Retrospective on Cholesterol Homeostasis: The Central Role of Scap.

Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.

Proteostatic Tactics in the Strategy of Sterol Regulation.

In eukaryotes, the synthesis and uptake of sterols undergo stringent multivalent regulation. Both individual enzymes and transcriptional networks are controlled to meet changing needs of the many sterol pathway products. Regulation is tailored by evolution to match regulatory constraints, which can be very different in distinct species. Nevertheless, a broadly conserved feature of many aspects of sterol regulation is employment of proteostasis mechanisms to bring about control of individual proteins. Proteostasis is the set of processes that maintain homeostasis of a dynamic proteome. Proteostasis includes protein quality control pathways for the detection, and then the correction or destruction, of the many misfolded proteins that arise as an unavoidable feature of protein-based life. Protein quality control displays not only the remarkable breadth needed to manage the wide variety of client molecules, but also extreme specificity toward the misfolded variants of a given protein. These features are amenable to evolutionary usurpation as a means to regulate proteins, and this approach has been used in sterol regulation. We describe both well-trod and less familiar versions of the interface between proteostasis and sterol regulation and suggest some underlying ideas with broad biological and clinical applicability.

FAM120A couples SREBP-dependent transcription and splicing of lipogenesis enzymes downstream of mTORC1.

The mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that stimulates macromolecule synthesis through transcription, RNA processing, and post-translational modification of metabolic enzymes. However, the mechanisms of how mTORC1 orchestrates multiple steps of gene expression programs remain unclear. Here, we identify family with sequence similarity 120A (FAM120A) as a transcription co-activator that couples transcription and splicing of de novo lipid synthesis enzymes downstream of mTORC1-serine/arginine-rich protein kinase 2 (SRPK2) signaling. The mTORC1-activated SRPK2 phosphorylates splicing factor serine/arginine-rich splicing factor 1 (SRSF1), enhancing its binding to FAM120A. FAM120A directly interacts with a lipogenic transcription factor SREBP1 at active promoters, thereby bridging the newly transcribed lipogenic genes from RNA polymerase II to the SRSF1 and U1-70K-containing RNA-splicing machinery. This mTORC1-regulated, multi-protein complex promotes efficient splicing and stability of lipogenic transcripts, resulting in fatty acid synthesis and cancer cell proliferation. These results elucidate FAM120A as a critical transcription co-factor that connects mTORC1-dependent gene regulation programs for anabolic cell growth.

Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration.

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here, we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease.

Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses.

Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism, and suppression of inflammatory-response genes, observed in macrophage foam cells. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, proinflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.

Mutant p53 disrupts mammary tissue architecture via the mevalonate pathway.

p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.

Lipid signalling enforces functional specialization of Treg cells in tumours.

Regulatory T cells (Treg cells) are essential for immune tolerance1, but also drive immunosuppression in the tumour microenvironment2. Therapeutic targeting of Treg cells in cancer will therefore require the identification of context-specific mechanisms that affect their function. Here we show that inhibiting lipid synthesis and metabolic signalling that are dependent on sterol-regulatory-element-binding proteins (SREBPs) in Treg cells unleashes effective antitumour immune responses without autoimmune toxicity. We find that the activity of SREBPs is upregulated in intratumoral Treg cells. Moreover, deletion of SREBP-cleavage-activating protein (SCAP)-a factor required for SREBP activity-in these cells inhibits tumour growth and boosts immunotherapy that is triggered by targeting the immune-checkpoint protein PD-1. These effects of SCAP deletion are associated with uncontrolled production of interferon-γ and impaired function of intratumoral Treg cells. Mechanistically, signalling through SCAP and SREBPs coordinates cellular programs for lipid synthesis and inhibitory receptor signalling in these cells. First, de novo fatty-acid synthesis mediated by fatty-acid synthase (FASN) contributes to functional maturation of Treg cells, and loss of FASN from Treg cells inhibits tumour growth. Second, Treg cells in tumours show enhanced expression of the PD-1 gene, through a process that depends on SREBP activity and signals via mevalonate metabolism to protein geranylgeranylation. Blocking PD-1 or SREBP signalling results in dysregulated activation of phosphatidylinositol-3-kinase in intratumoral Treg cells. Our findings show that metabolic reprogramming enforces the functional specialization of Treg cells in tumours, pointing to new ways of targeting these cells for cancer therapy.

Effects of Fermented Extracts of Wuniuzao Dark Loose Tea on Hepatic Sterol Regulatory Element-Binding Protein Pathway and Gut Microbiota Disorder in Obese Mice.

