Degradation Of Reductase Research Articles

Experimental study of the protective effects of SYVN1 against diabetic retinopathy

Genetic factors play an important role in the pathogenesis of diabetic retinopathy (DR). While many studies have focused on genes that increase susceptibility to DR, herein, we aimed to explore genes that confer DR resistance. Previously, we identified Hmg CoA reductase degradation protein 1 (SYVN1) as a putative DR protective gene via gene expression analysis. Transgenic mice overexpressing SYVN1 and wild-type (WT) mice with streptozotocin-induced diabetes were used in this experiment. Retinal damage and vascular leakage were investigated 6 months after induction of diabetes by histopathological and retinal cell apoptosis analyses and by retinal perfusion of fluorescein isothiocyanate-conjugated dextran. Compared with diabetic WT mice, diabetic SYVN1 mice had significantly more cells and reduced apoptosis in the retinal ganglion layer. Retinal vascular leakage was significantly lower in diabetic SYVN1 mice than in diabetic WT mice. The expression levels of endoplasmic reticulum (ER) stress-related, pro-inflammatory, and pro-angiogenic genes were also analyzed. Lower expression levels were observed in diabetic SYVN1 mice than in WT controls, suggesting that SYVN1 may play an important role in inhibiting ER stress, chronic inflammation, and vascular overgrowth associated with DR. Thus, these results strongly supported our hypothesis that SYVN1 confers DR resistance.

Hrd1 and ER-Associated Protein Degradation, ERAD, are Critical Elements of the Adaptive ER Stress Response in Cardiac Myocytes.

Hydroxymethyl glutaryl-coenzyme A reductase degradation protein 1 (Hrd1) is an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase that has been studied in yeast, where it contributes to ER protein quality control by ER-associated degradation (ERAD) of misfolded proteins that accumulate during ER stress. Neither Hrd1 nor ERAD has been studied in the heart, or in cardiac myocytes, where protein quality control is critical for proper heart function. The objective of this study were to elucidate roles for Hrd1 in ER stress, ERAD, and viability in cultured cardiac myocytes and in the mouse heart, in vivo. The effects of small interfering RNA-mediated Hrd1 knockdown were examined in cultured neonatal rat ventricular myocytes. The effects of adeno-associated virus-mediated Hrd1 knockdown and overexpression were examined in the hearts of mice subjected to pressure overload-induced pathological cardiac hypertrophy, which challenges protein-folding capacity. In cardiac myocytes, the ER stressors, thapsigargin and tunicamycin increased ERAD, as well as adaptive ER stress proteins, and minimally affected cell death. However, when Hrd1 was knocked down, thapsigargin and tunicamycin dramatically decreased ERAD, while increasing maladaptive ER stress proteins and cell death. In vivo, Hrd1 knockdown exacerbated cardiac dysfunction and increased apoptosis and cardiac hypertrophy, whereas Hrd1 overexpression preserved cardiac function and decreased apoptosis and attenuated cardiac hypertrophy in the hearts of mice subjected to pressure overload. Hrd1 and ERAD are essential components of the adaptive ER stress response in cardiac myocytes. Hrd1 contributes to preserving heart structure and function in a mouse model of pathological cardiac hypertrophy.

Synergistic Impact of d ‐δ‐Tocotrienol and Geranylgeraniol in Human DU145 Prostate Carcinoma Cells

The mevalonate pathway produces essential intermediates for the post-translational prenylation and dolichylation of growth-associated proteins including Ras, nuclear lamins and growth factor receptors. Dysregulation and overexpression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of this pathway, in malignant cells supports tumor growth and survival. d-δ-Tocotrienol, a vitamin E molecule with farnesyl side chain, and geranylgeraniol, a diterpene, accelerate the ubiquitination and proteasome-mediated degradation of HMG CoA reductase via complementary and collaborative mechanisms. We evaluated the impacts of these two isoprenoids in human DU145 prostate carcinoma cells. d-δ-Tocotrienol (IC50 = 15 μmol/L) and geranylgeraniol (IC50 = 60 μmol/L) individually induced concentration-dependent suppression of the viability of DU145 cells as measured by CellTiter 96® AQueous One Solution. Isobologram using CompuSyn software demonstrated the synergistic impact of the two agents in combination. Blends of d-δ-tocotrienol (7.5 μmol/L) and geranylgeraniol (30 μmol/L), each at half of their respective IC50 concentrations, cumulatively suppressed the level of membrane HMG CoA reductase and putatively as a consequence of reduced prenylation, that of membrane Ras protein. Synergistic impact of diverse isoprenoids on the mevalonate pathway and tumor cell viability may offer a potential therapeutic or preventive approach to prostate cancer. Supported by TWU REP and American River Nutrition, Inc.

