Epsilon Isoforms Research Articles

Stabilization of 14-3-3 protein-protein interactions with Fusicoccin-A decreases alpha-synuclein dependent cell-autonomous death in neuronal and mouse models

Synucleinopathies are a group of neurodegenerative diseases without effective treatment characterized by the abnormal aggregation of alpha-synuclein (aSyn) protein. Changes in levels or in the amino acid sequence of aSyn (by duplication/triplication of the aSyn gene or point mutations in the encoding region) cause familial cases of synucleinopathies. However, the specific molecular mechanisms of aSyn-dependent toxicity remain unclear. Increased aSyn protein levels or pathological mutations may favor abnormal protein-protein interactions (PPIs) that could either promote neuronal death or belong to a coping response program against neurotoxicity. Therefore, the identification and modulation of aSyn-dependent PPIs can provide new therapeutic targets for these diseases. To identify aSyn-dependent PPIs we performed a proximity biotinylation assay based on the promiscuous biotinylase BioID2. When expressed as a fusion protein, BioID2 biotinylates by proximity stable and transient interacting partners, allowing their identification by streptavidin affinity purification and mass spectrometry. The aSyn interactome was analyzed using BioID2-tagged wild-type (WT) and pathological mutant E46K aSyn versions in HEK293 cells. We found the 14-3-3 epsilon isoform as a common protein interactor for WT and E46K aSyn. 14‐3-3 epsilon correlates with aSyn protein levels in brain regions of a transgenic mouse model overexpressing WT human aSyn. Using a neuronal model in which aSyn cell-autonomous toxicity is quantitatively scored by longitudinal survival analysis, we found that stabilization of 14‐3-3 protein-proteins interactions with Fusicoccin-A (FC-A) decreases aSyn-dependent toxicity. Furthermore, FC-A treatment protects dopaminergic neuronal somas in the substantia nigra of a Parkinson's disease mouse model. Based on these results, we propose that the stabilization of 14‐3-3 epsilon interaction with aSyn might reduce aSyn toxicity, and highlight FC-A as a potential therapeutic compound for synucleinopathies.

The human RAP1 and GFAPɛ proteins increase γ-secretase activity in a yeast model system.

Alzheimer's disease (AD) is an age-related disorder that results in progressive cognitive impairment and memory loss. Deposition of amyloid β (Aβ) peptides in senile plaques is a hallmark of AD. γ-secretase produces Aβ peptides, mostly as the soluble Aβ40 with fewer insoluble Aβ42 peptides. Rare, early-onset AD (EOAD) occurs in individuals under 60 years of age. Most EOAD cases are due to unknown genetic causes, but a subset is due to mutations in the genes encoding the amyloid precursor protein that is processed into Aβ peptides or the presenilins (PS1 and PS2) that process APP. PS1 interacts with the epsilon isoform of glial fibrillary acidic protein (GFAPɛ), a protein found in the subventricular zone of the brain. We have found that GFAPɛ interacts with the telomere protection factor RAP1 (TERF2IP). RAP1 can also interact with PS1 alone or with GFAPɛ in vitro. Our data show that the nuclear protein RAP1 has an extratelomeric role in the cytoplasm through its interactions with GFAPɛ and PS1. GFAPɛ coprecipitated with RAP1 from human cell extracts. RAP1, GFAPɛ, and PS1 all colocalized in human SH-SY5Y cells. Using a genetic model of the γ-secretase complex in Saccharomyces cerevisiae, RAP1 increased γ-secretase activity, and this was potentiated by GFAPɛ. Our studies are the first to connect RAP1 with an age-related disorder.

Protein Kinase C Epsilon Contributes to the Exaggerated Mechanoreflex in Rats with Heart Failure

