass isotopomer distribution (MID) of key TCA cycle intermediates. Relative palmitate utilization (VFA) and relative anaplerosis (y) were determined by fitting the model to PID or MID. The two methods yielded similar VFA: 0.76±0.09, 0.76±0.09. y determined from MID fitting was higher than from PID fitting: 0.13±0.03 vs 0.08±0.02. Compare to model simulated MID using parameters from PID fitting, GC‐MS measured MID showed a large fraction of M0 isotopmer: 18±4% vs 9±4%. Our results suggest that NMR and GC‐MS are similar in determining substrate utilization. However, the cause of discrepancy in determining anaplerosis needs further investigation.This work is supported by NIH R0173315 and R01HL086935.
Crystal structures of the GCaMP calcium sensor protein reveal the mechanism of fluorescence signal change and aid rational design
Authors: Schreiter Eric Robert , Akerboom Jasper , Vélez Rivera Jonathan D. , Rodríguez Guilbe María M. , Alfaro Malavé Elisa C. , Hernandez Hector H. , Tian Lin , Hires S. Andrew , Marvin Jonathan S. , Looger Loren L.
Published: June 2021

The genetically encoded calcium indicator (GECI) protein GCaMP2 shows promise for neural network activity imaging, but is currently limited by low signal‐to‐noise ratio. We describe X‐ray crystal structures of GCaMP2 in the calcium‐free dark state, and in two calcium‐bound bright states: a monomeric form that dominates at intracellular concentrations observed during imaging experiments and an unexpected domain‐swapped dimer with decreased fluorescence. Based on these structures, we engineered a series of GCaMP2 point mutants and characterized one mutant with significantly improved signal‐to‐noise. The mutation is located at a domain interface and its effect on sensor function could not have been predicted in the absence of structural data.
Inhibition of cellular lipid biosynthesis by sulfated oxysterol is mediated via the LXR pathway
Authors: MA YONGJIE , Xu Leyuan , Pandak William M , Hylemon Phillip , Ren Shunlin
Published: June 2021

Nuclear oxysterol receptor, Liver X receptor (LXR) is a key transcriptional regulator of liver lipid metabolism. We recently identified the oxysterol 25‐diol 3‐sulfate (25HC3S) as an important regulatory molecule in liver. We now have studied the effects of 25HC3S and its precursor, 25‐hydroxycholesterol (25HC) on the LXR/SREBP‐1 signaling pathway in microphages. Addition of 25HC3S to human macrophages markedly decreased nuclear LXR protein levels. Real time PCR and western blot results showed 25HC3S inhibited SREBP‐1 mRNA and protein mature form.

25HC3S decreased the expression of SREBP‐1‐responsive genes including acetyl CoA carboxylase‐1 and fatty acid synthase, as well as HMGR and LDLR, which are key proteins involved in lipids metabolism.Subsequently, Oil‐Red O staining and TLC found 25HC3S inhibited intracellular lipids accumulation.25HC as a LXR ligand had opposite effects, increasing ABCA1, ABCG1, SREBP‐1, and FAS mRNA levels in the macrophages. In the presence of 25HC3S, 25HC and LXR agonist T0901317 failed to increase LXR targeting gene expression. We conclude that 25HC3S acts in macrophage as cholesterol satiety signal, down‐regulating cholesterol and fatty acid biosynthetic pathways via inhibition of LXR/SREBP signaling. These studies suggest that 25HC3S is a potent regulator of lipid metabolism in macrophages.
The glycerol 3‐phosphate acyltransferases Gat1p and Gat2p are microsomal phosphoproteins with differential contributions to polarized cell growth
Authors: Zaremberg Vanina , Alvarez Ana , Burrowes David , Bratschi Martin W , Cheung Jing , Kulaga Adam , Kearley Jennifer
Published: June 2021

