Structural basis for catalysis and substrate specificity of human ACAT1.

As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer's disease and cancer2-7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8.

SOAT1 in gallbladder cancer: Clinicopathological significance and avasimibe therapeutics.

The aim of this investigation was to evaluate the differential expression of the sterol O-acyltransferase 1 (SOAT1) protein in gallbladder cancer tissues and cells, investigate the impact of Avastin on the proliferation, migration, invasion capabilities of gallbladder cancer cells, and its potential to induce cell apoptosis. Immunohistochemical analysis of samples from 145 gallbladder cancer patients was conducted, along with analysis of SOAT1 protein, mRNA expression levels, and cholesterol content in gallbladder cancer cell lines SGC-996, NOZ, and gallbladder cancer (GBC)-SD using Western blot and q-PCR techniques. Furthermore, the effects of Avastin on the proliferation, migration, and invasion capabilities of these gallbladder cancer cell lines were studied, and its ability to induce cell apoptosis was evaluated using flow cytometry, Western blot, and immunohistochemical methods. Additionally, gene expression and pathway analysis were performed, and the synergistic therapeutic effects of Avastin combined with gemcitabine were tested in a gallbladder cancer xenograft model. The study found that SOAT1 expression was significantly upregulated in GBC tissues and positively correlated with lymph node metastasis and TNM staging. In vitro experiments demonstrated that Avastin significantly inhibited the proliferation, migration, and invasion capabilities of SGC-996 and GBC-SD cell lines and induced apoptosis. RNA sequencing analysis revealed multiple differentially expressed genes in cells treated with Avastin, primarily enriched in biological pathways such as signaling transduction, malignant tumors, and the immune system. In vivo, experiments confirmed that Avastin could effectively suppress tumor growth in a gallbladder cancer xenograft model and enhanced the treatment efficacy when used in combination with gemcitabine. Overall, these findings provide new insights and strategies for targeted therapy in gallbladder cancer.

Discovery of selective ACAT2 antagonist via a combination strategy based on deep docking, pharmacophore modelling, and molecular dynamics simulation.

Acyl-CoA: cholesterol acyltransferase (ACAT), a pivotal enzyme in the absorption and metabolism of cholesterol, is primarily responsible for intracellular esterification. ACAT inhibition is expected to diminish plasma lipid levels by impeding intestinal cholesterol absorption, thereby preventing the progression of atherosclerotic lesions. A previous study shows that selective inhibition of ACAT2 significantly mitigated hypercholesterolaemia and atherosclerosis in mouse models. Therefore, the need for ACAT2 selective inhibitors becomes particularly urgent. In this study, we established a multilayer virtual screening workflow and subjected biologically evaluated representative compounds to enzyme inhibitory assays. The experimental results indicated that the two compounds, STL565001 (inhibition rate at 25 µM: 75.7 ± 27.8%, selectivity = 6) and STL528213 (inhibition rate at 25 µM: 87.8 ± 12.4%, selectivity = 13), demonstrated robust activity against ACAT2, displaying greater selectivity for ACAT2 than for ACAT1. The molecular mechanisms governing the inhibitory activities of the selected compounds were systematically elucidated using computational approaches. In addition, hotspot residues in ACAT2 that are crucial for ligand binding were successfully identified. In summary, we devised a multilayer screening scheme to expeditiously and efficiently identify compounds with enzyme inhibitory activity, offering novel scaffolds for subsequent drug design centred on ACAT2 targets.

Sterol O-Acyltransferase 1 (SOAT1): A Genetic Modifier of Niemann-Pick Disease, Type C1.

Niemann-Pick disease type C1 (NPC1) is a lysosomal disorder due to impaired intracellular cholesterol transport out of the endolysosomal compartment.. Marked heterogeneity has been observed in individuals with the same NPC1 genotype, thus suggesting a significant effect of modifier genes. Prior work demonstrated that decreased SOAT1 activity decreased disease severity in an NPC1 mouse model. Thus, we hypothesized that a polymorphism associated with decreased SOAT1 expression might influence the NPC1 phenotype. Phenotyping and genomic sequencing of 117 individuals with NPC1 was performed as part of a Natural History trial. Phenotyping included determination of disease severity and disease burden. Significant clinical heterogeneity is present in individuals homozygous for the NPC1I1061T variant and in siblings. Analysis of the SOAT1 polymorphism, rs1044925 (A>C), showed a significant association of the C-allele with earlier age of neurological onset. The C-allele may be associated with a higher Annualized Severity Index Score as well as increased frequency of liver disease and seizures. A polymorphism associated with decreased expression of SOAT1 appears to be a genetic modifier of the NPC1 phenotype. This finding is consistent with prior data showing decreased phenotypic severity in Npc1-/-:Soat1-/- mice and supports efforts to investigate the potential of SOAT1 inhibitors as a potential therapy for NPC1.