BackgroundDuring exercise, skeletal muscle contraction stimulates mechanically sensitive channels on the sensory endings of thin fiber muscle afferents. Stimulation of these mechanically sensitive channels initiates a reflex, termed the mechanoreflex, which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF‐rEF), mechanoreflex activation contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated mechanoreflex in HF‐rEF is partially attributed to an increased responsiveness of thin fiber skeletal muscle afferents to mechanical stimulation. Several lines of evidence demonstrate that signaling within sensory neurons mediated by the non‐traditional protein kinase c epsilon (PKCε) isoform augments mechanically activated channel function and produces mechanical hyperalgesia. Whether PKCε‐mediated signaling within sensory neurons contributes the exaggerated mechanoreflex in HF‐rEF is unknown.Purpose & HypothesisWe determined the role played by PKCε within sensory endings of thin fiber muscle afferents in evoking the mechanoreflex in rats with HF‐rEF and healthy control rats. We tested the hypothesis that injection of the selective PKCε inhibitor PKCε141 (44μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked during 30 seconds of 1 Hz dynamic hindlimb muscle stretch (a model of mechanoreflex activation) in HF‐rEF rats, but not in healthy control rats.MethodsExperiments were performed on Sprague‐Dawley rats subjected to coronary artery ligation surgery to produce myocardial infarction and HF‐rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared (Student’s t‐tests) the mechanoreflex evoked by hindlimb skeletal muscle stretch before and after injection of PKCε141 into the arterial supply of the hindlimb.ResultsEjection fraction (determined via echocardiography) was significantly reduced in HF‐rEF rats (48±1%) compared to sham rats (80±2%, P<0.01). Intra‐arterial PKCε141 injection reduced the pressor and RSNA response to hindlimb muscle stretch in HF‐rEF rats (n=6, 4M/2F, peak ΔMAP pre: 34±8; post: 19±7 mmHg; P=0.04; peak ΔRSNA pre: 121±32; post: 70±24%; P=0.04), but not SHAM rats (n=4, 3M/1F, peak ΔMAP pre: 14±4; post: 14±3 mmHg; P=0.87; peak ΔRSNA pre: 62±32; post: 58±26%; P=0.80) rats. In control experiments (n=3 HF‐rEF rats, 2M/1F), injection of PKCε141 into the jugular vein had no effect on the pressor or RSNA response to muscle stretch demonstrating a lack of central nervous system effects.ConclusionsThese data suggest that that PKCε signaling within the sensory neuron endings contributes to the exaggerated mechanoreflex in HF‐rEF and enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF‐rEF.

Analysis of m6A RNA Methylation-Related Genes in Liver Hepatocellular Carcinoma and Their Correlation with Survival.

N6-methyladenosine (m6A) modification on RNA plays an important role in tumorigenesis and metastasis, which could change gene expression and even function at multiple levels such as RNA splicing, stability, translocation, and translation. In this study, we aim to conduct a comprehensive analysis on m6A RNA methylation-related genes, including m6A RNA methylation regulators and m6A RNA methylation-modified genes, in liver hepatocellular carcinoma, and their relationship with survival and clinical features. Data, which consist of the expression of widely reported m6A RNA methylation-related genes in liver hepatocellular carcinoma from The Cancer Genome Atlas (TCGA), were analyzed by one-way ANOVA, Univariate Cox regression, a protein–protein interaction network, gene enrichment analysis, feature screening, a risk prognostic model, correlation analysis, and consensus clustering analysis. In total, 405 of the m6A RNA methylation-related genes were found based on one-way ANOVA. Among them, DNA topoisomerase 2-alpha (TOP2A), exodeoxyribonuclease 1 (EXO1), ser-ine/threonine-protein kinase Nek2 (NEK2), baculoviral IAP repeat-containing protein 5 (BIRC5), hyaluronan mediated motility receptor (HMMR), structural maintenance of chromosomes protein 4 (SMC4), bloom syndrome protein (BLM), ca-sein kinase I isoform epsilon (CSNK1E), cytoskeleton-associated protein 5 (CKAP5), and inner centromere protein (INCENP), which were m6A RNA methylation-modified genes, were recognized as the hub genes based on the protein–protein interaction analysis. The risk prognostic model showed that gender, AJCC stage, grade, T, and N were significantly different between the subgroup with the high and low risk groups. The AUC, the evaluation parameter of the prediction model which was built by RandomForest, was 0.7. Furthermore, two subgroups were divided by consensus clustering analysis, in which stage, grade, and T differed. We identified the important genes expressed significantly among two clusters, including uridine-cytidine kinase 2 (UCK2), filensin (BFSP1), tubulin-specific chaperone D (TBCD), histone-lysine N-methyltransferase PRDM16 (PRDM16), phosphorylase b ki-nase regulatory subunit alpha (PHKA2), serine/threonine-protein kinase BRSK2 (BRSK2), Arf-GAP with coiled-coil (ACAP3), general transcription factor 3C polypep-tide 2 (GTF3C2), and guanine nucleotide exchange factor MSS4 (RABIF). In our study, the m6A RNA methylation-related genes in liver hepatocellular carcinoma were analyzed systematically, including the expression, interaction, function, and prognostic values, which provided an important theoretical basis for m6A RNA methylation in liver cancer. The nine important m6A-related genes could be prognostic markers in the survival time of patients.

Anti-aging Klotho Protects SH-SY5Y Cells Against Amyloid β1-42 Neurotoxicity: Involvement of Wnt1/pCREB/Nrf2/HO-1 Signaling.