Glycerol‐3‐phosphate acyltransferase (GPAT) catalyses the initial commited step in the synthesis of all glycerolipids. It is a redundant step in yeast, mammals and plants. S. cerevisiae possesses two GPATs, encoded by GAT1 and GAT2. Metabolic analysis of yeast lacking either GAT1 or GAT2 indicated partitioning of the two main branches of phospholipid synthesis at the initial and rate limiting GPAT step. We are interested in identifying molecular determinants mediating lipid metabolic pathway partitioning, thus as a start point we have performed a detailed study of Gat1p and Gat2p cellular localization. We have compared Gat1p and Gat2p localization by fluorescence microscopy and subcellular fractionation using equilibrium density gradients. Our results indicate i) Gat1p and Gat2p mostly overlap in their localization and are in fact microsomal GPATs, and ii) there is a differential enrichment of Gat1p and Gat2p in distinct ER fractions. Overexpression of these enzymes in the absence of endogenous GPATs, induces proliferation of distinct ER arrays, differentially affecting cortical ER morphology and polarized cell growth. In addition, our studies also uncovered a dynamic post‐translational regulation of Gat1p and Gat2p, and a compensation mechanism through phosphorylation that responds to cellular GPAT imbalance. This work has been supported by a grant from NSERC.
Phosphatidylcholine transfer protein (PC‐TP) regulates thermogenesis by limiting mitochondrial fatty acid oxidation in brown fat
Authors: Kang Hye Won , Ribich Scott , Kim Brian W. , Hagen Susan J. , Bianco Antonio C. , Cohen David E.
Published: June 2021

Mice with homozygous disruption of the gene encoding PC‐TP (Pctp−/−) exhibit a marked shift towards utilization of fatty acids for oxidative phosphorylation. Based on the observation in vitro that it stimulates the acyl‐CoA thioesterase activity of thioesterase superfamily member 2 (Them2), we have proposed that PC‐TP may regulate entry of fatty acyl‐CoAs into mitochondria for oxidation. To explore this hypothesis, we examined the function and structure of brown fat using Pctp−/− mice. Consistent with increased thermogenesis, Pctp−/− mice exhibited increased core body temperatures at room temperature. During a cold challenge, these mice defended core body temperature, but with reduced upregulation of thermogenic genes compared to wild type controls. Also in the setting of reduced thermogenic gene expression, oxygen consumption rates in cultured brown adipocytes lacking PC‐TP were hyper‐responsive to norepinephrine. Electron microscopy revealed mitochondrial changes suggestive of increased fatty acyl‐CoA uptake, as well as increased turnover of brown adipocytes. Collectively, these findings support a key role for PC‐TP in regulating thermogenesis by limiting mitochondrial oxidation of fatty acids. Supported by the NIH.
Genome‐wide Mapping of SREBP‐1 Distribution on Mouse liver‐chromatin in Refeeding Condition by using ChIP‐seq Microarray
Authors: Seo Young‐Kyo , Chong Hansook Kim , Osborne Timothy F.
Published: June 2021

Lipid homeostasis in vertebrates is regulated by a family of transcription factors designated sterol regulatory element binding proteins (SREBPs) implicated in diabetes and other diet‐related disorders. In order to identify the distribution of the SREBP‐1 when nutritional conditions are changed, mice were subjected to a fasting/refeeding protocol. This leads to a high level of SREBP‐1 protein in the liver which we crosslinked to chromatin prepared from livers of the refed mice. The SREBP‐1‐DNA complexes were isolated and subjected to ChIP‐seq procedure. With 6.7 million sequence reads from the anti‐SREBP‐1 chromatin, we used bioinformatics tools to finally identify 427 peaks of SREBP‐1 enrichment versus IgG. We found that SREBP‐1 occupied not only many well characterized SREBP‐1 target genes previously identified but several others putative target genes that are involved in diverse biological pathways, including novel targets with roles in lipid and carbohydrate metabolism. Using the sequences from the peaks, we also identified a preferred SREBP‐1 DNA‐binding motif that was present in 30% of the identified binding sites. This motif ACTACANNTCCC closely matches the consensus SRE site proposed by our lab previously. Our report should be useful for understanding to maintain metabolic homeostasis in response to shifting nutritional fluctuations.
Transgenic Overexpression of AlphaBeta1 Integrin Stimulates p70S6K Phosphorylation in Mice with a Severe Form of Muscular Dystrophy
Authors: Boppart Marni Della , Kaufman Stephen J
Published: June 2021

MCK‐driven transgenic expression of the alpha7 integrin can ameliorate pathology in a mouse model of Duchenne muscular dystrophy (mdx/utrn‐/‐) and thus compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the alpha7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remain to be established.

PURPOSE

To determine a role for signaling in the amelioration of muscular dystrophy by alpha7 integrin.

METHODS

Five wk wild type, mdx/utrn‐/‐, and alpha7BX2‐mdx/utrn‐/‐ gastrocnemius muscles (n=3‐8/group) were dissected and extracted. Activation of PI3K, ILK, AKT, mTOR, p70S6K, GSK‐3, and p38 was measured using in vitro activity assays or phosphospecific antibodies and western blotting.