Characterization of Stealth Liposome-Based Nanoparticles Encapsulating the ACAT1/SOAT1 Inhibitor F26: Efficacy and Toxicity Studies In Vitro and in Wild-Type Mice.

Cholesterol homeostasis is pivotal for cellular function. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), also abbreviated as SOAT1, is an enzyme responsible for catalyzing the storage of excess cholesterol to cholesteryl esters. ACAT1 is an emerging target to treat diverse diseases including atherosclerosis, cancer, and neurodegenerative diseases. F12511 is a high-affinity ACAT1 inhibitor. Previously, we developed a stealth liposome-based nanoparticle to encapsulate F12511 to enhance its delivery to the brain and showed its efficacy in treating a mouse model for Alzheimer's disease (AD). In this study, we introduce F26, a close derivative of F12511 metabolite in rats. F26 was encapsulated in the same DSPE-PEG2000/phosphatidylcholine (PC) liposome-based nanoparticle system. We employed various in vitro and in vivo methodologies to assess F26's efficacy and toxicity compared to F12511. The results demonstrate that F26 is more effective and durable than F12511 in inhibiting ACAT1, in both mouse embryonic fibroblasts (MEFs), and in multiple mouse tissues including the brain tissues, without exhibiting any overt systemic or neurotoxic effects. This study demonstrates the superior pharmacokinetic and safety profile of F26 in wild-type mice, and suggests its therapeutic potential against various neurodegenerative diseases including AD.

Sevoflurane attenuates proliferative and migratory activity of lung cancer cells via mediating the microRNA-100-3p/sterol O-Acyltransferase 1 axis.

Recently, evidence has shown that microRNA-100-3p (miR-100-3p) has been revealed as a tumor suppressor in diverse human diseases, while its capability in lung cancer warrants further validation. In this work, we aimed to discuss the impact of sevoflurane on biological functions of lung cancer cells by modulating the miR-100-3p/sterol O-acyltransferase 1 (SOAT1) axis. Lung cancer cell lines (A549 and H460) were treated with various concentrations of sevoflurane. Cell viability, proliferation, migration, and invasion were evaluated using MTT, colony formation, wound healing, and transwell assays. Moreover, miR-100-3p and SOAT1 expressions were evaluated by reverse transcription-quantitative polymerase chain reaction in lung cancer cells. The target interaction between miR-100-3p and SOAT1 was predicted by bioinformatics analysis and verified by the dual-luciferase reporter gene assay. The findings of our work demonstrated that sevoflurane impeded the abilities on viability, proliferation, migration, and invasion of A549 and H460 cells. The expression of miR-100-3p was reduced, and SOAT1 expression was elevated in lung cancer cells. miR-100-3p targeted SOAT1. Besides, sevoflurane could lead to expressed improvement of miR-100-3p or limitation of SOAT1. Downregulation of miR-100-3p or upregulation of SOAT1 restored the suppression of sevoflurane on abilities of viability, proliferation, migration, and invasion in A549 and H460 cells. In the rescue experiment, downregulation of SOAT1 reversed the impacts of downregulation of miR-100-3p on sevoflurane on lung cancer cells. Collectively, our study provides evidence that sevoflurane restrained the proliferation and invasion in lung cancer cells by modulating the miR-100-3p/SOAT1 axis. This article provides a new idea for further study of the pathogenesis of lung cancer.

Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans.

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0-37.8] mg/dl) and slightly reduced in heterozygotes (218 [153-234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.

High-affinity SOAT1 ligands remodeled cholesterol metabolism program to inhibit tumor growth.