Alzheimer's disease (AD) is considered a prevalent neurological disorder with a neurodegenerative nature in elderly people. Oxidative stress and neuroinflammation due to amyloid β (Aβ) peptides are strongly involved in AD pathogenesis. Klotho is an anti-aging protein with multiple protective effects that its deficiency is involved in development of age-related disorders. In this study, we investigated the beneficial effect of Klotho pretreatment at different concentrations of 0.5, 1, and 2nM against Aβ1-42 toxicity at a concentration of 20μM in human SH-SY5Y neuroblastoma cells. Our findings showed that Klotho could significantly and partially restore cell viability and decrease reactive oxygen species (known as ROS) and improve superoxide dismutase activity (SOD) in addition to reduction of caspase 3 activity and DNA fragmentation following Aβ1-42 challenge. In addition, exogenous Klotho also reduced inflammatory biomarkers consisting of nuclear factor-kB (NF-kB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in Aβ-exposed cells. Besides, Klotho caused downregulation of Wnt1 level, upregulation of phosphorylated cyclic AMP response element binding (pCREB), and mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) with no significant alteration of epsilon isoform of protein kinase C (PKCε) after Aβ toxicity. In summary, Klotho could alleviate apoptosis, oxidative stress, and inflammation in human neuroblastoma cells after Aβ challenge and its beneficial effect is partially exerted through appropriate modulation of Wnt1/pCREB/Nrf2/HO-1 signaling.

The Effects of Novel Myristoylated PKC Epsilon Peptide Modulators on Real‐Time Nitric Oxide Release in Human Umbilical Vein Endothelial Cells

Endothelial dysfunction is a major consequence of ischemia‐reperfusion (I/R) injury, such as during the re‐establishment of blood flow after endovascular thrombus removal. It is characterized by limited nitric oxide (NO) bioavailability and production of superoxide (SO) instead of NO, in part from uncoupled endothelial NO synthase (eNOS) activity when the dihydrobiopterin (BH2) to tetrahydrobiopterin (BH4) ratio is elevated during reperfusion following prolonged ischemia. The protein kinase C epsilon (PKCɛ) isoform is known to participate in pre‐conditioning (PKCɛ activation treatment prior to ischemia) and post‐conditioning (PKCɛ inhibition treatment during reperfusion) to attenuate rat myocardial I/R injury. Normally following diacylglycerol‐dependent activation, PKCɛ binds to isoform‐specific receptors for activated C‐kinase (RACK) and translocates from the cytosol to the cell membrane to phosphorylate its targets, such as eNOS at Ser‐1177 to augment NO release when the BH2 to BH4 ratio is reduced to promote coupled eNOS activity that is normally seen prior to prolonged ischemia. Cell permeable myristic acid conjugated PKCɛ peptide activator (myr‐HDAPIGYD; myr‐PKCɛ+) and inhibitor (N‐myr‐EAVSLKPT; myr‐PKCɛ‐) has been shown to increase and decrease NO release, respectively, in rat aortic tissue by regulating PKCɛ translocation to eNOS. However, modulation of PKCɛ‐mediated NO release with these peptides in human cells remains undetermined. We hypothesize that myr‐PKCɛ+ will augment and myr‐PKCɛ‐ will attenuate NO release in cultured human umbilical vein endothelial cells (HUVECs). Real‐time HUVEC NO release (pmol) was measured using a calibrated NO electrode following administration of myr‐PKCɛ+ or myr‐PKCɛ‐ ± acetylcholine (Ach; positive control). Data were analyzed by ANOVA using Fishers post‐hoc analysis. Basal NO levels (66±6; n=35) were determined as the pmol difference between cell‐populated (106 cells/well) and reagent only wells. Control Ach (10 μM) enhanced NO release (102±7; n=37), myr‐PKCɛ+ (10 μM) enhanced NO release with Ach (107±16; n=8) and without Ach (107±13; n=7), and myr‐PKCɛ‐ (10 μM) attenuated NO release with Ach (37±9; n=13) and without Ach (7±29; n=9) compared to basal levels (all p<0.05). Results suggest that myr‐PKCɛ +/− effects on NO release is translational across mammalian species, presumably through activation and inhibition of PKCɛ‐mediated phosphorylation of eNOS. Thus, therapeutic intervention that targets inhibition of PKCɛ to reduce uncoupled eNOS activity during reperfusion may yield protective effects via attenuating the extent of I/R injury seen in clinical myocardial infarction, coronary bypass, coronary angioplasty, and organ transplantation.Support or Funding InformationThis research was supported by the Division of Research, Department of Biomedical Sciences, and the Center for Chronic Disorders of Aging at Philadelphia College of Osteopathic Medicine. Current research license is supported by Young Therapeutics, LLC.