RESULTS

Significant increases in ILK activity (2.0‐fold), AKT‐(P) (2.3‐fold), mTOR‐(P) (57%), p70S6K‐(P) (11.7‐fold), and ERK‐(P) (66%) were observed in dystrophic mdx/utrn‐/‐ muscle compared to wild type. Significant decreases in GSK‐3‐(P) (57%) and p38‐(P)(2.9‐fold) were also observed. Most of these signaling events were similar in dystrophic mice over‐expressing the alpha7 integrin. However, a further increase in p70S6K‐(P) (18‐fold) and decrease in GSK‐3‐(P)(3.7‐fold) were detected in alpha7BX2‐mdx/utrn‐/‐ compared to wild type mice and these changes were significant compared to mdx/utrn‐/‐ mice.

CONCLUSION

The alpha7beta1 integrin confers a protective effect in dystrophic mice and increased p70S6K activity appears important to this. Supported by the NIH and MDA.
Adenosine A3 receptor suppresses prostate cancer metastasis by inhibiting NADPH oxidase activity
Authors: Jajoo Sarvesh , Mukherjea Debashree , Watabe Kounosuke , Ramkumar Vickram
Published: June 2021

Prostate cancer metastasis is the major cause of mortality resulting from a lack of effective treatment. Studies have shown that the adenosine A3 receptor (A3AR) suppresses proliferation of variety of tumors. However, their role in prostate tumor metastases is still not known. In the present study, we determined the effect of A3AR agonist N6‐(3‐iodobenzyl) adenosine‐5′‐N‐methyluronamide (IB‐MECA) on prostate cancer cells invasion in vitro and metastasis in vivo. In severe combined immunodeficient (SCID) mice, IB‐MECA inhibited the metastases of AT6.1 rat prostate cancer cells. In vitro, IB‐MECA inhibited AT6.1 cells invasion by reducing high basal levels of reactive oxygen species (ROS) generation via NADPH oxidase. Furthermore, inhibition of Rac1 and p47phox subunits of this enzyme by the dominant negative RacN17 or short interfering (si)RNA against p47phox, reduced ROS generation and invasion of AT6.1 cells and inhibited the expression of p47phox and Rac1 subunits. The inhibition of NADPH oxidase involves reduction in p47phox translocation to the plasma membrane as a result of inhibition of extracellular signal‐regulated kinase (ERK)1/2‐dependent pathway. These results suggest that activation of A3AR in prostate cancer cells reduces ERK1/2‐dependent NADPH oxidase activity, which could account for reduced cancer cell invasiveness. This study was supported by a grant from SIU School of Medicine.
Src kinase expression, phosphorylation and activation in human and bovine left ventricles
Authors: Goldman Todd L , Du Yanmei , Buttrick Peter M , Walker Lori A
Published: June 2021

Several biochemical signaling pathways contribute to heart failure. Using protein microarrays, we previously identified proteins differentially expressed in human and bovine models of heart failure. One protein upregulated in failing left ventricles (LVs) was CSK (C‐terminal Src kinase), a tyrosine kinase that negatively regulates c‐Src. It has been suggested that Src is important in Ang‐II‐induced signal transduction; that myocardial ischemia may activate Src; and that Src signaling is involved in cardiac hypertrophy. Therefore, we examined the role Src plays in failing LVs. Studies showed that when compared to controls, Src was markedly upregulated in failing LVs, and that another smaller protein product immunologically bound Src antibody. This doublet was isolated using 2‐D gels, and sent to Mass spec for identification. To measure Src phosphorylation, we used phosphospecific antibodies to the Src tyrosine 527 site (negatively regulates Src) and tyrosine 416 site (upregulates Src). Data showed failing samples were not phosphorylated at the Tyr 527 site compared to control. Finally, using a Src kinase activity assay we showed greater Src activity in failing compared to control LVs. These data show there are significant differences in expression, activation and phosphorylation of Src in failing LVs and suggest that Src may be a nodal upstream regulator of progressive LV dysfunction. NIH grant #RO1HL077195
Inflammatory stress and incipient apoptosis in primary venous and aortic endothelial cells (ECs) of type 1 diabetic mice
Authors: Bucciarelli Loredana G. , Pollreisz Andreas , Kebschull Moritz , Ganda Anjali , Kalea Anastasia Z. , Hudson Barry I. , Zou Yu Shan , Lalla Evanthia , Ramasamy Ravichandran , Colombo Paolo C. , Schmidt Ann Marie , Yan Shi Fang
Published: June 2021

The progression of diabetic vasculopathy is associated with profound endothelial dysfunction. We tested the hypothesis that markers of cellular stress would be detectable in ECs retrieved from both arteries and veins of type 1 diabetic mice.