BACKGROUND: Although cholesterol metabolism is a common pathway for the development of antitumor drugs, there are no specific targets and drugs for clinical use. Here, based on our previous study of sterol O-acyltransferase 1 (SOAT1) in hepatocelluar carcinoma, we sought to screen an effective targeted drug for precise treatment of hepatocelluar carcinoma and, from the perspective of cholesterol metabolism, clarify the relationship between cholesterol regulation and tumorigenesis and development. METHODS: In this study, we developed a virtual screening integrated affinity screening technology for target protein drug screening. A series of in vitro and in vivo experiments were used for drug activity verification. Multi-omics analysis and flow cytometry analysis were used to explore antitumor mechanisms. Comparative analysis of proteome and transcriptome combined with survival follow-up information of patients reveals the clinical therapeutic potential of screened drugs. RESULTS: We screened three compounds, nilotinib, ABT-737, and evacetrapib, that exhibited optimal binding with SOAT1. In particular, nilotinib displayed a high affinity for SOAT1 protein and significantly inhibited tumor activity both in vitro and in vivo. Multi-omics analysis and flow cytometry analysis indicated that SOAT1-targeting compounds reprogrammed the cholesterol metabolism in tumors and enhanced CD8+ T cells and neutrophils to suppress tumor growth. CONCLUSIONS: Taken together, we reported several high-affinity SOAT1 ligands and demonstrated their clinical potential in the precision therapy of liver cancer, and also reveal the potential antitumor mechanism of SOAT1-targeting compounds.

Soat2 ties cholesterol metabolism to ß-oxidation and glucose tolerance in male mice.

BACKGROUND: Sterol O-acyltransferase 2 (Soat2) encodes acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2), which synthesizes cholesteryl esters in hepatocytes and enterocytes fated either to storage or to secretion into nascent triglyceride-rich lipoproteins. OBJECTIVES: We aimed to unravel the molecular mechanisms leading to reduced hepatic steatosis when Soat2 is depleted in mice. METHODS: Soat2-/- and wild-type mice were fed a high-fat, a high-carbohydrate, or a chow diet, and parameters of lipid and glucose metabolism were assessed. RESULTS: Glucose, insulin, homeostatic model assessment for insulin resistance (HOMA-IR), oral glucose tolerance (OGTT), and insulin tolerance tests significantly improved in Soat2-/- mice, irrespective of the dietary regimes (2-way ANOVA). The significant positive correlations between area under the curve (AUC) OGTT (r = 0.66, p < 0.05), serum fasting insulin (r = 0.86, p < 0.05), HOMA-IR (r = 0.86, p < 0.05), Adipo-IR (0.87, p < 0.05), hepatic triglycerides (TGs) (r = 0.89, p < 0.05), very-low-density lipoprotein (VLDL)-TG (r = 0.87, p < 0.05) and the hepatic cholesteryl esters in wild-type mice disappeared in Soat2-/- mice. Genetic depletion of Soat2 also increased whole-body oxidation by 30% (p < 0.05) compared to wild-type mice. CONCLUSION: Our data demonstrate that ACAT2-generated cholesteryl esters negatively affect the metabolic control by retaining TG in the liver and that genetic inhibition of Soat2 improves liver steatosis via partitioning of lipids into secretory (VLDL-TG) and oxidative (fatty acids) pathways.

Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids.

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA', and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

