acyl-CoA:cholesterol Acyltransferase Research Articles

System Bioinformatic Approach Through Molecular Docking, Network Pharmacology and Microarray Data Analysis to Determine the Molecular Mechanism Underlying the Effects of Rehmanniae Radix Praeparata on Cardiovascular Diseases.

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide. Rehmanniae Radix Praeparata (RRP) is a popular medicinal herb widely used in traditional Chinese medicine (TCM) to treat CVDs. However, the development of this novel therapeutic product has been stagnant, and its molecular mechanism of action remains unclear. This study aims to explore the effective ingredients of RRP against CVDs, especially atherosclerosis (AS). Using the AutoDock Vina software, the RRP's ingredients were docked with the targets which can be collected by RCSB and UniProt. Then the screened ingredients and targets could be used to dispose the pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG). We used GEO, GCBI and DAVID databases to analyze the microarray data of AS which could be used to verify the results of molecular docking, all of which could show the molecular mechanism of RRP on CVDs. We also constructed a compound-target interaction network of CVD with 85 nodes and 272 edges on the basis of molecular docking analysis through Cytoscape. The network showed that forsythiaside, acteoside and stigmasterol are the most important compounds and 2HRR (ACAT (Acyl-CoA cholesterol acyl transferase) protein), 4ATB (MMP13) and 1JBQ (cystathionine beta-synthase) are the most valuable targets in the action of RRP against CVD. We also examined the biological functions involved in the biological process, molecular function and cellular components. In accordance with the analysis of GSE6054 microarray data of AS disease, the 20 most specifically expressed genes (differentially expressed genes [DEGs]) and the top 10 pathways of DEGs were discovered. Five key pathways, including non-alcoholic fatty liver disease (NAFLD), pathways in cancer and PI3K-Akt signalling pathway were also explored. Amongst these pathways, the top three were the pathways in cancer, MAPK signalling pathway and human T-cell lymphotropic virus infection. The pathways in cancer and PI3K-Akt signalling pathway were found simultaneously in the pathway analysis for CVD on RRP and for AS on microarray data. This study provided a new potential herbal medicine against CVD and has increased the understanding on the molecular mechanisms of RRPmediated protection against CVD, especially AS.

A Secreted Phospholipase A2 Induces Formation of Smooth Muscle Foam Cells Which Transdifferentiate to Macrophage-Like State.

Vascular smooth muscle cells (VSMCs) loaded with lipid droplets (LDs) are markers of atherosclerosis. In this disease, inflammatory Group IIA-secreted phospholipase A2s (GIIA sPLA2s) are highly expressed in VSMCs, but their actions in these cells are unknown. Here, we investigated the ability of myotoxin III (MT-III), an ophidian GIIA sPLA2 sharing structural and functional features with mammalian GIIA sPLA2s, to induce LD formation and lipid metabolism factors involved in this effect. Modulation of VSMC phenotypes by this sPLA2 was also evaluated. Incubation of VSMCs with MT-III significantly increased the number of LDs. MT-III upregulated scavenger receptor type 1 (SR-A1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) protein expression and enhanced acetylated-low density lipoprotein (acLDL) uptake by VSMCs, revealing the ability of a GIIA PLA2 to modulate scavenger receptor activities. MT-III induced translocation and protein expression of PPAR-γ and -β/δ. Inhibition of peroxisome proliferator-activated receptors (PPARs) and diacylglycerol O-acyltransferase (DGAT) and acyl-CoA:cholesterolacyltransferase (ACAT) enzymes abrogated MT-III-induced LD formation. Moreover, in response to MT-III, VSMCs acquired phagocytic activity and expressed macrophage markers CD68 and MAC-2. In conclusion, MT-III is able to stimulate VSMCs and recruit factors involved in lipid uptake and metabolism, leading to the formation of VSMC-derived foam cells with acquisition of macrophage-like markers and functions.

