Regulatory Element-binding Protein Cleavage Research Articles

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 μmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 μmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

Progesterone Receptor Membrane Component 1 and its Accomplice: Emerging Therapeutic Targets in Lung Cancer.

Progesterone receptor membrane component 1 (PGRMC1) is a trans-membrane evolutionarily conserved protein with a cytochrome b5 like heme/steroid binding domain. PGRMC1 clinical levels are strongly suggested to correlate with poor patient survival and lung cancer prognosis. PGRMC1 has been reported to possess pleiotropic functions, such as participating in cellular and membrane trafficking, steroid hormone signaling, cholesterol metabolism and steroidogenesis, glycolysis and mitochondrial energy metabolism, heme transport and homeostasis, neuronal movement and synaptic function, autophagy, anti-apoptosis, stem cell survival and the list is still expanding. PGRMC1 mediates its pleiotropic functions through its ability to interact with multiple binding partners, such as epidermal growth factor receptor (EGFR), sterol regulatory element binding protein cleavage activating protein (SCAP), insulin induced gene-1 protein (Insig-1), heme binding proteins (hepcidin, ferrochelatase and cyp450 members), plasminogen activator inhibitor 1 RNA binding protein (PAIR-BP1). In this review, we provide a comprehensive overview of PGRMC1 and its associated pleiotropic functions that are indispensable for lung cancer promotion and progression, suggesting it as a prospective therapeutic target for intervention. Notably, we have compiled and reported various preclinical studies wherein prospective agonists and antagonists had been tested against PGRMC1 expressing cancer cell lines, suggesting it as a prospective therapeutic target for cancer intervention.

Cholesterol-lowering activity of natural mono- and sesquiterpenoid compounds in essential oils: A review and investigation of mechanisms using in silico protein-ligand docking.

Mono- and sesquiterpenoids are the main chemical constituents of essential oils. Essential oils and their constituents have received increasing attention for lipid-lowering properties in both cell and animal models. Despite the chemical diversity of essential oil compounds, the effects of many of these compounds on cholesterol metabolism are highly similar. In this report, we review the literature regarding the effects of essential oils and their terpenoid constituents on cholesterol homeostasis, and explore likely mechanisms using protein-ligand docking. We identified 98 experimental and seven clinical studies on essential oils, isolated compounds, and blends; 100 of these described improvements either in blood cholesterol levels or in sterol metabolic pathways. Our review and docking analysis confirmed two likely mechanisms common to many essential oil compounds: (1) direct agonism of peroxisome-proliferator-activated receptors, and (2) direct interaction with sterol-sensing domains, motifs found in key sterol regulatory proteins including sterol regulatory element binding protein cleavage activating protein and HMG-CoA reductase. Notably, these direct interactions lead to decreased transcription and accelerated degradation of HMG-CoA reductase. Our work suggests that terpene derivatives in essential oils have cholesterol-lowering activity and could potentially work synergistically with statins, however, further high quality studies are needed to establish their clinical efficacy.

Ginsenoside F2 Reduces Hair Loss by Controlling Apoptosis through the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein and Transforming Growth Factor-β Pathways in a Dihydrotestosterone-Induced Mouse Model

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

Inflammatory Stress Induces Statin Resistance by Disrupting 3-Hydroxy-3-Methylglutaryl-CoA Reductase Feedback Regulation

The risk of cardiovascular disease is increased by up to 33 to 50× in chronic inflammatory states and convention doses of statins may not provide the same cardiovascular protection as in noninflamed patients. This study investigated whether the increase in 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA-R)-mediated cholesterol synthesis observed under inflammatory stress was resistant to the action of statins and if so, whether this was because of interference with the sterol regulatory element binding protein cleavage-activating protein pathway. Inflammatory stress was induced by adding cytokines (interleukin-1β, tumor necrosis factor-α, and interleukin-6) and lipopolysaccharides to vascular smooth muscle cells in vitro and by subcutaneous casein injection in apolipoprotein E/scavenger receptors class A/CD36 triple knockout mice in vivo. Inflammatory stress exacerbated cholesterol ester accumulation and was accompanied in vitro and in vivo by increased HMGCoA-R mRNA and protein expression mediated via activation of the sterol regulatory element binding protein cleavage-activating protein/sterol regulatory element binding protein-2 pathway. Atorvastatin reduced HMGCoA-R enzymatic activity and intracellular cholesterol synthesis in vitro. However, inflammatory stress weakened these suppressive effects. Atorvastatin at concentrations of 16 μmol/L inhibited HMGCoA-R activity by 50% in vascular smooth muscle cells, but the same concentration resulted in only 30% of HMGCoA-R activity in vascular smooth muscle cells in the presence of interleukin-1β. Knocking down sterol regulatory element binding protein cleavage-activating protein prevented statin resistance induced by interleukin-1β, and overexpression of sterol regulatory element binding protein cleavage-activating protein induced statin resistance even without inflammatory stress. In vivo, the amount of atorvastatin required to lower serum cholesterol and decrease aortic lipid accumulation rose from 2 to 10 mg/kg per day in the presence of inflammatory stress. Increased cholesterol synthesis mediated by HMGCoA-R under inflammatory stress may be one of the mechanisms for intracellular lipid accumulation and statin resistance.

Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression

Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many cancers. However, the precise molecular composition of lipids in tumors remains generally poorly characterized. The aim of the present study was to analyze the global lipid profiles of breast cancer, integrate the results to protein expression, and validate the findings by functional experiments. Comprehensive lipidomics was conducted in 267 human breast tissues using ultraperformance liquid chromatography/ mass spectrometry. The products of de novo fatty acid synthesis incorporated into membrane phospholipids, such as palmitate-containing phosphatidylcholines, were increased in tumors as compared with normal breast tissues. These lipids were associated with cancer progression and patient survival, as their concentration was highest in estrogen receptor-negative and grade 3 tumors. In silico transcriptomics database was utilized in investigating the expression of lipid metabolism related genes in breast cancer, and on the basis of these results, the expression of specific proteins was studied by immunohistochemistry. Immunohistochemical analyses showed that several genes regulating lipid metabolism were highly expressed in clinical breast cancer samples and supported also the lipidomics results. Gene silencing experiments with seven genes [ACACA (acetyl-CoA carboxylase α), ELOVL1 (elongation of very long chain fatty acid-like 1), FASN (fatty acid synthase), INSIG1 (insulin-induced gene 1), SCAP (sterol regulatory element-binding protein cleavage-activating protein), SCD (stearoyl-CoA desaturase), and THRSP (thyroid hormone-responsive protein)] indicated that silencing of multiple lipid metabolism-regulating genes reduced the lipidomic profiles and viability of the breast cancer cells. Taken together, our results imply that phospholipids may have diagnostic potential as well as that modulation of their metabolism may provide therapeutic opportunities in breast cancer treatment.

Progesterone Receptor Membrane Component 1 (Pgrmc1): A Heme-1 Domain Protein That Promotes Tumorigenesis and Is Inhibited by a Small Molecule

Tumorigenesis requires the concerted action of multiple pathways, including pathways that stimulate proliferation and increase metabolism. Progesterone receptor membrane component 1 (Pgrmc1) is related to cytochrome b5, binds to heme, and is associated with DNA damage resistance and apoptotic suppression. Pgrmc1 is induced by carcinogens, including dioxin, and is up-regulated in multiple types of cancer. In the present study, we found that Pgrmc1 increased in vivo tumor growth, anchorage-independent growth, and migration. Pgrmc1 also promoted proliferation in the absence of serum in A549 non-small cell lung cancer cells but enhanced proliferation regardless of serum concentration in MDA-MB-468 breast cancer cells. Pgrmc1 promotes cholesterol synthesis and binds to Insig (insulin-induced gene), Scap (sterol regulatory element binding protein cleavage activating protein), and P450 proteins, but Pgrmc1 did not affect cholesterol synthesis in lung cancer cells. Pgrmc1 is also associated with progesterone signaling and plasminogen activator inhibitor (PAI1) RNA binding protein, but neither progesterone activity nor PAI1 transcript levels were altered in Pgrmc1-knockdown lung cancer cells. Pgrmc1 homologues bind to aryl ligands identified in an in silico screen, and we have found that a Pgrmc1 ligand induced cell death in a Pgrmc1-specific manner in multiple breast and lung tumor cell lines. Our data support a role for Pgrmc1 in promoting cancer-associated phenotypes and provide a therapeutic approach for targeting Pgrmc1 with a small molecule in lung and breast cancer.

Cyclodextrin overcomes deficient lysosome-to-endoplasmic reticulum transport of cholesterol in Niemann-Pick type C cells

A handoff model has been proposed to explain the egress from lysosomes of cholesterol derived from receptor-mediated endocytosis of LDL. Cholesterol is first bound by soluble Niemann-Pick C2 (NPC2) protein, which hands off the cholesterol to the N-terminal domain of membrane-bound NPC1. Cells lacking NPC1 or NPC2 accumulate LDL-derived cholesterol in lysosomes and fail to deliver LDL cholesterol to the endoplasmic reticulum (ER) for esterification by acyl-CoA acyltransferase (ACAT) and for inhibition of sterol regulatory element-binding protein cleavage. Here, we support this model by showing that the cholesterol transport defect in NPC1 mutant cells is restricted to lysosomal export. Other cholesterol transport pathways appear normal, including the movement of cholesterol from the plasma membrane to the ER after treatment of cells with 25-hydroxycholesterol or sphingomyelinase. The NPC1 or NPC2 block in cholesterol delivery to the ER can be overcome by 2-hydroxypropyl-beta-cyclodextrin, which leads to a marked increase in ACAT-mediated cholesterol esterification. The buildup of cholesteryl esters in the cytosol is expected to be much less toxic than the buildup of free cholesterol in the lysosomes of patients with mutations in NPC1 or NPC2.