Construction of a Novel Fatty Liver Cell Model with Low Density Lipoprotein Receptor Gene Knockout Based On Cas9/Single Guide RNA Technique

Clustered regularly interspaced short palindromic repeats/Cas9 gene editing technique was used to construct the human liver Lo2 stable cell line with low density lipoprotein receptor gene deletion. According to the design principle of clustered regularly interspaced short palindromic repeats/Cas9 target, the single guide RNA specifically target low density lipoprotein receptor gene was designed and the recombinant plasmid lenticlustered regularly interspaced short palindromic repeats v2 was used to construct the recombinant plasmid which can express this single guide RNA and Cas9 protein. After sequencing identification, the recombinant plasmid was transferred into Human embryonic kidney 293T cells together with retroviral packaging plasmids VSVG and PAX2. 48 h after transfection, the virus supernatant was collected and directly infected with human liver Lo2 cell. Puromycin was used to screen low density lipoprotein receptor-deficient Lo2 cells, which was verified by polymerase chain reaction, western blotting, and immunofluorescence staining. Then polymerase chain reaction and western blotting were used to verify the effects of low density lipoprotein receptor knockout on the function, lipid metabolism, glucose metabolism pathway, and insulin resistance of the Lo2 cell line. In this paper, the clustered regularly interspaced short palindromic repeats/Cas9 plasmid targeting low density lipoprotein receptor was successfully constructed. The results of polymerase chain reaction and western blotting showed that the human liver Lo2 cell with a stable knockout of low density lipoprotein receptor was obtained. The results of immunofluorescence staining showed that compared with the control group, the content of prealbumin in low density lipoprotein receptor knockout cells decreased significantly, while the content of diacylglycerol increased. At the same time, low density lipoprotein receptor gene knockout can increase the expression level of fatty acid synthase, peroxisome proliferator-activated receptor-γ, sterol regulatory element binding protein 1c, and inhibit the expression of carnitine palmitoyltransferase 1a. In addition, compared with the control group, there was no significant difference in mRNA expression and protein level of mammalian target of rapamycin and phosphatidylinositol-3-kinase, and there was no significant change in epsilon isoform of protein kinase C and insulin receptor substrates 1 expression level. The Lo2 cell line with low density lipoprotein receptor gene knockout was constructed by clustered regularly interspaced short palindromic repeats/Cas9 technique, and the role of low density lipoprotein receptor in lipid metabolism and glucose metabolism was proved by Polymerase chain reaction and Western blotting. The low density lipoprotein receptor knockout polyclonal and monoclonal cells constructed in this study can provide a useful tool for future research.

14-3-3ε protein-immobilized PCL-HA electrospun scaffolds with enhanced osteogenicity.

Adipose-derived mesenchymal stem cells (ASCs) accelerate the osteointegration of bone grafts and improve the efficiency in the formation of uniform bone tissue, providing a practical and clinically attractive approach in bone tissue regeneration. In this work, the effect of nanofibrous biomimetic matrices composed of poly(ε-caprolactone) (PCL), nanometric hydroxyapatite (nHA) particles and 14-3-3 protein isoform epsilon on the initial stages of human ASCs (hASCs) osteogenic differentiation was investigated. The cells were characterized by flow cytometry and induction to differentiation to adipogenic and osteogenic lineages. The isolated hASCs were induced to differentiate to osteoblasts over all scaffolds, and adhesion and viability of the hASCs were found to be similar. However, the activity of alkaline phosphatase (ALP) as early osteogenic marker in the PCL-nHA/protein scaffold was four times higher than in PCL-nHA and more than five times than the measured in neat PCL.

Klotho Ameliorates Cellular Inflammation via Suppression of Cytokine Release and Upregulation of miR-29a in the PBMCs of Diagnosed Alzheimer's Disease Patients.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by neural inflammation and oxidative stress. In the current study, the protective effects of klotho and linagliptin treatment on human peripheral blood mononuclear cells (PBMCs) of AD patients and healthy controls (HCs) are assessed through measurement of inflammatory cytokines, signaling proteins, and miRNA expression. Sixteen diagnosed AD patients and sixteen HCs were enrolled in the study. Blood samples were obtained and PBMCs were isolated. PBMCs were treated with klotho at different concentrations (0.5, 1, and 2nM) and linagliptin (50μM). The concentration of interleukin-1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), epsilon isoform of protein kinase C (PKCε), phosphorylated cyclic AMP response element binding (pCREB), and Wnt1 were measured by ELISA. The expression of miR-29a and miR-195 was detected by real-time PCR. The results showed that klotho significantly reduced IL-1β, IL-6, and TNF-α levels in both groups of the experiment. Linagliptin also remarkably reduced TNF-α levels in the AD group. Moreover, klotho caused the downregulation of Wnt1 in the PBMCs of both groups and the upregulation of the pCREB in HCs. Meanwhile, klotho induced miR-29a expression in the PBMCs of HCs, while miR-29a expression was induced in the AD group by klotho and linagliptin. The current findings revealed that klotho alleviates inflammation in human PBMCs, probably through the suppression of inflammatory cytokines and the upregulation of miR-29a, and part of its beneficial effect is mediated through appropriate modulation of the Wnt1/pCREB signaling cascade. In addition, linagliptin exerts protective effects by reducing TNF-α and inducing miR-29a expression in PBMCs.