The prenyltransferase UBIAD1 is the target of geranylgeraniol in degradation of HMG CoA reductase.

Schnyder corneal dystrophy (SCD) is an autosomal dominant disorder in humans characterized by abnormal accumulation of cholesterol in the cornea. SCD-associated mutations have been identified in the gene encoding UBIAD1, a prenyltransferase that synthesizes vitamin K2. Here, we show that sterols stimulate binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase, which is subject to sterol-accelerated, endoplasmic reticulum (ER)-associated degradation augmented by the nonsterol isoprenoid geranylgeraniol through an unknown mechanism. Geranylgeraniol inhibits binding of UBIAD1 to reductase, allowing its degradation and promoting transport of UBIAD1 from the ER to the Golgi. CRISPR-CAS9-mediated knockout of UBIAD1 relieves the geranylgeraniol requirement for reductase degradation. SCD-associated mutations in UBIAD1 block its displacement from reductase in the presence of geranylgeraniol, thereby preventing degradation of reductase. The current results identify UBIAD1 as the elusive target of geranylgeraniol in reductase degradation, the inhibition of which may contribute to accumulation of cholesterol in SCD.

The interplay of Hrd3 and the molecular chaperone system ensures efficient degradation of malfolded secretory proteins.

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD-ligase), and degraded by cytosolic 26S proteasomes. This process is termed ER-associated protein degradation (ERAD). We previously showed that the membrane protein Der1, which is a subunit of the HRD-ligase, is involved in the export of aberrant polypeptides from the ER. Unexpectedly, we also uncovered a close spatial proximity of Der1 and the substrate receptor Hrd3 in the ER lumen. We report here on a mutant Hrd3KR that is selectively defective for ERAD of soluble proteins. Hrd3KR displays subtle structural changes that affect its positioning toward Der1. Furthermore, increased quantities of the ER-resident Hsp70-type chaperone Kar2 and the Hsp40-type cochaperone Scj1 bind to Hrd3KR. Of note, deletion of SCJ1 impairs ERAD of model substrates and causes the accumulation of client proteins at Hrd3. Our data imply a function of Scj1 in the removal of malfolded proteins from the receptor Hrd3, which facilitates their delivery to downstream-acting components like Der1.

Endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 1 targets misfolded HLA-B27 dimers for endoplasmic reticulum-associated degradation.

HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease.

GW25-e3139 A Novel Polymorphism of the GP78 Gene is Associated with Coronary Artery Disease in Han Population in China

GP78 is a membrane-anchored ubiquitin ligase mediating the degradation of HMG-CoA reductase (HMGCR) and Insig-1, which was very essential for the synthesis of cholesterol process. The cholesterol has a causal role in the development of cardiovascular disease. The aim of the present study was to

D‐δ‐Tocotrienol suppresses the differentiation of murine 3T3‐F442A preadipocytes (641.8)

Suppression of adipogenesis is one of the potential approaches in obesity prevention. The statins, competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, suppress the differentiation of adipocytes via mevalonate deprivation. We hypothesize that d‐δ‐tocotrienol, a vitamin E molecule shown to accelerate the degradation of HMG CoA reductase, suppresses adipocyte differentiation and adipogenic gene expression. Adipo‐Red assay and oil Red O staining showed that an 8‐d incubation with 2.5 ‐ 10 μmol/L d‐δ‐tocotrienol dose‐dependently reduced the intracellular triglyceride content of murine 3T3‐F442A adipocytes, reminiscent of the lovastatin (1.25 μmol/L) effect. Concomitantly, d‐δ‐tocotrienol dose‐dependently inhibited glucose uptake by 3T3‐F442A cells and the expression of GluT4, pAKT, and HMG CoA reductase proteins. Rosiglitazone (1 µmol/L), an agonist of peroxisome proliferator activator receptor‐activated receptor γ ‐ a key mediator of adipocyte differentiation ‐ reversed the effects of d‐δ‐tocotrienol on cellular lipid content and glucose uptake but not those of lovastatin. In contrast, mevalonate (100 µmol/L) reversed the effects of lovastatin. d‐α‐Tocopherol at 10 µmol/L did not have any impact on 3T3‐F442A differentiation. Trypan blue staining showed no changes in cell viability following 48‐h incubation of 3T3‐F442A cells with d‐δ‐tocotrienol (0‐80 µmol/L), suggesting that the adipogenesis‐suppressive activity of d‐δ‐tocotrienol was independent of cytotoxicity. Dietary d‐δ‐tocotrienol may have potential as anti‐adipogenesis compounds.Grant Funding Source: Supported by Texas Department of Agriculture and TWU Research Enhancement Program

Anti‐cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae

KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N-terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109Δ mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double-strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.