Comparative Analysis of the Characteristics of Triterpenoid Transcriptome from Different Strains of Wolfiporia cocos.

The dried sclerotia of Wolfiporia cocos (Schwein.) Ryvarden & Gilb., a traditional Chinese medicine, has triterpenoid as its main active component. Breeding high-yield triterpenoid in W. cocos is an important research topic at present. We screened out two monosporal strains from the same W. cocos 5.78, high-yielding DZAC-Wp-H-29 (H) and low-yielding DZAC-Wp-L-123 (L), and cultured mycelia for 17 days, 34 days, and 51 days, respectively. Transcriptome analysis results showed that triterpenoid synthesis is closely related to gene expression in triterpenoid synthesis pathways (hydroxymethyl glutaryl-CoA reductase (HMGCR), farnesyl diphosphate synthase (FDPS), 4-hydroxybenzoate polyprenyltransferase (COQ2), C-8 sterol isomerase (ERG2), sterol O-acyltransferase (ACAT), tyrosine aminotransferase (TAT), torulene dioxygenase (CAO2), and sterol-4alpha-carboxylate 3-dehydrogenase (erg26)), and is limited by the expression of enzyme M20 combined with domain protein peptide (Pm20d2), aryl-alcohol dehydrogenase (norA), ISWI chromatin-remodeling complex ATPase ISW2, GroES-like protein (adh), cytochrome P450 (ftmP450-1), and unknown proteins unigene0001029 and unigene0011374. In addition, maintaining high triterpenoid accumulation in W. cocos may require a stable membrane structure, so the accumulation ability may be related to the high synthesis ability of sterols. The low accumulation of triterpenoid in W. cocos may be due to the products of key enzymes increasing flow to other pathways.

Physiological status of silver carp (Hypophthalmichthys molitrix) in the Illinois River: An assessment of fish at the leading edge of the invasion front

Silver carp (Hypophthalmichthys molitrix) are invasive to North America, and their range has expanded within the Mississippi River Basin, seemingly unchecked, since their introduction in the late 1970s, with the exception of the upper reaches of the Illinois River. With the imminent threat of their movement into the Great Lakes, the goal of the present study was to assess whether differences in the physiological status between silver carp at the leading edge of their invasion front and core population sites could explain their lack of expansion upstream toward Lake Michigan over the past decade. A transcriptomic approach using RNA sequencing and analysis of plasma variables were used to quantify differences among fish at the leading edge and two downstream core population sites. Leading-edge fish exhibited upregulation of genes associated with xenobiotic defense (e.g., ATP-binding cassette C1 [abcc1], abcc2, abcc6), decreased cell integrity (i.e., macroautophagy and apoptosis; autophagy-related protein 9A [atg9a], caspase 3b [casp3b]), and cholesterol metabolism (e.g., abca1, apolipoprotein A1 [apoa1], sterol O-acyltransferase [soat1]) and downregulation of genes associated with DNA repair (e.g., tumor suppressor p53-binding protein 1 [tp53bp1]) compared to core population sites. Transcriptomic profiles of leading-edge fish were consistent with fish inhabiting a polluted environment and suggest that poorer water quality conditions upstream of the leading edge may represent a non-permanent barrier to silver carp range expansion. The present study provides potential molecular targets for monitoring the physiological status of silver carp over time and in response to future improvements in water quality upstream of their leading edge.

ATP-binding cassette A1 deficiency causes cardiolipin-driven mitochondrial dysfunction in podocytes.