Membrane Physical Properties Regulate the Rate and Acyl Chain Specificity of One of the Steps of the Phosphatidylinositol Cycle

The plethora of biological functions regulated by phosphatidylinositol (PI) and its phosphorylated forms makes this lipid class one of the most important in cell physiology. The major metabolic pathway for the synthesis of this lipid class is the PI‐cycle. One of the steps of the PI‐cycle is the ATP‐dependent phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), a reaction catalyzed by the epsilon isoform of diacylglycerol kinases (DGKɛ). The enzyme when assayed with mixed‐micelles exhibits a high degree of acyl chain specificity for its DAG substrate, with 1‐stearoyl‐2‐arachidonoyl glycerol (SAG) being the preferred substrate. These acyl chains are the same as those found in the lipid intermediates of the PI‐cycle, which was proposed to be in part due to the action of DGKɛ. Opposite to other metabolic cycles, steps of the PI‐cycle spans two different locations (membranes) within a cell, the plasma membrane (PM) and endoplasmic reticulum (ER), the same membranes where DGKɛ has been reported to be found. However, it is currently unknown whether DGKɛ catalyze its reaction within the PI‐cycle in the PM and/or ER. Since DGKɛ lacks a putative regulatory domain and these two membranes have several different physical and chemical properties, we hypothesized that either DGKɛ is constitutively active or it is regulated by the properties of the membrane it's bound to. To address this question we use an assay with purified DGKɛ and liposomes. This strategy allowed us to systematically vary the membrane physical and chemical properties while avoiding complications coming from other levels of regulation. In the present work we show that the rate of DGKɛ ‐ catalyzed reaction and its substrate acyl chain specificity are regulated by membrane physical properties. In a locally flat membrane (in relation to the size of the enzyme and its membrane binding platform) the enzyme works at very low basal levels without discriminating the nature of the substrate acyl chains. In these membrane structures the majority of biological negatively charged phospholipids showed an inhibitory effect on the enzyme. However, its product (PA), which is also negatively charged, exhibited the contrary effect leading to an enhancement in enzyme activity and acyl chain specificity. This result suggested a positive feedback mechanism, contrary to what is currently believed. Likewise, the presence of highly curved membranes, specifically those membrane structures bearing negative Gaussian (saddle‐point like) curvature greatly enhanced the rate of reaction as well as the enzyme substrate acyl chain specificity, which was also dependent on the degree of curvature. To sum up, our results indicate that DGKɛ is not likely to catalyze its reaction within the PI‐cycle in stable regions of either the ER or the PM, but rather at the junction between these two organelles where highly curved membrane structures have been reported and/or at PA‐enriched domains.Support or Funding InformationThis work was supported by the Canadian Natural Sciences and Engineering Research Council grant RGPIN‐2018‐05585.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of membrane shape in regulating the phosphatidylinositol cycle at contact sites.

The extensive number of metabolic processes regulated by phosphatidylinositol (PI) phospholipids highlights their physiological importance. The major metabolic pathway for their biosynthesis in cells is the PI-cycle. Contrary to most metabolic cycles, reactions of the PI-cycle occur in two different locations; those are, the plasma membrane (PM) and endoplasmic reticulum (ER). Lipid movement between the two organelles is, therefore, a requirement of the cyclical process. Moreover, in mammals the PI-cycle yield PI molecular species enriched in specific acyl chains, namely 1-stearoyl-2-arachidonoyl acyl chains. Hence, to ensure cycle efficiency and specificity it should take place in specialized regions of PM and ER rather than being randomly distributed among those membranes. Along these lines, ER-PM contact sites have emerged as the location where a number of proteins related to the PI-cycle have been reported to localize. Of importance to this review is the presence of the epsilon isoform of diacylglycerol kinase (DGKε) at ER-PM contact sites. In the PI-cycle DGKε is in part responsible for the acyl chain enrichment of the PI molecular species. However, it has recently been shown that the enzyme can only engage in the PI-cycle upon membrane morphological changes. In this review we will discuss the PI-cycle at ER-PM contact sites and how the generation of membrane negative Gaussian curvature nearby those regions could regulate the cycle. We will focus our discussion on the hypothesis that actin polymerization provides the mechanical force needed to change membrane shape nearby ER-PM contact sites, which will transiently trigger DGKε and, therefore, link enzymatic catalysis and lipid transfer in the PI-cycle.