Correlation of cholesteryl ester metabolism to pathogenesis, progression and disparities in colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide characterized by disparities in age, gender, race and anatomic sites. The mechanism underlying pathogenesis, progression and disparities of CRC remains unclear. This study aims to reveal the association of expression levels of enzymes related to cholesteryl ester (CE) metabolism with pathogenesis, progression and disparities of CRC. METHODS: The differences in gene expression levels were analyzed for enzymes in CE synthesis (acyl CoA: cholesterol acyltransferase 1 and 2, ACAT1, and ACAT2), and in CE hydrolysis (neutral cholesterol ester hydrolase, NCEH1 and lysosomal acid lipase, LAL) on TNMplot platform between CRC and normal colorectal tissues (NCT) in a large cohort. The differences in protein expression levels for these enzymes were determined by Immunochemistry (IHC) performed on tissue microarray containing 96 pairs of CRC and benign colorectal tissues (BCT) from different patient populations. The expression level represented as IHC score of each enzyme was compared between CRC and BCT in entire population and populations stratified by race, gender and anatomic sites. Student's t-test, Fisher exact test and ANOVA were used for data analysis. Significant p value was set at P<0.05. RESULTS: The gene expression level of ACAT1 was significantly lower in CRC than in NCT (P = 2.15e-119). The gene expression level of ACAT2 was not statistically different between CRC and NCT. The gene expression level of LIPA (encoding LAL) was significantly higher in CRC than in NCT (P = 2.01e-14). No data was found for the gene expression level of NCEH1. The IHC score of ACAT1was significantly lower in CRC than in BCT in all studied populations and in sub site of colon, but not in that of rectum. The IHC score of ACAT2 was not statistically different between CRC and BCT. IHC score of NCEH1 was significantly higher in CRC than in BCT only in African American (AA) population. The IHC score of LAL was significantly higher in CRC than in BCT in all studied populations and in all sub sites. In addition, decreased ACAT1 in CRC significantly correlated to progression of CRC: the lower IHC score of ACAT1, the more advanced clinical stage of CRC will be. CONCLUSIONS: This study revealed that altered expression levels in enzymes related to CE metabolism highly correlate to the pathogenesis, clinical progression and disparities of CRC. The results will add revenue in elucidating mechanisms underlying progression of CRC, and shed light on seeking biomarkers and exploring therapeutic targets for CRC in a new direction.

SOAT1: A Suitable Target for Therapy in High-Grade Astrocytic Glioma?

Targeting molecular alterations as an effective treatment for isocitrate dehydrogenase-wildtype glioblastoma (GBM) patients has not yet been established. Sterol-O-Acyl Transferase 1 (SOAT1), a key enzyme in the conversion of endoplasmic reticulum cholesterol to esters for storage in lipid droplets (LD), serves as a target for the orphan drug mitotane to treat adrenocortical carcinoma. Inhibition of SOAT1 also suppresses GBM growth. Here, we refined SOAT1-expression in GBM and IDH-mutant astrocytoma, CNS WHO grade 4 (HGA), and assessed the distribution of LD in these tumors. Twenty-seven GBM and three HGA specimens were evaluated by multiple GFAP, Iba1, IDH1 R132H, and SOAT1 immunofluorescence labeling as well as Oil Red O staining. To a small extent SOAT1 was expressed by tumor cells in both tumor entities. In contrast, strong expression was observed in glioma-associated macrophages. Triple immunofluorescence labeling revealed, for the first time, evidence for SOAT1 colocalization with Iba1 and IDH1 R132H, respectively. Furthermore, a notable difference in the amount of LD between GBM and HGA was observed. Therefore, SOAT1 suppression might be a therapeutic option to target GBM and HGA growth and invasiveness. In addition, the high expression in cells related to neuroinflammation could be beneficial for a concomitant suppression of protumoral microglia/macrophages.

Sterol O-Acyltransferase Inhibition Ameliorates High-Fat Diet-Induced Renal Fibrosis and Tertiary Lymphoid Tissue Maturation after Ischemic Reperfusion Injury.

Metabolic syndrome is associated with the development of chronic kidney disease (CKD). We previously demonstrated that aged kidneys are prone to developing tertiary lymphoid tissues (TLTs) and sustain inflammation after injury, leading to CKD progression; however, the relationship between renal TLT and metabolic syndrome is unknown. In this study, we demonstrated that a high-fat diet (HFD) promoted renal TLT formation and inflammation via sterol O-acyltransferase (SOAT) 1-dependent mechanism. Mice fed a HFD prior to ischemic reperfusion injury (IRI) exhibited pronounced renal TLT formation and sustained inflammation compared to the controls. Untargeted lipidomics revealed the increased levels of cholesteryl esters (CEs) in aged kidneys with TLT formation after IRI, and, consistently, the Soat1 gene expression increased. Treatment with avasimibe, a SOAT inhibitor, attenuated TLT maturation and renal inflammation in HFD-fed mice subjected to IRI. Our findings suggest the importance of SOAT1-dependent CE accumulation in the pathophysiology of CKDs associated with TLT.

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

The 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.

Circ_RPL23A acts as a miR-1233 sponge to suppress the progression of clear cell renal cell carcinoma by promoting ACAT2.