The relationship of sterol regulatory element–binding protein cleavage–activation protein and apolipoprotein E gene polymorphisms with metabolic changes during weight reduction

Sterol regulatory element–binding protein cleavage–activating protein (SCAP) and apolipoprotein E (apo E) regulate cellular and plasma lipid metabolism. Therefore, variations in the corresponding genes might influence weight reduction and obesity-associated metabolic changes. We investigated the relationships of SCAP (Ile796Val) and apo E polymorphisms on metabolic changes during weight reduction by using a 12-week very low-energy diet. Body composition, serum lipids, plasma glucose, and insulin were assessed in 78 healthy premenopausal women (initial body mass index, 34 ± 4 kg/m 2; age, 40 ± 4 years) before and after the intervention. The SCAP genotype groups did not differ in the responses of any parameters measured during weight reduction. Apo E did not differentiate the weight loss, but the changes in total and low-density lipoprotein cholesterol for the genotype groups apo E ε2/3, ε3/3, as well as ε3/4 and ε4/4 combined were −0.94 ± 0.56 and −0.59 ± 0.32, −0.71 ± 0.49 and −0.49 ± 0.45, and −0.55 ± 0.47 and −0.37 ± 0.39 mmol/L, respectively ( P < .05 for both). In conclusion, neither the SCAP Ile796Val nor the apo E polymorphism was associated with weight loss in obese premenopausal women. However, the apo E—but not SCAP genotype—seems to be one of the modifying factors for serum cholesterol concentrations during very low-energy diet in obese premenopausal women.

Evidence That the “NF” Motif in Transmembrane Domain 4 of Presenilin 1 Is Critical for Binding with PEN-2

Macromolecular complexes containing presenilins (PS1 and PS2), nicastrin, anterior pharynx defective phenotype 1 (APH-1), and PS enhancer 2 (PEN-2) mediate the intramembranous, gamma-secretase cleavage of beta-amyloid precursor protein (APP), Notch, and a variety of type 1 membrane proteins. We previously demonstrated that PEN-2 is critical for promoting endoproteolysis of PS1 and that the proximal two-thirds of transmembrane domain (TMD) 1 of PEN-2 is required for binding with PS1. In this study, we sought to identify the structural domains of PS1 that are necessary for binding with PEN-2. To address this issue, we generated a series of constructs encoding PS1 mutants harboring deletions or replacements of specific TMDs of PS1-NTF, and examined the effects of encoded molecules on interactions with PEN-2, stabilization and endoproteolysis of PS1, and gamma-secretase activity. We now show that PS1 TMDs 1 and 2 and the intervening hydrophilic loop are dispensable for binding to PEN-2. Furthermore, analysis of chimeric PS1 molecules that harbor replacements of each TMD with corresponding transmembrane segments from the sterol regulatory element-binding protein cleavage activating protein (SCAP) revealed that the PS1-SCAP TMD4 mutant failed to coimmunoprecipitate endogenous PEN-2, strongly suggesting that the fourth TMD of PS1 is required for interaction with PEN-2. Further mutational analyses revealed that the "NF" sequence within the TMD4 of PS1 is the minimal motif that is required for binding with PEN-2, promoting PS1 endoproteolysis and gamma-secretase activity.

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway.

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.

Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway

Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway

Inhibition of sterol regulatory element-binding protein cleavage by the prodomain of human SKI-1 protease

Inhibition of sterol regulatory element-binding protein cleavage by the prodomain of human SKI-1 protease

Related membrane domains in proteins of sterol sensing and cell signaling provide a glimpse of treasures still buried within the dynamic realm of intracellular metabolic regulation.

Recent discoveries in the regulation of cholesterol metabolism have documented a two step proteolytic pathway that regulates nuclear targeting of the sterol regulatory element binding proteins. Sterol regulatory element binding protein cleavage activating protein is a newly identified protein that modulates the proteolytic maturation of the sterol regulatory element binding proteins. It contains a domain that is quite similar in sequence to the membrane spanning region of the rate controlling enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. The membrane domain of the reductase is involved in its post-translational regulation by cholesterol. The molecular defect in the intracellular cholesterol storage disease, Niemann-Pick type C, has also recently been identified. Surprisingly, the affected gene encodes a protein with similarity to the membrane domains that are conserved in 3-hydroxy-3-methylglutaryl reductase and sterol regulatory element binding protein cleavage activating protein. Furthermore, the cell surface receptor for the sterol modified hedgehog morphogen, Patched, also contains a membrane domain with significant similarity to this putative sterol monitoring domain. These recent developments suggest a common mechanism for sensing intracellular sterol levels and cell signaling, which is based on the function of related membrane domains that are contained in key regulatory proteins.