PKC\u03b5 phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration

BackgroundGlobal cerebral ischemia triggers neurodegeneration in the hippocampal CA1 region, but the mechanism of neuronal death remains elusive. The epsilon isoform of protein kinase C (PKCε) has recently been identified as a master switch that controls the nucleocytoplasmic trafficking of ATF2 and the survival of melanoma cells. It is of interest to assess the role of PKCε–ATF2 signaling in neurodegeneration.ResultsPhosphorylation of ATF2 at Thr-52 was reduced in the hippocampus of PKCε null mice, suggesting that ATF2 is a phosphorylation substrate of PKCε. PKCε protein concentrations were significantly reduced 4, 24, 48 and 72 h after transient global cerebral ischemia, resulting in translocation of nuclear ATF2 to the mitochondria. Degenerating neurons staining positively with Fluoro-Jade C exhibited cytoplasmic ATF2.ConclusionsOur results support the hypothesis that PKCε regulates phosphorylation and nuclear sequestration of ATF2 in hippocampal neurons during ischemia-induced neurodegeneration.

Potential involvement of heat shock proteins in pancreatic-duodenal homeobox-1-mediated effects on the genesis of gastric cancer: A 2D gel-based proteomic study.

AIMTo identify functional proteins involved in pancreatic-duodenal homeobox-1 (PDX1)-mediated effects on gastric carcinogenesis.METHODSA PDX1-overexpressed model was established by transfecting gastric cancer cell line SGC7901 with pcDNA3.1(+)-PDX1 vector (SGC-PDX1). Transfection with empty pcDNA3.1 vector (SGC-pcDNA) served as control. Comparative protein profiles of the two groups were analyzed by two-dimensional electrophoresis based-proteomics (2DE gel-based proteomics). The differential proteins identified by 2DE were further validated by qRT-PCR and immunoblotting. Finally, co-immunoprecipitation was used to determine any direct interactions between PDX1 and the differential proteins.RESULTS2DE gel proteomics identified seven differential proteins in SGC-PDX1 when compared with those in SGC-pcDNA. These included four heat shock proteins (HSPs; HSP70p1B, HSP70p8, HSP60, HSP27) and three other proteins (ER60, laminin receptor 1, similar to epsilon isoform of 14-3-3 protein). Immunoblotting validated the expression of the HSPs (HSP70, HSP60, HSP27). Furthermore, their expressions were lowered to 80%, 20% and 24%, respectively, in SGC-PDX1, while PDX1 exhibited a 9-fold increase, compared to SGC-pcDNA. However, qRT-PCR analysis revealed that mRNA levels of the HSPs were increased in SGC-PDX1, suggesting that the expression of the HSPs was post-translationally regulated by the PDX1 protein. Finally, co-immunoprecipitation failed to identify any direct interaction between PDX1 and HSP70 proteins.CONCLUSIONThis study demonstrates the potential involvement of HSPs in PDX1-mediated effects on the genesis of gastric cancer.

C.1289G>A (p.Arg430His) variant in the epsilon isoform of the GFAP gene in a patient with adult onset Alexander disease

Alexander disease (AD) is a rare form of leukodystrophy caused by pathogenic variants in the GFAP gene. In young children the condition is fatal, while adults have variable neurological symptoms and prognosis. On magnetic resonance imaging, a pattern of atrophy of the medulla oblongata and cervical spinal cord with a ‘tadpole’ appearance is highly suggestive of adult-onset Alexander disease (AOAD). GFAP gene sequencing is used to confirm the diagnosis. Pre-mRNA of this gene undergoes alternative splicing resulting in formation of at least 8 different protein isoforms. Most patients with AD described to date have a pathogenic variant in the coding sequence of the main and the most abundant gene isoform, the GFAPα. Recently, two half-siblings with neurological symptoms and radiological signs of AOAD were reported and were not found to have any pathogenic variants in the GFAPα gene while further genetic testing by next generation sequencing revealed a c.1289G>A (p.Arg430His) variant in the alternative exon 7A of the GFAPε isoform.Here we present a case of another patient with symptoms and brain MRI pattern suggestive of AOAD. Similarly to the previously described patients, this patient did not have any pathogenic variants in the main gene isoform and had the same c.1289G>A (p.Arg430His) variant in the GFAPε. This report contributes to evidence of pathogenicity of the c.1289G>A (p.Arg430His) variant in the GFAPε.

Constitutive downregulation protein kinase C epsilon in hSOD1G93A astrocytes influences mGluR5 signaling and the regulation of glutamate uptake.