Scavenger receptor CD36 mediates inhibition of cholesterol synthesisviaactivation of the PPARγ/PGC‐1α pathway and Insig1/2 expression in hepatocytes

The scavenger receptor CD36 plays a central role in lipid metabolism by promoting macrophage cholesterol efflux with the potential to reduce atherosclerotic lesions. However, the effect of CD36 on de novo cholesterol synthesis is not known. Here, we describe the cellular mechanism by which CD36 activation induces cholesterol depletion in HepG2 cells. Using the CD36 ligand hexarelin, we found a rapid phosphorylation of HMG-CoA reductase Ser-872 in treated cells, resulting in inactivation of the rate-limiting enzyme in sterol synthesis. Degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway was also enhanced by hexarelin, through an increased recruitment of the anchor proteins insulin-induced gene (Insig)-1 and Insig-2. Genes encoding key enzymes involved in cholesterol synthesis and under the control of transcription factor sterol regulatory element-binding protein (SREBP)-2 remained unresponsive to sterol depletion, due to retention of the SREBP-2 escort protein Scap by Insig-1/2. Insig1 and Insig2 gene expression was also increased through activation of nuclear receptor peroxisome-proliferator activating receptor γ (PPARγ) by CD36, which lifted the inhibitory effect of PPARγ1 Ser-84 phosphorylation. Recruitment of coactivator peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) to activated AMPKα was also promoted, resulting in PGC-1α transcriptional activation through Sirt1-mediated deacetylation, increased recruitment of PPARγ, and up-regulation of Insig-1/2, revealing a regulatory role of CD36 on PGC-1α signaling. Our data identify CD36 as a novel regulator of HMG-CoA reductase function and Insig-1/2 expression, 2 critical steps regulating cholesterol synthesis in hepatocytes.

Der1 promotes movement of misfolded proteins through the endoplasmic reticulum membrane

Misfolded proteins of the secretory pathway are extracted from the endoplasmic reticulum (ER), polyubiquitylated by a protein complex termed the Hmg-CoA reductase degradation ligase (HRD ligase) and degraded by cytosolic 26S proteasomes. The movement of these proteins through the lipid bilayer is assumed to occur via a protein-conducting channel of unknown nature. We show that the integral membrane protein Der1 oligomerizes, which relies on its interaction with the scaffolding protein Usa1. Mutations in the transmembrane domains of Der1 block the passage of soluble proteins across the ER membrane. As determined by site-specific photocrosslinking, the ER-luminal exposed parts of Der1 are in spatial proximity to the substrate receptor Hrd3, whereas the membrane-embedded domains reside adjacent to the ubiquitin ligase Hrd1. Intriguingly, both regions also form crosslinks to client proteins. Our data imply that Der1 initiates the export of aberrant polypeptides from the ER lumen by threading such molecules into the ER membrane and routing them to Hrd1 for ubiquitylation.

Gamma-Tocotrienol and Simvastatin Synergistically Induce Cytotoxicity In Leukemia Cell Lines, K-562 and HL-60