BACKGROUND: Artificially fermented dark loose tea is a type of novel dark tea prepared via fermentation by Eurotium cristatum. The effects of artificially fermented dark loose tea on lipid metabolism are still unclear. OBJECTIVES: This study aimed to explore if artificially fermented dark loose tea has the same effects as naturally fermented dark loose tea in regulating hepatic lipid metabolism. METHODS: Thirty-six 8-wk-old male C57BL/6 mice were randomly divided into 6 treatment groups, including normal control (NC), high-fat diet (HFD), positive control (PC), Wuniuzao dark raw tea (WDT), Wuniuzao naturally fermented dark loose tea (NFLT), and Wuniuzao artificially fermented dark loose tea (AFLT) groups. The HFD, PC, WDT, NFLT, and AFLT groups were fed a HFD. The PC group was supplemented with atorvastatin (10 mg/kg). The WDT group was supplemented with WDT (300 mg/kg), the NFLT group with NFLT (300 mg/kg), and the AFLT group with AFLT (300 mg/kg). RESULTS: The study compared the effect of WDT, NFLT, and AFLT on liver steatosis and gut microbiota disorder in obese mice. All 3 tea extracts reduced body weight, glucose tolerance, and serum lipid concentrations. Via sterol-regulatory element binding protein (SREBP)-mediated lipid metabolism, all 3 tea extracts alleviated hepatic steatosis in mice with obesity. Furthermore, NFLT and AFLT intervened in the abundance of Firmicutes, Bacteroidetes, Clostridia, Muribaculaceae, and Lachnospiraceae. CONCLUSION: In mice with obesity induced by a HFD, WDT, NFLT, and AFLT may improve hepatic steatosis through an SREBP-mediated lipid metabolism. Moreover, NFLT and AFLT improved the composition of gut microbiota.

Identification of the novel role of sterol regulatory element binding proteins (SREBPs) in mechanotransduction and intraocular pressure regulation.

Trabecular meshwork (TM) cells are contractile and mechanosensitive, and they aid in maintaining intraocular pressure (IOP) homeostasis. Lipids are attributed to modulating TM contractility, with poor mechanistic understanding. In this study using human TM cells, we identify the mechanosensing role of the transcription factors sterol regulatory element binding proteins (SREBPs) involved in lipogenesis. By constitutively activating SREBPs and pharmacologically inactivating SREBPs, we have mechanistically deciphered the attributes of SREBPs in regulating the contractile properties of TM. The pharmacological inhibition of SREBPs by fatostatin and molecular inactivation of SREBPs ex vivo and in vivo, respectively, results in significant IOP lowering. As a proof of concept, fatostatin significantly decreased the SREBPs responsive genes and enzymes involved in lipogenic pathways as well as the levels of the phospholipid, cholesterol, and triglyceride. Further, we show that fatostatin mitigated actin polymerization machinery and stabilization, and decreased ECM synthesis and secretion. We thus postulate that lowering lipogenesis in the TM outflow pathway can hold the key to lowering IOP by modifying the TM biomechanics.

MicroRNAs in metabolism and metabolic disorders.

MicroRNAs (miRNAs) have recently emerged as key regulators of metabolism. For example, miR-33a and miR-33b have a crucial role in controlling cholesterol and lipid metabolism in concert with their host genes, the sterol-regulatory element-binding protein (SREBP) transcription factors. Other metabolic miRNAs, such as miR-103 and miR-107, regulate insulin and glucose homeostasis, whereas miRNAs such as miR-34a are emerging as key regulators of hepatic lipid homeostasis. The discovery of circulating miRNAs has highlighted their potential as both endocrine signalling molecules and disease markers. Dysregulation of miRNAs may contribute to metabolic abnormalities, suggesting that miRNAs may potentially serve as therapeutic targets for ameliorating cardiometabolic disorders.

High fatty acid accumulation and coloration molecular mechanism of the elm mushroom (Pleurotus citrinopileatus).

Pleurotus citrinopileatus is a low-cholesterol, protein-rich, and high-nutrient food. The molecular mechanisms of the compounds and coloration have not been reported. Metabolome and transcriptome were used to clarify the molecular mechanisms of key compounds biosynthesis. K-means analysis identified 19 compounds in P. citrinopileatus, mainly lipids and alkaloids in class 8. In addition, 84 lipids were higher and that the different compounds were mainly enriched in linoleic acid metabolism. A total of 14 compounds detected in the linoleic acid metabolism pathway were significantly up-regulated, while 3 sterol regulatory element binding protein (SREBP) transcription factors were screened. Tryptophan metabolism and riboflavin biosynthesis pathway analysis indicated that 3 Unigenes had tryptophan decarboxylase similar elements, which belonged to tyrosine decarboxylase 1. Moreover, CL15618.Contig5_All had high homology with MFS. In conclusion, the expression of 3 SREBP, the synthesis of isobavachalcone D, and the regulation of riboflavin transport by MCH5 were the reasons for fatty acid accumulation and yellow cap formation in the P. citrinopileatus.