Clear cell renal cell carcinoma (ccRCC) is a prevalent urological carcinoma with high metastatic risk. Circular RNAs (circRNAs) have been identified as effective diagnostic and therapeutic biomarkers for ccRCC. This research aims to disclose the effect and regulatory mechanism of circRNA ribosomal protein L23a (circ_RPL23A) in ccRCC. We performed quantitative real-time polymerase chain reaction (qRT-PCR) to examine circ_RPL23A, microRNA-1233 (miR-1233) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). Cell cycle progression, apoptosis, cell viability, invasion and migration, which were respectively conducted by using flow cytometry, 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT), transwell assays. The levels of ACAT2 protein and cell cycle proteins, proliferation-associated protein, and epithelial-mesenchymal transition (EMT) associated proteins were measured by western blot. Target relationship was analyzed via dual-luciferase reporter assay and RNA pull down assay. The animal model was used to study how circ_RPL23A affects in vivo. Circ_RPL23A was lower expressed in ccRCC tissues and cells. The elevated circ_RPL23A suppressed cell cycle progression, proliferation, migration and invasion but promoted apoptosis in ccRCC cells. MiR-1233 was a target of circ_RPL23A and direct targeted to ACAT2. Besides, circ_RPL23A exerted its anti-tumor effect by sponging miR-1233, and then relieved the inhibition effect of miR-1233 on ACAT2. Overexpression of circ_RPL23A also curbed ccRCC tumor growth in vivo. Circ_RPL23A inhibited ccRCC progression by upregulating ACAT2 expression by competitively binding miR-1233, which might provide an in-depth cognition for ccRCC pathogenesis and circ_RPL23A might be a promising biomarker in ccRCC diagnosis and treatment.

Impacts of the SOAT1 genetic variants and protein expression on HBV-related hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) remains a major public health problem and its pathogenesis remains unresolved. A recent proteomics study discovered a lipid enzyme Sterol O-acyltransferase (SOAT1) involvement in the progression of HCC. We aimed to explore the association between SOAT1 genetic variation and HCC. METHODS: We genotyped three exonic SOAT1 variants (rs10753191, V323V; rs3753526, L475L; rs13306731, Q526R) tagging most variations in the gene, in 221 HCC patients and 229 healthy individuals, to assess the impact of SOAT1 gene variation on risk of HCC occurrence. We further conducted immunohistochemistry to compare SOAT1 protein expression levels in 42 paired tumor and adjacent non-tumor tissues. RESULTS: We found that rs10753191 (Odds ratio (OR) = 0.58, P = 0.04) and a haplotype TGA (OR = 0.40, P = 0.01) were associated with reduced HCC risk after adjusting for lipid levels. In the immunohistochemistry experiment, we found that the protein expression of SOAT1 was significantly increased in the tumor compared with adjacent tissue (P < 0.001). CONCLUSION: This study revealed for the first time SOAT1 genetic variation that associates with host susceptibility to HCC occurrence. Our results suggest a role of SOAT1 in the HCC development, which warrants further elucidation.

Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos.

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

Novel lecithin: cholesterol acyltransferase-based therapeutic approaches.

PURPOSE OF REVIEW: To review recent lecithin:cholesterol acyltransferas (LCAT)-based therapeutic approaches for atherosclerosis, acute coronary syndrome, and LCAT deficiency disorders. RECENT FINDINGS: A wide variety of approaches to using LCAT as a novel therapeutic target have been proposed. Enzyme replacement therapy with recombinant human LCAT is the most clinically advanced therapy for atherosclerosis and familial LCAT deficiency (FLD), with Phase I and Phase 2A clinical trials recently completed. Liver-directed LCAT gene therapy and engineered cell therapies are also another promising approach. Peptide and small molecule activators have shown efficacy in early-stage preclinical studies. Finally, lifestyle modifications, such as fat-restricted diets, cessation of cigarette smoking, and a diet rich in antioxidants may potentially suppress lipoprotein abnormalities in FLD patients and help preserve LCAT activity and renal function but have not been adequately tested. SUMMARY: Preclinical and early-stage clinical trials demonstrate the promise of novel LCAT therapies as HDL-raising agents that may be used to treat not only FLD but potentially also atherosclerosis and other disorders with low or dysfunctional HDL.

Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions.

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1-M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE-/-) mouse model for early lesions, Acat1-M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1-M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1-M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1-M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE-/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.