Fibroblasts from patients with Tangier disease carrying ATP-binding cassette A1 (ABCA1) loss-of-function mutations are characterized by cardiolipin accumulation, a mitochondrial-specific phospholipid. Suppression of ABCA1 expression occurs in glomeruli from patients with diabetic kidney disease (DKD) and in human podocytes exposed to DKD sera collected prior to the development of DKD. We demonstrated that siRNA ABCA1 knockdown in podocytes led to reduced oxygen consumption capabilities associated with alterations in the oxidative phosphorylation (OXPHOS) complexes and with cardiolipin accumulation. Podocyte-specific deletion of Abca1 (Abca1fl/fl) rendered mice susceptible to DKD, and pharmacological induction of ABCA1 improved established DKD. This was not mediated by free cholesterol, as genetic deletion of sterol-o-acyltransferase-1 (SOAT1) in Abca1fl/fl mice was sufficient to cause free cholesterol accumulation but did not cause glomerular injury. Instead, cardiolipin mediates ABCA1-dependent susceptibility to podocyte injury, as inhibition of cardiolipin peroxidation with elamipretide improved DKD in vivo and prevented ABCA1-dependent podocyte injury in vitro and in vivo. Collectively, we describe a pathway definitively linking ABCA1 deficiency to cardiolipin-driven mitochondrial dysfunction. We demonstrated that this pathway is relevant to DKD and that ABCA1 inducers or inhibitors of cardiolipin peroxidation may each represent therapeutic strategies for the treatment of established DKD.

FNDC5 inhibits foam cell formation and monocyte adhesion in vascular smooth muscle cells via suppressing NFκB-mediated NLRP3 upregulation

Foam cell formation and monocytes adhesion are key events in pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMCs) are an important origin of foam cells besides macrophages. Fibronectin type III domain containing protein 5 (FNDC5) is a protein, which induces browning of fat and attenuates glucose/lipid metabolic derangements in obese mice. The present study was designed to determine the roles of FNDC5 in inhibiting foam cell formation and monocyte adhesion in VSMCs and its underlying mechanisms. Oxidized low-density lipoprotein (oxLDL) was used to induce foam cell formation and monocyte adhesion in human aortic VSMCs. Foam cell formation was evaluated by intracellular lipid droplets, cholesterol contents, and mRNA levels of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT-1) and ATP binding cassette transporter A-1 (ABCA-1). Monocyte adhesion was evaluated by the number of monocytes adhered to VSMCs and mRNA levels of monocyte chemotactic protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1). FNDC5 inhibited oxLDL-induced foam cell formation, monocyte adhesion, ABCA-1 mRNA downregulation, and ACAT-1, MCP-1 and VCAM-1 mRNA upregulation in VSMCs. It inhibited oxLDL-induced p65-NFκB nuclear translocation, NLRP3 upregulation, caspase-1 and IL-1β production. Inhibition of NFκB with BMS-345541 or inhibition of NLRP3 inflammasome with MCC950 showed similar effects to FNDC5 in attenuating the oxLDL-induced foam cell formation, monocyte adhesion, and caspase-1 and IL-1β production. The oxLDL-induced NLRP3 upregulation was prevented by BMS-345541 rather than MCC950. These results indicate that FNDC5 inhibits oxLDL-induced foam cell formation and monocyte adhesion in VSMCs via suppressing NFκB-mediated NLRP3 upregulation and IL-1β production.

Acyl-Coenzyme A: Cholesterol Acyltransferase Inhibition in Cancer Treatment.

Overexpression of acyl-coenzyme A:cholesterol acyltransferase (ACAT) results in increased cholesteryl ester levels and has been involved in a variety of cancer types. As a consequence, cholesterol metabolism has raised interest as a potential target for cancer treatment. Inhibition of ACAT results in suppression of proliferation in a range of cancer cell types both in vitro and in vivo. The exact mechanism of this phenomenon is being investigated, and the most important findings are presented in this review.

Inhibition of neutral lipid synthesis by avarols from a marine sponge.