Accumulating evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is a non-cell-autonomous process and that impaired glutamate clearance by astrocytes, leading to excitotoxicity, could participate in progression of the disease. In astrocytes derived from an animal model of ALS (hSOD1G93A rats), activation of type 5 metabotropic glutamate receptor (mGluR5) fails to increase glutamate uptake, impeding a putative dynamic neuroprotective mechanism involving astrocytes. Using astrocyte cultures from hSOD1G93A rats, we have demonstrated that the typical Ca2+ oscillations associated with mGluR5 activation were reduced, and that the majority of cells responded with a sustained elevation of intracellular Ca2+ concentration. Since the expression of protein kinase C epsilon isoform (PKCɛ) has been found to be considerably reduced in astrocytes from hSOD1G93A rats, the consequences of manipulating its activity and expression on mGluR5 signaling and on the regulation of glutamate uptake have been examined. Increasing PKCɛ expression was found to restore Ca2+ oscillations induced by mGluR5 activation in hSOD1G93A -expressing astrocytes. This was also associated with an increase in glutamate uptake capacity in response to mGluR5 activation. Conversely, reducing PKCɛ expression in astrocytes from wild-type animals with specific PKCɛ-shRNAs was found to alter the mGluR5 associated oscillatory signaling profile, and consistently reduced the regulation of the glutamate uptake-mediated by mGluR5 activation. These results suggest that PKCɛ is required to generate Ca2+ oscillations following mGluR5 activation, which support the regulation of astrocytic glutamate uptake. Reduced expression of astrocytic PKCɛ could impair this neuroprotective process and participate in the progression of ALS.

Flow-Responsive Vascular Endothelial Growth Factor Receptor-Protein Kinase C Isoform Epsilon Signaling Mediates Glycolytic Metabolites for Vascular Repair.

Hemodynamic shear stress participates in maintaining vascular redox status. Elucidating flow-mediated endothelial metabolites enables us to discover metabolic biomarkers and therapeutic targets. We posited that flow-responsive vascular endothelial growth factor receptor (VEGFR)-protein kinase C isoform epsilon (PKCɛ)-6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) signaling modulates glycolytic metabolites for vascular repair. Bidirectional oscillatory flow (oscillatory shear stress [OSS]: 0.1 ± 3 dyne·cm-2 at 1 Hz) upregulated VEGFR-dependent PKCɛ expression to a greater degree than did unidirectional pulsatile flow (pulsatile shear stress [PSS]: 23 ± 8 dyne·cm-2 at 1 Hz) in human aortic endothelial cells (p < 0.05, n = 3). PSS and OSS further upregulated PKCɛ-dependent PFKFB3 expression for glycolysis (p < 0.05, n = 4). Constitutively active PKCɛ increased, whereas dominant-negative PKCɛ reduced both basal and maximal extracellular acidification rates for glycolytic flux (p < 0.01, n = 4). Metabolomic analysis demonstrated an increase in PKCɛ-dependent glycolytic metabolite, dihydroxyacetone (DHA), but a decrease in gluconeogenic metabolite, aspartic acid (p < 0.05 vs. control, n = 6). In a New Zealand White rabbit model, both PKCɛ and PFKFB3 immunostaining was prominent in the PSS- and OSS-exposed aortic arch and descending aorta. In a transgenic Tg(flk-1:EGFP) zebrafish model, GATA-1a morpholino oligonucleotide injection (to reduce viscosity-dependent shear stress) impaired vascular regeneration after tail amputation (p < 0.01, n = 20), which was restored with PKCɛ messenger RNA (mRNA) rescue (p < 0.05, n = 5). As a corollary, siPKCɛ inhibited tube formation and vascular repair, which were restored by DHA treatment in our Matrigel and zebrafish models. Innovation and Conclusion: Flow-sensitive VEGFR-PKCɛ-PFKFB3 signaling increases the glycolytic metabolite, dihydroxyacetone, to promote vascular repair. Antioxid. Redox Signal. 28, 31-43.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Diacylglycerol kinase epsilon suppresses expression of p53 and glycerol kinase in mouse embryo fibroblasts

The incorporation of glycerol into lipid was measured using SV40 transformed mouse embryo fibroblasts (MEFs) from either wild-type (WT) mice or from mice in which the epsilon isoform of diacylglycerol kinase (DGKε) was knocked out (DGKε−/−). We present an explanation for our finding that DGKε−/− MEFs exhibited greater uptake of 3H-glycerol into the cell and a greater incorporation into lipids compared with their WT counterparts, with no change in the relative amounts of various lipids between the DGKε−/− and WT MEFs. Glycerol kinase is more highly expressed in the DGKε−/− cells than in their WT counterparts. In addition, the activity of glycerol kinase is greater in the DGKε−/− cells than in their WT counterparts. Other substrates that enter the cell independent of glycerol kinase, such as pyruvate or acetate, are incorporated into lipid to the same extent between DGKε−/− and WT cell lines. We also show that expression of p53, a transcription factor that increases the synthesis of glycerol kinase, is increased in DGKε−/− MEFs in comparison to WT cells. We conclude that the increased incorporation of glycerol into lipids in DGKε−/− cells is a consequence of up-regulation of glycerol kinase and not a result of an increase in the rate of lipid synthesis. Furthermore, increased expression of the pro-survival gene, p53, in cells knocked out for DGKε suggests that cells over-expressing DGKε would have a greater propensity to become tumorigenic.

Protein kinase Cɛ activity regulates mGluR5 surface expression in the rat nucleus accumbens.

