Identification of Potential New Genes Related to the SREBP Pathway in Xanthophyllomyces dendrorhous.

The sterol regulatory element-binding protein (SREBP) pathway is an integral cellular mechanism that regulates lipid homeostasis, in which transcriptional activator SREBPs regulate the expression of various genes. In the carotenogenic yeast Xanthophyllomyces dendrorhous, Sre1 (the yeast SREBP homolog) regulates lipid biosynthesis and carotenogenesis, among other processes. Despite the characterization of several components of the SREBP pathway across various eukaryotes, the specific elements of this pathway in X. dendrorhous remain largely unknown. This study aimed to explore the potential regulatory mechanisms of the SREBP pathway in X. dendrorhous using the strain CBS.cyp61- as a model, which is known to have Sre1 in its active state under standard culture conditions, resulting in a carotenoid-overproducing phenotype. This strain was subjected to random mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), followed by a screening methodology that focused on identifying mutants with altered Sre1 activation phenotypes. Single-nucleotide polymorphism (SNP) analysis of 20 selected mutants detected 5439 single-nucleotide variants (SNVs), narrowing them down to 1327 SNPs of interest after a series of filters. Classification based on SNP impact identified 116 candidate genes, including 49 genes with high impact and 68 genes with deleterious moderate-impact mutations. BLAST, InterProScan, and gene ontology enrichment analyses highlighted 25 genes as potential participants in regulating Sre1 in X. dendrorhous. The key findings of this study include the identification of genes potentially encoding proteins involved in protein import/export to the nucleus, sterol biosynthesis, the ubiquitin-proteasome system, protein regulatory activities such as deacetylases, a subset of kinases and proteases, as well as transcription factors that could be influential in SREBP regulation. These findings are expected to significantly contribute to the current understanding of the intricate regulation of the transcription factor Sre1 in X. dendrorhous, providing valuable groundwork for future research and potential biotechnological applications.

In vivo CRISPR screening identifies geranylgeranyl diphosphate as a pancreatic cancer tumor growth dependency.

OBJECTIVE: Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. In this study, we examined the requirement of SREBP target genes for pancreatic ductal adenocarcinoma (PDAC) tumor growth. METHODS: Here we constructed a custom CRISPR knockout library containing known SREBP target genes and performed in vitro 2D culture and in vivo orthotopic xenograft CRISPR screens using a patient-derived PDAC cell line. In vitro, we grew cells in medium supplemented with 10% fetal bovine serum (FBS) or 10% lipoprotein-deficient serum (LPDS) to examine differences in gene essentiality in different lipid environments. In vivo, we injected cells into the pancreata of nude mice and collected tumors after 4 weeks. RESULTS: We identified terpenoid backbone biosynthesis genes as essential for PDAC tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that geranylgeranyl diphosphate synthase 1 (GGPS1) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model. CONCLUSIONS: These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.

Hyperactivation of SREBP induces pannexin-1-dependent lytic cell death.

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.

SPRING is a Dedicated Licensing Factor for SREBP-Specific Activation by S1P.

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PA→C and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.

Sterol Regulatory Element-Binding Protein, BbSre1, Controls Oxidative Stress Response, Peroxisome Division, and Lipid Homeostasis in an Insect Fungal Pathogen.

Sterol regulatory element-binding protein, Sre1, regulates sterol biosynthesis, lipid metabolism, hypoxia adaptation, and virulence in some fungi, even though its roles are varied in fungal species. However, few studies report its other functions in fungi. Here, we report novel roles of Sre1 homolog, BbSre1, in the insect fungal pathogen, Beauveria bassiana, that regulates oxidative stress response, peroxisome division, and redox homeostasis. The gene disruption stain showed increased sensitivity to oxidative stress, which was in line with oxidative stress-induced-BbSre1 nuclear import and control of antioxidant and detoxification-involved genes. The gene mutation also inhibited peroxisome division, affected redox homeostasis, and impaired lipid/fatty acid metabolism and sterol biosynthesis, which was verified by downregulation of their associated genes. These data broaden our understanding of role of Sre1, which regulates peroxisome division, antioxidant, and detoxification-involved genes for control of redox homeostasis and oxidative stress response that links to lipid/fatty acid metabolism and sterol biosynthesis.