The effects of 14 sesquiterpene hydroquinones, including 8 marine sponge-derived avarols (1-8) and 6 semisynthetic derivatives (9-14), on lipid droplet accumulation and neutral lipid synthesis in Chinese hamster ovary (CHO) K1 cells were investigated. In intact CHO-K1 cell assays, avarol (1) markedly decreased the number and size of lipid droplets in CHO-K1 cells and exhibited the most potent inhibitory activity on the synthesis of cholesteryl ester (CE) and triglyceride (TG) with IC50 values of 5.74 and 6.80 µM, respectively. In enzyme assays, sterol O-acyltransferase (SOAT), the final enzyme involved in CE biosynthesis, and diacylglycerol acyltransferase (DGAT), the final enzyme involved in TG biosynthesis, were inhibited by 1 with IC50 values of 7.31 and 20.0 µM, respectively, which correlated well with those obtained in the intact cell assay. These results strongly suggest that 1 inhibited SOAT and DGAT activities in CHO-K1 cells, leading to a reduction in the accumulation of CE and TG in lipid droplets.

Biological activity of some ACAT inhibitors in the light of DFT-based quantum descriptors

A detailed investigation is performed for a series of 24 aminosulfonyl carbamates as acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitors. The biological activity of aminosulfonyl carbamates in terms of log (IC50) has been estimated by developing suitable quantum chemical descriptors in the light of density functional theory. In order to understand inhibitor-biosystem interactions and to identify potential descriptors, a model biosystem is considered which comprises five different nucleic acid bases. Accordingly, two global descriptors, viz. electron affinity (EA) and energy transfer during the interaction between inhibitors and biosystems (∆E), and a local descriptor, viz. group atomic charges on sulfonyl moiety (∑QSul), are developed toward understanding bio-activity of aminosulfonyl carbamates. It is noteworthy to achieve that the three-parameter regression model for considered parameters EA, ∆E, and ∑QSul is observed to predict about 90% activities of aminosulfonyl carbamates in terms of log(IC50). The developed regression model is also statistically validated and successfully tested for unknown compounds.

The effects of Crataegus pinnatifida (Chinese hawthorn) on metabolic syndrome: A review.

Metabolic syndrome is described as a group of risk factors in which at least three unhealthy medical conditions, including obesity, high blood sugar, hypertension or dyslipidemia occur simultaneously in a patient. These conditions raise the risk for diabetes mellitus and cardiovascular diseases. Many recent studies have focused on herbal remedies and their pharmacological effects on metabolic syndrome. Crataegus pinnatifida or Chinese hawthorn has been widely used in the treatment of hyperlipidemia and cardiovascular diseases. Its leaves, fruits and seeds have various active substances such as, flavonoids, triterpenic acids and sesquiterpenes, which through different mechanisms can be beneficial in metabolic syndrome. Flavonoids found in the leaves of hawthorn can significantly reduce atherosclerotic lesion areas, the fruit extracts contain two triterpenic acids (oleanolic acid and ursolic acid), that have the ability to inhibit the acyl-coA-cholesterol acyltransferase (ACAT) enzyme and as a result reduce very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterol levels. Another example regards a sesquiterpene found in the seeds of C. pinnatifida, which exhibits the ability to inhibit platelet aggregation, thus showing antithrombotic activity. Various studies have shown that C. pinnatifida can have beneficial effects on controlling and treating high blood sugar, dyslipidemia, obesity and atherosclerosis. The aim of this review is to highlight the interesting effects of C. pinnatifida on metabolic syndrome.

Nonglucuronidated Ezetimibe Disrupts CD13- and CD64-Coassembly in Membrane Microdomains and Decreases Cellular Cholesterol Content in Human Monocytes/Macrophages.

Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.

SAT-354 Characterization of Cell Death Induced by SOAT1 Inhibition in Adrenocortical Carcinoma Cells