IntroductionStatins and tocotrienols modulate the cholesterol biosynthesis pathway by inhibiting the 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA) reductase. Tocotrienols modulate HMG-CoA reductase by post-transcriptional downregulation. In addition, tocotrienols contain a farnesol moiety in its side-chain that triggers degradation of HMG-CoA reductase. These effects lead to suppression of cell proliferation, cell cycle arrest, and apoptosis. Several studies have shown that statins have suppressive effects in in vitro experiments on acute myelocytic leukemia cell lines. Since both statins and gamma-tocotrienol are associated with decreased cholesterol biosynthesis, we hypothesized that if the cytotoxicity of these drugs on cancer cells is related to impaired biosynthesis of cholesterol, combination of them could synergize in cytotoxicity on leukemic cells. Materials and MethodsK-562 and HL-60 leukemia cells were grown in Iscove's Modified Dulbecco's medium with penicillin/streptomycin, 10% fetal bovine serum and 20% fetal bovine serum added respectively. K-562 and HL-60 leukemia cells were seeded in 96 well plates, grown overnight, and treated for 24, 48 and 72 hours with simvastatin in concentrations of 1,2,4, and5 µM; gamma-tocotrienol in concentrations of 20, 40, and 80 µM; and a combination of the two drugs in the same concentrations. For 24 hour dose, cells were seeded at a density of 5000/well and for 48 and 72 hour doses, at 3500/well. Following the treatment, MTS/PMS reagent (Promega, Madison, WI), [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] mixed with and an electron coupling reagent (phenazine methosulfate), was added at 40 µg/well and incubated at 37°C for 2 hours. We measured the solubilized formazan crystals at 450 nm as an indicator of cytotoxicity. The presence of ATP as an indicator of cell viability was measured with the CellTiter Glo® assay (Promega). Cells were again seeded, grown overnight, and dosed as indicated above. Following treatment, the assay was conducted as specified by the manufacturer. ResultsBoth simvastatin and gamma-tocotrienol induce cytotoxicity in K-562 and HL-60 cell lines by the MTS and Cell Titer Glo Assays. The IC50 at 72 hour incubation are as follows 1) HL-60: simvastatin - 16.543 uM, gamma tocotrienol - 36.297 uM; 2) K-562: simvastatin - 5.235 uM, gamma tocotrienol - 34.947 uM. We used CompuSyn to calculate the IC50. When combined, simvastatin and gamma-tocotrienol exhibit synergy at lower concentrations when examined by isobologram analysis. [Display omitted] [Display omitted] ConclusionGamma-tocotrienol, an isoform of vitamin E, and simvastatin, a cholesterol lowering drug exhibit synergy in induction of cytotoxicity in K-562 and HL-60 leukemia cell lines. Rescue experiments and mechanistic pathway analysis are being explored to confirm these observations. Disclosures:No relevant conflicts of interest to declare.

Scavenger Receptor CD36 Mediates Inhibition of Cholesterol Synthesis via Activation of the LKB1-AMPK Pathway and Insig1/Insig2 Expression in Hepatocytes

Scavenger receptor CD36 is known to play a central role in regulating lipid metabolism, in part, through the uptake of oxidized LDL in macrophages and its ability to internalize long chain fatty acids in adipocytes. Here, we demonstrate that CD36 activity impacts cholesterol synthesis in hepatocytes. Using hexarelin, a growth hormone-releasing peptide that interacts with CD36, we found a rapid phosphorylation of LKB1, which led to subsequent AMPK phosphorylation in treated HepG2 cells. HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol synthesis, was phosphorylated and inactivated following hexarelin treatment. This was accompanied with a significant degradation of HMG-CoA reductase by the ubiquitin-proteasome pathway in response to CD36 activation and an enhanced recruitment of escort proteins Insig-1 and Insig-2. Key enzymes involved in cholesterol biosynthesis and under SREBP-2 control remained downregulated upon long-term hexarelin treatment despite sterol depletion. Increased expression of Insig-1 and Insig-2 in HepG2 cells was dependent on nuclear receptor PPARγ activation by hexarelin, providing a role for PPARγ in upregulating Insig expression. This study provides a mechanistic basis by which CD36 modulates HMG-CoA reductase function and Insig expression through activation of PPARγ, thereby supporting a novel role of scavenger receptor CD36 to regulate cholesterol synthesis in hepatocytes.

Sterol-induced dislocation of 3-hydroxy-3-methylglutaryl coenzyme A reductase from membranes of permeabilized cells

The polytopic endoplasmic reticulum (ER)-localized enzyme 3-hydroxy-3-methylglutaryl CoA reductase catalyzes a rate-limiting step in the synthesis of cholesterol and nonsterol isoprenoids. Excess sterols cause the reductase to bind to ER membrane proteins called Insig-1 and Insig-2, which are carriers for the ubiquitin ligases gp78 and Trc8. The resulting gp78/Trc8-mediated ubiquitination of reductase marks it for recognition by VCP/p97, an ATPase that mediates subsequent dislocation of reductase from ER membranes into the cytosol for proteasomal degradation. Here we report that in vitro additions of the oxysterol 25-hydroxycholesterol (25-HC), exogenous cytosol, and ATP trigger dislocation of ubiquitinated and full-length forms of reductase from membranes of permeabilized cells. In addition, the sterol-regulated reaction requires the action of Insigs, is stimulated by reagents that replace 25-HC in accelerating reductase degradation in intact cells, and is augmented by the nonsterol isoprenoid geranylgeraniol. Finally, pharmacologic inhibition of deubiquitinating enzymes markedly enhances sterol-dependent ubiquitination of reductase in membranes of permeabilized cells, leading to enhanced dislocation of the enzyme. Considered together, these results establish permeabilized cells as a viable system in which to elucidate mechanisms for postubiquitination steps in sterol-accelerated degradation of reductase.