Discovery of an insulin-induced gene binding compound that ameliorates nonalcoholic steatohepatitis by inhibiting sterol regulatory element-binding protein-mediated lipogenesis.

BACKGROUND AND AIMS: NASH is associated with high levels of cholesterol and triglyceride (TG) in the liver; however, there is still no approved pharmacological therapy. Synthesis of cholesterol and TG is controlled by sterol regulatory element-binding protein (SREBP), which is found to be abnormally activated in NASH patients. We aim to discover small molecules for treating NASH by inhibiting the SREBP pathway. APPROACH AND RESULTS: Here, we identify a potent SREBP inhibitor, 25-hydroxylanosterol (25-HL). 25-HL binds to insulin-induced gene (INSIG) proteins, stimulates the interaction between INSIG and SCAP, and retains them in the endoplasmic reticulum, thereby suppressing SREBP activation and inhibiting lipogenesis. In NASH mouse models, 25-HL lowers levels of cholesterol and TG in serum and the liver, enhances energy expenditure to prevent obesity, and improves insulin sensitivity. 25-HL dramatically ameliorates hepatic steatosis, inflammation, ballooning, and fibrosis through down-regulating the expression of lipogenic genes. Furthermore, 25-HL exhibits both prophylactic and therapeutic efficacies of alleviating NASH and atherosclerosis in amylin liver NASH model diet-treated Ldlr-/- mice, and reduces the formation of cholesterol crystals and associated crown-like structures of Kupffer cells. Notably, 25-HL lowers lipid contents in serum and the liver to a greater extent than lovastatin or obeticholic acid. 25-HL shows a good safety and pharmacokinetics profile. CONCLUSIONS: This study provides the proof of concept that inhibiting SREBP activation by targeting INSIG to lower lipids could be a promising strategy for treating NASH. It suggests the translational potential of 25-HL in human NASH and demonstrates the critical role of SREBP-controlled lipogenesis in the progression of NASH by pharmacological inhibition.

Coordination of lipid metabolism in membrane biogenesis.

Bilayer synthesis during membrane biogenesis involves the concerted assembly of multiple lipid species, requiring coordination of the level of lipid synthesis, uptake, turnover, and subcellular distribution. In this review, we discuss some of the salient conclusions regarding the coordination of lipid synthesis that have emerged from work in mammalian and yeast cells. The principal instruments of global control are a small number of transcription factors that target a wide range of genes encoding enzymes that operate in a given metabolic pathway. Critical in mammalian cells are sterol regulatory element binding proteins (SREBPs) that stimulate expression of genes for the uptake and synthesis of cholesterol and fatty acids. From work with Saccharomyces cerevisiae, much has been learned about glycerophospholipid and ergosterol regulation through Ino2p/Ino4p and Upc2p transcription factors, respectively. Lipid supply is fine-tuned through a multitude of negative feedback circuits initiated by both end products and intermediates of lipid synthesis pathways. Moreover, there is evidence that the diversity of membrane lipids is maintained through cross-regulatory effects, whereby classes of lipids activate the activity of enzymes operating in another metabolic branch.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp.

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

SREBP and MDT-15 protect C. elegans from glucose-induced accelerated aging by preventing accumulation of saturated fat.

Glucose-rich diets shorten the life spans of various organisms. However, the metabolic processes involved in this phenomenon remain unknown. Here, we show that sterol regulatory element-binding protein (SREBP) and mediator-15 (MDT-15) prevent the life-shortening effects of a glucose-rich diet by regulating fat-converting processes in Caenorhabditis elegans. Up-regulation of the SREBP/MDT-15 transcription factor complex was necessary and sufficient for alleviating the life-shortening effect of a glucose-rich diet. Glucose feeding induced key enzymes that convert saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), which are regulated by SREBP and MDT-15. Furthermore, SREBP/MDT-15 reduced the levels of SFAs and moderated glucose toxicity on life span. Our study may help to develop strategies against elevated blood glucose and free fatty acids, which cause glucolipotoxicity in diabetic patients.

SREBPs in Lipid Metabolism, Insulin Signaling, and Beyond.

Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that activate genes encoding enzymes required for synthesis of cholesterol and unsaturated fatty acids. SREBPs are controlled by multiple mechanisms at the level of mRNA synthesis, proteolytic activation, and transcriptional activity. In this review, we summarize the recent findings that contribute to the current understanding of the regulation of SREBPs and their physiologic roles in maintenance of lipid homeostasis, insulin signaling, innate immunity, and cancer development.

Abnormal lipid droplets accumulation induced cognitive deficits in obstructive sleep apnea syndrome mice via JNK/SREBP/ACC pathway but not through PDP1/PDC pathway.