Background: Mitotane is the only drug approved for treatment of adrenocortical carcinoma (ACC). We have found that mitotane inhibits Sterol-O-Acyl transferase 1 (SOAT1) which leads to depletion of cholesterol esters and increase of free cholesterol in the ACC cell line NCI-H295. We demonstrated activation of endoplasmic reticulum response pathway that resulted in decreased cell viability. Aim: To characterize the mechanisms underlying cell death resulting from SOAT1 inhibition with inhibitors mitotane, ATR101, AZD3988 and Sandoz58-035. Methods: SOAT1 inhibition was quantified in vitro, ER-stress marker expression by qPCR, cell viability by cell titer glo assay, lipid composition by GC-MS, and lipid peroxidation with BODIPY 581/591 C11. Cell death in NCIH295R cells induced by mitotane was determined by mitotane treatment alone and in combination with either the pan caspase inhibitor zvad-fmk, necroptose inhibitor necrostatin-1, ferroptosis inhibitor liproxstatin or ferroptosis activators erastin and RSL-3. Results: Mitotane and ATR101 but not AZD3988 and Sandoz58-035 induced lipid peroxidation in NCI-H295R cells in a concentration dependent manner. ATR101, AZD3988 and Sandoz58-035 were inhibitors of SOAT1 with IC50 of 3 nM, 13 nM and 0.9 nM. Correspondingly, expression of ER-stress marker CHOP and splicing of X-box protein 1 mRNA was activated by mitotane, ATR101 and poorly by AZD3988. Mitotane and ATR101 but not AZD3988 and Sandoz58-035 treatment induced the accumulation of free cholesterol in NCI-H295 cells after 6h. Cell viability was impaired with mitotane, ATR101 and AZD3988 but not Sandoz58-035. None of the inhibitors could reverse the effects of mitotane while synergistic effects were observed for RSL3. Erastin was shown to reverse the effects of mitotane treatment. Conclusion: Although SOAT1 inhibition was confirmed for all compounds, downstream effects on ER-stress markers and cell viability exhibit marked differences. Hence it is likely that targets different from SOAT1 are relevant for in vitro cytotoxic activity of mitotane and ATR101. Caspases do not seem to be predominantly involved in mitotane-induced cell death. Although we observed a synergistic effect of mitotane with RSL3, ferroptosis inhibitors were not able to block mitotane-induced cell death. The precise mechanism of cell death and immunogenic cell death pathways require further investigation.

SUN-350 Sterol-O-Acyl Transferase 1 Protein Expression Alone Is Not Sufficient to Predict Response to Mitotane Treatment in Adrenocortical Carcinoma

Background: Objective response rate to mitotane in advanced ACC is only ~20% and adverse drug effects are frequent and potentially serious. Markers of treatment response are not established. We have previously discovered that mitotane is an inhibitor of sterol-O-Acyl transferase 1 (SOAT1) which leads to depletion of cholesterol esters, endoplasmic reticulum stress and cell death in the ACC cell line NCI-H295. Data from a small cohort of patients with advanced ACC treated with mitotane monotherapy suggested longer progression-free survival in patients with high SOAT1 expression in ACC tissue. Aim: To investigate SOAT1 protein expression as a marker of treatment response to mitotane. Methods: SOAT1 protein expression was semiquantitatively determined by immunohistochemistry in full sections of 223 ACC treated with mitotane monotherapy in an adjuvant (n=151) and palliative setting (n=72) from eleven ENSAT centers. Expression was classified as high (H-score ≥2) and low (H-score <2) and correlated with recurrence-free (RFS) and progression-free survival (PFS), overall survival (OS), and mitotane levels after three and six months. Results: After multivariate adjustment for sex, ENSAT stage and Ki67 index, RFS (HR=1.3, log rank p=0.09) and OS (HR=1.6, log rank p=0.14) in adjuvantly treated ACC patients did not differ significantly between tumours with high and low SOAT1 expression. Similarly in the palliative setting, OS (HR=1.0, p=0.47) and PFS (HR=0.6, p=0.66) were not different. In line, high SOAT1 expression did not predict if patients reach the therapeutic window of mitotane. Conclusion: SOAT1 expression alone was not correlated with clinically significantly RFS, PFS, and OS in ACC patients neither in the adjuvant nor in the palliative setting.

Resveratrol protects against hepatic insulin resistance in a rat's model of non-alcoholic fatty liver disease by down-regulation of GPAT-1 and DGAT2 expression and inhibition of PKC membranous translocation.

Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance (IR). Resveratrol (RES) a potent hypolipidemic dietary polyphenol has been identified for its ability to prevent hepatic steatosis and hepatic IR in high-fat diet (HFD)-fed murine models of NAFLD. In the present study, we have carried an in vivo animal experiment to identify a novel mechanism for RES protective action. Sub-chronic (45days) RES pretreatment in 3days HFD-fed adult Wistar rats prevented early hepatic IR through inhibiting PKC/JNK activation; decreasing p-IRS (Ser307 ) and increasing p-IRS(Tyr612 ), p-Akt(Ser473 ) and p-GSK3(Ser9 ). These effects of RES were associated with reduced expression of acyl-CoA:glycerol-sn-3-phosphate acyltransferase (GPAT-1) and diacylglycerol:acyl-CoA acyltransferase (DGAT2), two critical enzymes in the glycerol-3-phosphate pathway for de novo triglycerides synthesis. These data indicate that RES protects against NAFLD, initially, by inhibiting the early development of hepatic IR.

MiRNAs regulate the expression of sterol‐O acyltransferase 1 through inhibiting the 3′UTR of TGFβ receptor type 1 during embryonic development

Nutrients are utilized and re‐constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol‐O acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester, and for packaging into very‐low‐density lipoproteins for further delivering to growing embryos. During embryonic development, the quick absorption of yolk is matched with the significant changes of SOAT1 mRNA and enzymatic activity. MicroRNAs (miRNAs), groups of highly conserved small non‐coding RNA molecules, are usually ~23 nucleotides across species, serving classical biofunctions as mRNA‐targeting cleavage, translation repression and mRNA deadenylation. miRNAs have been identified to regulate angiogenesis, invasion and metabolism during mammalian development. However, the involvement and importance of miRNAs in lipid utilization during avian embryogenesis remains unclear.Using a RNA sequencing technique, we found many miRNAs expressed during quail embryonic development. We confirmed the expression pattern with real time PCR and several miRNAs (gga‐miR‐181a‐5p, gga‐miR‐133a‐5p, and gga‐miR‐429‐3p) were selected because they are candidates targeting to the receptor (TGFβ receptor type 1, TGFBR1) that regulates the expression of SOAT1. Similar to SOAT1 mRNA concentration, the gga‐miR‐181a‐5p expressions were gradually elevated during development, but gga‐miR‐429‐3p were presented in the opposite direction. Moreover, transfection with gga‐miR‐181a‐5p, gga‐133a‐5p, or gga‐429‐3p inhibited TGFBR1 and SOAT1 mRNA expressions in EECs. The 3′ untranslated region (3′UTR) of TGFBR1 is speculated to be one of the targets of miRNAs (gga‐miR‐181a‐5p, gga‐miR‐133a‐5p, and gga‐miR‐429‐3p).Taken together, miRNAs expression during embryonic development can regulate SOAT1 expression through inhibiting the 3′UTR of TGFBR1. This is indicative of possible regulation of avian yolk lipid utilization and modification of hatchability by modifying miRNA expression.Support or Funding InformationMinistry of Science and Technology, Taiwan (ROC)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mouse lipogenic proteins promote the co-accumulation of triacylglycerols and sesquiterpenes in plant cells.

Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.

Effect of DGAT1 variant (K232A) on milk traits and milk fat composition in outdoor pasture-grazed dairy cattle