Interactive effect of salicylic acid on some physiological features and antioxidant enzymes activity in ginger (Zingiber officinale Roscoe).

The effect of foliar salicylic acid (SA) applications (10−3 and 10−5 M) on activities of nitrate reductase, guaiacol peroxidase (POD), superoxide dismutases (SOD), catalase (CAT) and proline enzymes and physiological parameters was evaluated in two ginger varieties (Halia Bentong and Halia Bara) under greenhouse conditions. In both varieties, tested treatments generally enhanced photosynthetic rate and total dry weight. Photosynthetic rate increases were generally accompanied by increased or unchanged stomatal conductance levels, although intercellular CO2 concentrations of treated plants were typically lower than in controls. Lower SA concentrations were generally more effective in enhancing photosynthetic rate and plant growth. Exogenous application of SA increased antioxidant enzyme activities and proline content; the greatest responses were obtained in plants sprayed with 10–5 M SA, with significant increases observed in CAT (20.1%), POD (45.2%), SOD (44.1%) and proline (43.1%) activities. Increased CAT activity in leaves is naturally expected to increase photosynthetic efficiency and thus net photosynthesis by maintaining a constant CO2 supply. Our results support the idea that low SA concentrations (10–5 M) may induce nitrite reductase synthesis by mobilizing intracellular NO3− and can provide protection to nitrite reductase degradation in vivo in the absence of NO3–. Observed positive correlations among proline, SOD, CAT and POD activities in the studied varieties suggest that increased SOD activity was accompanied by increases in CAT and POD activities because of the high demands of H2O2 quenching.

β-Ionone Induces Cell Cycle Arrest and Apoptosis in Human Prostate Tumor Cells

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the rate-limiting activity in the mevalonate pathway that provides essential intermediates for posttranslational modification of growth-associated proteins. Assorted dietary isoprenoids found in plant foods suppress HMG CoA reductase and have cancer chemopreventive activity. β-Ionone, a cyclic sesquiterpene and an end-ring analog of β-carotene, induced concentration-dependent inhibition of the proliferation of human DU145 (IC50 = 210 μmol/L) and LNCaP (IC50 = 130 μmol/L) prostate carcinoma cells and PC-3 prostate adenocarcinoma cells (IC50 = 130 μmol/L). Concomitantly, β-ionone-induced apoptosis and cell cycle arrest at the G1 phase in DU145 and PC-3 cells were shown by fluorescence microscopy, flow cytometry, and TUNEL reaction, and downregulation of cyclin-dependent kinase 4 (Cdk4) and cyclin D1 proteins. Growth suppression was accompanied by β-ionone-induced downregulation of reductase protein. A blend of β-ionone (150 μmol/L) and trans, trans-farnesol (25 μmol/L), an acyclic sesquiterpene that putatively initiates the degradation of reductase, suppressed the net growth of DU145 cells by 73%, an impact exceeding the sum of those of β-ionone (36%) and farnesol (22%), suggesting a synergistic effect. β-ionone, individually or in combination with other HMG CoA reductase suppressors, may have potential in prostate cancer chemoprevention and/or therapy.

Abstract 2106: Gamma-tocotrienol upregulates the ceramide transporter, Arv-1, in pancreatic cancer cells.