The mechanisms of chronic intermittent hypoxia (CIH)-induced cognitive deficits remain unclear. Here, our study found that about 3 months CIH treatment induced lipid droplets (LDs) accumulation in hippocampal nerve and glia cells of C57BL/6 mice, and caused severe neuro damage including neuron lesions, neuroblast (NB) apoptosis and abnormal glial activation. Studies have shown that the neuronal metabolism disorders might contribute to the CIH induced-hippocampal impairment. Mechanistically, the results showed that pyruvate dehydrogenase complex E1ɑ subunit (PDHA1) and the pyruvate dehydrogenase complex (PDC) activator pyruvate dehydrogenase phosphatase 1 (PDP1) did not noticeable change after intermittent hypoxia. Consistent with those results, the level of Acetyl-CoA in hippocampus did not significantly change after CIH exposure. Interestingly, we found that CIH produced large quantities of ROS, which activated the JNK/SREBP/ACC pathway in nerve and glia cells. ACC catalyzed the carboxylation of Acetyl-CoA to malonyl-CoA and then more lipid acids were synthesized, which finally caused aberrant LDs accumulation. Therefore, the JNK/SREBP/ACC pathway played a crucial role in the cognitive deficits caused by LDs accumulation after CIH exposure. Additionally, LDs were peroxidized by the high level of ROS under CIH conditions. Together, lipid metabolic disorders contributed to nerve and glia cells damage, which ultimately caused behavioral dysfunction. An active component of Salvia miltiorrhiza, SMND-309, dramatically alleviated these injuries and improved cognitive deficits of CIH mice.

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Modulation of sensitivity to gaseous signaling by sterol-regulatory hypoxic transcription factors in Aspergillus nidulans biofilm cells.

Fungal biofilm founder cells experience self-generated hypoxia leading to dramatic changes in their cell biology. For example, during Aspergillus nidulans biofilm formation microtubule (MT) disassembly is triggered causing dispersal of EB1 from MT tips. This process is dependent on SrbA, a sterol regulatory element-binding transcription factor required for adaptation to hypoxia. We show that SrbA, an ER resident protein prior to activation, is proteolytically activated during early stages of biofilm formation and that, like SrbA itself, its activating proteases are also required for normal biofilm MT disassembly. In addition to SrbA, the AtrR transcription factor is also found to be required to modulate cellular responses to gaseous signaling during biofilm development. Using co-cultures, we further show that cells lacking srbA or atrR are capable of responding to biofilm generated gaseous microenvironments but are actually more sensitive to this signal than wild type cells. SrbA is a regulator of ergosterol biosynthetic genes and we find that the levels of seven GFP-tagged Erg proteins differentially accumulate during biofilm formation with various dependencies on SrbA for their accumulation. This uncovers a complex pattern of regulation with biofilm accumulation of only some Erg proteins being dependent on SrbA with others accumulating to higher levels in its absence. Because different membrane sterols are known to influence cell permeability to gaseous molecules, including oxygen, we propose that differential regulation of ergosterol biosynthetic proteins by SrbA potentially calibrates the cell's responsiveness to gaseous signaling which in turn modifies the cell biology of developing biofilm cells.

Feedback modulation of cholesterol metabolism by the lipid-responsive non-coding RNA LeXis.

Liver X receptors (LXRs) are transcriptional regulators of cellular and systemic cholesterol homeostasis. Under conditions of excess cholesterol, LXR activation induces the expression of several genes involved in cholesterol efflux, facilitates cholesterol esterification by promoting fatty acid synthesis, and inhibits cholesterol uptake by the low-density lipoprotein receptor. The fact that sterol content is maintained in a narrow range in most cell types and in the organism as a whole suggests that extensive crosstalk between regulatory pathways must exist. However, the molecular mechanisms that integrate LXRs with other lipid metabolic pathways are incompletely understood. Here we show that ligand activation of LXRs in mouse liver not only promotes cholesterol efflux, but also simultaneously inhibits cholesterol biosynthesis. We further identify the long non-coding RNA LeXis as a mediator of this effect. Hepatic LeXis expression is robustly induced in response to a Western diet (high in fat and cholesterol) or to pharmacological LXR activation. Raising or lowering LeXis levels in the liver affects the expression of genes involved in cholesterol biosynthesis and alters the cholesterol levels in the liver and plasma. LeXis interacts with and affects the DNA interactions of RALY, a heterogeneous ribonucleoprotein that acts as a transcriptional cofactor for cholesterol biosynthetic genes in the mouse liver. These findings outline a regulatory role for a non-coding RNA in lipid metabolism and advance our understanding of the mechanisms that coordinate sterol homeostasis.