We directly isolated aortic and venous ECs from type 1 diabetic mice (streptozotocin) and age‐matched C57BL/6 controls. EC purity was supported by comparing levels of smooth muscle and leukocyte markers (amplified mRNA) between whole aorta (present) and aortic ECs (negligible).

Gene expression profiling was performed using Mouse Endothelial Cell Biology RT² Profiler(tm) PCR Array and confirmed by real‐time PCR. At 18 weeks of age, Interleukin‐1b was 80x higher in aortic ECs and 15x higher in venous ECs of diabetic mice vs. controls (p=0.03, p=0.01, respectively). At 36 weeks of age, Caspase‐1 was 369x higher in aortic ECs and 618x higher in venous ECs of diabetic mice vs. controls (p=0.04, p<0.05, respectively). By western blot, trends towards higher levels of Cleaved Caspase‐3 protein were seen in aortic and venous ECs retrieved from diabetic mice vs. control mice (p=0.05).

Given new minimally‐invasive techniques to harvest human venous ECs to track disease activity and gauge response to treatment, these results are clinically relevant to studies of human subjects with diabetes.

Funded by United States Public Health Service hl60901 and JDRF.
Neuroprotectin D1 Modulates Bcl‐xL Phosphorylation during Oxidative Stress in Retinal Pigment Epithelial Cells
Authors: Antony Rajee , Bazan Nicolas
Published: June 2021

Age‐related macular degeneration (AMD) is the leading cause of "central vision" loss in people 60 years of age or older. In AMD there is progressive degeneration of retinal pigment epithelium (RPE) and the photoreceptors in the macular area of the retina. Docosahexaenoic acid, the precursor of neuroprotectin D1 (NPD1), is concentrated in photoreceptor outer segments. Our laboratory has reported that NPD1 inhibits oxidative‐stress‐mediated proinflammatory gene induction and apoptosis, and consequently promotes RPE cell survival. Recent studies have shown that oxidative stress, leading to apoptosis, neovascularization and lipid peroxidation, participates in AMD pathogenesis.

Apoptosis is mediated by a well‐orchestrated interaction between the anti‐apoptotic and pro‐apoptotic members of the Bcl‐2 family. Bcl‐xL is one such anti‐apoptotic protein involved in this intracellular signal trans‐duction. Bcl‐xL ‐/‐ mice are prone to early death, succumbing to a massive loss of neurons in the brain. In vitro studies have shown that the neuronal yield was increased in cultures of mouse embryonic stem cells when Bcl‐xL was over‐expressed. In this study, we describe the regulation of Bcl‐xL through mitogen activated protein kinase pathway. We also show that NPD1 significantly downregulated the activation of phos‐phorylated Bcl‐xL when induced with oxidative stress. The long term goal of this project is to better understand mechanisms of neuroprotection mediated by NPD1.

NIH/NEI Grant RO1 EY05121.
Roles of DNA‐PKcs and TORC1 in CpG‐ODN‐ induced cell survival in response to vincristine
Authors: Cao Cong , Dargoi Ana Maria , Li Gloria , Su Bing , Wan Yinsheng , Chu Wen‐Ming
Published: June 2021

CpG‐ODN induces cell survival, but the mechanism of its action remains elusive. Here we show that CpG‐ODN activates the TORC1 pathway in a DNA‐PKcs‐ and Akt‐ dependent manner. Loss of DNA‐PKcs impairs phosphorylation of Akt473S, mTOR2448S, and TORC1 effectors 4E‐BP1(65S), S6K(389T) and S6(235/236S) in response to CpG‐ODN, whereas lack of TLR9, MyD88 or IRAK4 only has a negligible effect in this phosphorylation process. Ablation of both Akt1 and Akt2 or inhibition of Akt abrogates TORC1 activation. As a consequence, deletion of DNA‐PKcs and inhibition of Akt and TORC1 results in failure of CpG‐ODN to dissociate 4E‐BP1 from eIF4E. Immunoprecipitation experimental results also show that DNA‐PKcs pre‐associates with mTOR, and CpG‐ODN stimulation enhances this association. Finally, DNA‐PKcs deficiency and TORC1 inhibition lead to a defect in CpG‐ODN‐ induced protection of cells against the anti‐leukemia agent vincristine. Overall, our findings indicate that the TORC1 pathway is downstream of the CpG‐ODN/DNA‐PKcs/Akt axis and its activation contributes to the CpG‐ODN‐ induced anti‐apoptotic function.
Cholesterol Secoaldehyde Induces Apoptosis in H9c2 Cardiomyocytes through Reactive Oxygen Species Involving Mitochondrial and Death Receptor Pathways
Authors: Raghavamenon Achuthan C , Sathishkumar Kunjureddiar , Gao Xueli , Parinandi Narasimham L , Pryor William A , Uppu Rao M
Published: June 2021