ABSTRACT An amino acid substitution (K232A) in the enzyme diacylglycerol acyl-CoA acyltransferase 1 (DGAT1) has been reported to explain variation in milk traits and milk fatty acid (FA) profile. In this study, associations between K232A, and milk traits including milk FA composition, were studied in wholly pasture-fed New Zealand Holstein-Friesian × Jersey cross-bred (KiwicrossTM) dairy cows. With a high frequency of K variant (61.9%), the KK cows produced more milk fat on average (5.41 ± 0.04%), than the AA cows (4.43 ± 0.06%). The milk volume, fat concentration and protein concentration for the AK cows were between the values for the genotypes AA and KK. More C16 : 0 (saturated fatty acid), conjugated linoleic acid (CLA) and polyunsaturated fatty acid (C18:3 cis-9, 12, 15) were found in the milk of the KiwicrossTM cows, and the influence of DGAT1 K232A on these FAs during the mid- and late- stage of lactation were significant. The AA cows produced more (P < 0.001) CLA (1.137 ± 0.047 g/100 g) and C18:3 cis-9, 12, 15 (0.855 ± 0.015 g/100 g), but less C16:0 (35.170 ± 0.355 g/100 g) than the KK cows (0.934 ± 0.025 g/100 g, 0.778 ± 0.009 g/100 g and 38.010 ± 0.250 g/100 g respectively).

Proteomics identifies new therapeutic targets of early-stage hepatocellular carcinoma.

Hepatocellular carcinoma is the third leading cause of deaths from cancer worldwide. Infection with the hepatitis B virus is one of the leading risk factors for developing hepatocellular carcinoma, particularly in East Asia1. Although surgical treatment may be effective in the early stages, the five-year overall rate of survival after developing this cancer is only 50-70%2. Here, using proteomic and phospho-proteomic profiling, we characterize 110 paired tumour and non-tumour tissues of clinical early-stage hepatocellular carcinoma related to hepatitis B virus infection. Our quantitative proteomic data highlight heterogeneity in early-stage hepatocellular carcinoma: we used this to stratify the cohort into the subtypes S-I, S-II and S-III, each of which has a different clinical outcome. S-III, which is characterized by disrupted cholesterol homeostasis, is associated with the lowest overall rate of survival and the greatest risk of a poor prognosis after first-line surgery. The knockdown of sterol O-acyltransferase 1 (SOAT1)-high expression of which is a signature specific to the S-III subtype-alters the distribution of cellular cholesterol, and effectively suppresses the proliferation and migration of hepatocellular carcinoma. Finally, on the basis of a patient-derived tumour xenograft mouse model of hepatocellular carcinoma, we found that treatment with avasimibe, an inhibitor of SOAT1, markedly reduced the size of tumours that had high levels of SOAT1 expression. The proteomic stratification of early-stage hepatocellular carcinoma presented in this study provides insight into the tumour biology of this cancer, and suggests opportunities for personalized therapies that target it.

The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis.

ABSTRACTThe eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.This article has an associated First Person interview with the first author of the paper.

Changes of Biochemical Components Contents and Gene Expression Profiles in Tissues of Litopenaeus vannamei During Spermatophore Regeneration

Spermatophore regeneration is an important biological process in Litopenaeus vannamei. In this study, changes of biochemical components contents during spermatophore regeneration showed that in hemolymph, the glucose content significantly increased from day 2 to day 8 and then decreased from day 10 to day 12; triglyceride (TG) and total cholesterol (TC) contents were relatively stable. In the hepatopancreas, glucose, and TG contents significantly decreased during regeneration; TC contents significantly increased at day 4 and then decreased. In the testes, glucose content significantly increased while TC content significantly decreased during whole stage; TG content at day 4 was highest. In terminal ampoule, changes of glucose and TC contents were similar, the contents at day 4 were highest; TG content at day 6 was significantly lower than day 2. During spermatophore regeneration, expression level of pyruvate kinase (PK) was significantly higher during day 2 to day 4 than at other times, and expression levels of three lipogenesis-related genes diacylglycerol O-acyltransferase homolog 1 (DGAT1), sterol regulatory element-binding protein 1 (SREBP1) and sterol O-acyltransferase 2 (SOAT2) were significantly higher during day 2 to day 8 than at other times. The results indicated that the hepatopancreas could be the energy source that provides glucose and TG; glucose, TG, and TC play important roles in spermatophore regeneration