Type 5 metabotropic glutamate receptors (mGluR5) activate protein kinase C (PKC) via coupling to Gαq/11 protein signaling. We have previously demonstrated that the epsilon isoform of PKC (PKCɛ) is a critical downstream target of mGluR5 in regulating behavioral and biochemical responses to alcohol. Recent evidence suggests that PKC-mediated phosphorylation of mGluR5 can lead to receptor desensitization and internalization. We therefore sought to examine the specific involvement of PKCɛ in the regulation of mGluR5 surface expression in the nucleus accumbens (NAc), a key regulator of alcohol-associated behaviors. Coronal brain sections from male Wistar rats were analyzed for either colocalization of mGluR5 and PKCɛ via immunohistochemistry or changes in mGluR5 surface expression and PKCɛ phosphorylation following local application of PKCɛ translocation activator or inhibitor peptides and/or an orthosteric mGluR5 agonist. We observed colocalization of mGluR5 and PKCɛ in the NAc. We also showed that intra-NAc infusion of the PKCɛ translocation inhibitor ɛV1-2 increased mGluR5 surface expression under baseline conditions. Stimulation of mGluR5 with an orthosteric agonist DHPG, dose dependently increased ERK1/2 and PKCɛ phosphorylation as well as mGluR5 internalization in acute NAc slices. Finally, we observed that activation of PKCɛ translocation with Tat-ΨɛRACK peptide mediates agonist-independent mGluR5 internalization, whereas PKCɛ translocation inhibitor ɛV1-2 prevents agonist-dependent internalization of mGluR5 in NAc slice preparations. These findings suggest that the subcellular localization of mGluR5 in the NAc is regulated by PKCɛ under basal and stimulation conditions, which may influence the role of mGluR5-PKCɛ signaling in alcohol-related behaviors. © 2016 Wiley Periodicals, Inc.

Abstract 940: The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines

Abstract Background: Hepatocellular carcinoma (HCC) is the third largest contributor to cancer mortality in the world. Alphafetoprotein (AFP) is synthetized and secreted by the majority of HCCs. Despite its controversial value as classical HCC diagnostic and follow-up marker, the AFP was lately shown to correlate with the volume of liver cancer. It is also useful as a predictive marker for radiotherapy treatment response and patients prognosis. On the molecular level, AFP has a proliferative role in HCC since it acts on its own receptor on the surface of hepatocytes; however, its mechanism of action remains unclear. Our laboratory have previously demonstrated that telomerase and protein kinase C (PKC) modulate AFP secretion in HCC cell lines and this effect is additive. The aim of this study is to elucidate the PKC isoforms involved in AFP secretion modulation and telomerase expression. Methods: Two AFP secretory cell lines, HepG2/C3a and PLC/PRF/5, and one non-secretory AFP cell line, SNU-387, were cultured in DMEM (RPMI-1640 for SNU-387) media with 10% FBS and 1% Penicillin/Streptomycin and incubated in humid 5% CO2 incubator. The RNA was extracted; the RT-PCR was performed to characterize the PKC isoforms and the modulation of hTERT by these isoforms. The role of each isoform on AFP secretion was elucidated using different PKC isoform modulators; the AFP concentration was measured using ELISA technique. The viability and toxicity of cells were assessed using WST-1. Results: The three cell lines express the PKC alpha, delta and epsilon isoforms. Whereas the other isoforms Beta 1 and 2 are slightly expressed in C3a, the gamma and beta isoforms are slightly expressed in PLC and SNU respectively. Dose-dependent inhibition of PKC alpha and beta by GO6976 decreased AFP secretion by 40% at 10 uM by the HepG2/C3a cell line. The inhibition of PKC delta by Rottlerin dose-dependently decreased AFP secretion by 30% at 5 uM. The PKC epsilon modulator FR236924 dose-dependently inhibited AFP secretion by 50% at 1 uM. Pan PKC inhibitor, GO6983, decreased AFP secretion by 45% at 5 uM. The same profile was obtained for PLC/PRF/5 with a maximum 30% of AFP down-regulation except for Rottlerin which showed a 75% of AFP inhibition. The catalytic subunit expression of telomerase, hTERT, was only inhibited by Rottlerin at 5 uM. Our experiments did not show any cell death nor cell proliferation modulation when incubating the three cell lines with PKC inhibitors. Conclusion: Combining the above results with our previous ones, we suggest for the first time, that PKC delta modulates AFP secretion through telomerase however the mechanism by which PKC alpha and epsilon modulate AFP secretion is to be elucidated. However, further experiments are needed to support this statement. Citation Format: Raia Doumit, Roula Tahtouh, Rita Ammoury, Riad Sarkis, Nada Alaaddine, George Hilal. The role of protein kinase C isoforms in telomerase activity and alphafetoprotein secretion by the hepatocellular carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 940.