Cholesterol secoaldehyde (ChSeco) has been shown to induce apoptosis in H9c2 cardiomyocytes. The present study investigated the involvement of apoptotic‐related proteins and gene expression using RT‐PCR, western blot, and biochemical assays. ChSeco activated the expression of genes involved in the extrinsic pathway. The significance of this pathway was also evident from the increased activity of caspase‐8. The over‐expression of Apaf‐1, loss of mitochondrial transmembrane potential, release of cytochrome c, and increased activity of caspase‐9, provided further evidence for the involvement of intrinsic pathway. Western blot analysis showed a decrease in the phosphorylated‐Bcl2 without any change in the level of Bcl2 or Bax. Time‐course analysis showed an upstream increase in the generation of ROS and the associated decrease in the cellular glutathione. N‐Acetyl‐L‐cysteine and Trolox significantly attenuated the ROS formation and cytotoxicity and also down‐regulated the expression of genes of all the players of either pathway. This study clearly demonstrated that ChSeco induces apoptosis in H9c2 cells through the ROS generation and activation of both the intrinsic and extrinsic pathways.
Oxidative stress response and trafficking of platelet‐activating factor acetylhydrolase type II (PAFAH‐II)
Authors: Fuzaylova Anastasia G. , Sedlack Elizabeth M. , Winget Jason M. , Bahnson Brian J.
Published: June 2021

Platelet activating factor acetylhydrolase type II (PAFAH‐II) is a member of the phospholipase A2 subfamily that can hydrolyze and inactivate platelet activating factor and oxidatively fragmented phospholipids at the sn‐2 position. This enzyme is a 44kDa, intracellular, myristoylated protein that becomes associated with the inner membranes under oxidative stress, thus maintaining cell membrane integrity. In order to elucidate the precise mechanism of the stress response and trafficking of this protein, the role of the myristoyl tail was investigated. Trafficking of myristoylated, wild type PAFAH‐II, and of a mutant (Gly‐2‐Ala) that cannot be myristoylated, was monitored in live cells using fluorescent protein fusion tags. Confocal microscopy indicates that unmyristoylated PAFAH‐II remains cytoplasmic under both stressed and unstressed conditions. In contrast, myristoylated PAFAH‐II, which is only partially localized to the inner membranes prior to stress, becomes more concentrated in the inner membranes upon stress. Therefore, this study shows that the myristoyl tail plays a key role in this oxidative stress response. The possible roles of additional regulatory mechanisms, such as residue modifications, which may be facilitating the exposure of the myristoyl tail for membrane anchoring, are also the focus of our current investigations.

Support: COBRE 2P20RR015588 and 1R01HL084366
Metabolism and Biological Activity of Sulindac and its Epimers
Authors: Brunell David , Marchetti Maria , Gamliel Edna , Sagher Daphna , Brot Nathan , Weissbach Herbert , Binninger David
Published: June 2021

Sulindac can be oxidized to sulindac sulfone and reduced to sulindac sulfide, an active cyclooxygenase (COX) inhibitor. However, the metabolism and biological activities of the individual R and S epimers are not known. In vivo, the R epimer yielded higher plasma levels of the sulfone, whereas the S epimer was more readily reduced to the sulfide. In a hepatoma cell line, both epimers activated the aryl hydrocarbon receptor and significantly induced cytochrome P450 enzymes. The R epimer was oxidized to sulindac sulfone more readily than the S epimer. A rat liver cell extract reduced both the R and S epimers of sulindac with either DTT or thioredoxin. The S epimer is primarily reduced by methionine sulfoxide reductase A (MsrA). The enzyme(s) responsible for reduction of the R epimer has been partially purified.

Sulindac enhances killing of human colon, skin, and lung cancer cells by oxidative stress. The effect does not involve COX inhibition or the Msr system. An increase in reactive oxygen species (ROS) and loss of mitochondrial membrane potential suggest that cell death is due to apoptosis. Both epimers were effective under the conditions used. In contrast, no enhanced killing was observed using either epimer with normal cells under the same conditions. In summary it appears that the R and S epimers of sulindac are metabolized by different pathways and that these epimers may have different pharmacological properties.