Abstract Pancreatic cancer is the fourth leading cause of cancer-related death in the United States. Treatment options are limited and novel therapeutic agents that can inhibit signaling pathways implicated in the proliferation and survival of pancreatic cancer cells are of interest. Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side-chain that confers superior and mechanistically different anti-cancer properties on pancreatic cancer cells. The ability of tocotrienols to selectively inhibit the HMG CoA reductase pathway through post-translational degradation of HMG CoA reductase, inhibit the PI3/Akt, ERK pathways through downregulation of Her2/ErbB2 receptors as well as suppression of the activity of transcription factor NF kappa B, could be the basis for some of these properties. Recent studies have shown increase in cellular ceramide levels in cancer cells treated with tocopherols, leading to arrest in tumor proliferation and apoptosis. The actual mechanism of increase in ceramide levels is not clearly understood and is thought to be due to increased synthesis. We explored the hypothesis that the increase in ceramide on the cellular membrane could be due to increased transport from the ER to the membrane. The ARV1 (ACAT-related enzyme-2 required for viability) gene encodes an ER- localized protein involved in lipid homeostasis and ceramide transport in yeast and mammals. Deletion of the ARV-1 gene in yeast causes accumulation of ceramide in cytoplasm. Our previous work indicated that gamma-tocotrienol (GT3) has potent anti-proliferative activity in k-ras mutated pancreatic cancer cell (MIA-Paca-2) through the inhibitory effect on Akt, Ras/Raf/Erk and ErBb2 receptor as well as induction of apoptotic pathways through Fox-03 and GSK-3b. Our current data shows GT3 may decrease expression of caveolin, a cell survival marker, and cause up-regulation of ARV-1 that encodes a ceramide transport protein. We further show via HPLC (Babraham Bioscience Technologies Ltd, Cambridge, U.K) that total ceramide levels remain unchanged in the cell. There may be a change in ceramide (monoclonal anti-ceramide IgM, Sigma Chemicals) cellular distribution as indicated by immunofluorescence data. Upregulation of ARV-1 may be involved in increase in cellular ceramide transport or redistribution of ceramide to the cell membrane. Transport of ceramide to membrane lipid rafts or mitochondrial membrane channels may cause loss of survival signaling and induce apoptosis. Thus it is likely that GT3 apoptotic effect on k-ras mutated MIA-Paca2 cells is related not to changes in the total ceramide levels, but rather to its increased transport and redistribution. Further work is necessary in order to better understand the role of GT3 in ceramide redistribution in pancreatic cancer cells. Citation Format: Kanishka Chakraborty, Victoria P. Ramsauer, Janet W. Lightner, William L. Stone, Koyamangalath Krishnan. Gamma-tocotrienol upregulates the ceramide transporter, Arv-1, in pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2013-2106

The impact of geranylgeraniol on the differentiation of murine 3T3‐F442A preadipocytes

The statins, competitive inhibitors of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG CoA) reductase, suppress the differentiation of adipocytes via mevalonate deprivation. We hypothesize that geranylgeraniol, a diterpene shown to accelerate the degradation of HMG CoA reductase, mimics the impact of lovastatin in adipocytes by suppressing adipocyte differentiation and adipogenic gene expression. Adipo‐Red assay and oil Red O staining showed that a 7‐d incubation with 2.5 – 20 μmol/L geranylgeraniol dose‐dependently reduced the intracellular triglyceride content of murine 3T3‐F442A adipocytes. Concomitantly, geranylgeraniol down‐regulated the expression of peroxisome proliferator‐activated receptor γ (PPARγ), a key regulator of adipocyte differentiation as analyzed by real‐time qPCR. The expression of adipocyte marker genes including adiponectin, leptin, fatty acid binding protein 4 and lipoprotein lipase was also suppressed by geranylgeraniol. No changes in cell viability were observed in 3T3‐F442A cells incubated with geranylgeraniol (0–400 μmol/L) for 24 and 48 h. Mevalonate‐derived metabolites have essential roles in promoting adipocyte differentiation and adipogenic gene expression. Dietary mevalonate suppressors may have potential as anti‐adipogenesis compounds.

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein's cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation

Endoplasmic reticulum-associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome. The accumulation of misfolded proteins can be catastrophic at both the cellular and organismal level. Although the players involved and mechanistic details of ERAD are being characterized, much remains to be learned. Because of the complexity of the pathway, experimental studies generally require labor-intensive biochemical techniques. Here, we report the development of a system to analyze ERAD based on mutants of split or intact Venus fluorescent protein for which fluorescence depends on enzymatic deglycosylation. We have generated variants that only become fluorescent when they are first glycosylated in the ER and subsequently deglycosylated after retrotranslocation into the cytosol. The E3 ubiquitin ligase HMG-coA reductase degradation 1 homolog (Hrd1) and, consistent with the demonstrated glycosylation/deglycosylation requirement, the cytosolic deglycosylating enzyme peptide:N'glycanase are both required for fluorescence. Furthermore, although these deglycosylation-dependent fluorescent proteins are themselves ERAD substrates, they can also be fused to additional ERAD substrates to interrogate substrate-specific pathways. To validate the system we performed a genomewide siRNA screen that successfully identified known ERAD factors such as Hrd1; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP); sel-1 suppressor of lin-12-like (SEL1L); and p97. These tools should greatly facilitate the identification of ERAD components and investigation of the mechanisms involved in this critical pathway.