The bHLH-zip transcription factor SREBP regulates triterpenoid and lipid metabolisms in the medicinal fungus Ganoderma lingzhi.

Ganoderic acids (GAs) are well recognized as important pharmacological components of the medicinal species belonging to the basidiomycete genus Ganoderma. However, transcription factors directly regulating the expression of GA biosynthesis genes remain poorly understood. Here, the genome of Ganoderma lingzhi is de novo sequenced. Using DNA affinity purification sequencing, we identify putative targets of the transcription factor sterol regulatory element-binding protein (SREBP), including the genes of triterpenoid synthesis and lipid metabolism. Interactions between SREBP and the targets are verified by electrophoretic mobility gel shift assay. RNA-seq shows that SREBP targets, mevalonate kinase and 3-hydroxy-3-methylglutaryl coenzyme A synthetase in mevalonate pathway, sterol isomerase and lanosterol 14-demethylase in ergosterol biosynthesis, are significantly upregulated in the SREBP overexpression (OE::SREBP) strain. In addition, 3 targets involved in glycerophospholipid/glycerolipid metabolism are upregulated. Then, the contents of mevalonic acid, lanosterol, ergosterol and 13 different GAs as well as a variety of lipids are significantly increased in this strain. Furthermore, the effects of SREBP overexpression on triterpenoid and lipid metabolisms are recovered when OE::SREBP strain are treated with exogenous fatostatin, a specific inhibitor of SREBP. Taken together, our genome-wide study clarify the role of SREBP in triterpenoid and lipid metabolisms of G. lingzhi.

Transcriptome Analysis of Particulate Matter 2.5-Induced Abnormal Effects on Human Sebocytes.

Particulate matter 2.5 (PM2.5), an atmospheric pollutant with an aerodynamic diameter of <2.5 µm, can cause serious human health problems, including skin damage. Since sebocytes are involved in the regulation of skin homeostasis, it is necessary to study the effects of PM2.5 on sebocytes. We examined the role of PM2.5 via the identification of differentially expressed genes, functional enrichment and canonical pathway analysis, upstream regulator analysis, and disease and biological function analysis through mRNA sequencing. Xenobiotic and lipid metabolism, inflammation, oxidative stress, and cell barrier damage-related pathways were enriched; additionally, PM2.5 altered steroid hormone biosynthesis and retinol metabolism-related pathways. Consequently, PM2.5 increased lipid synthesis, lipid peroxidation, inflammatory cytokine expression, and oxidative stress and altered the lipid composition and expression of factors that affect cell barriers. Furthermore, PM2.5 altered the activity of sterol regulatory element binding proteins, mitogen-activated protein kinases, transforming growth factor beta-SMAD, and forkhead box O3-mediated pathways. We also suggest that the alterations in retinol and estrogen metabolism by PM2.5 are related to the damage. These results were validated using the HairSkin® model. Thus, our results provide evidence of the harmful effects of PM2.5 on sebocytes as well as new targets for alleviating the skin damage it causes.

Effects of bisphenol AF on growth, behavior, histology and gene expression in marine medaka (Oryzias melastigma).

Bisphenol AF (BPAF) is one of the substitutes for bisphenol A (BPA), which has endocrine-disrupting, reproductive and neurological toxicity. BPAF has frequently been detected in the aquatic environment, which has been a long-term threat to the health of aquatic organisms. In this study, female marine medaka (Oryzias melastigma) were exposed to 6.7 µg/L, 73.4 µg/L, and 367.0 µg/L BPAF for 120 d. The effects of BPAF on behavior, growth, liver and ovarian histology, gene transcriptional profiles, and reproduction of marine medaka were determined. The results showed that with the increase of BPAF concentration, the swimming speed of female marine medaka showed an increasing trend and then decreasing trend. BPAF (367.0 µg/L) significantly increased body weight and condition factors in females. BPAF (73.4 µg/L and 367.0 µg/L) significantly delayed oocyte maturation. Exposure to 367.0 µg/L BPAF showed an increasing trend in the transcript levels of lipid synthesis and transport-related genes such as fatty acid synthase (fasn), sterol regulatory element binding protein (srebf), diacylglycerol acyltransferase (dgat), solute carrier family 27 member 4 (slc27a4), fatty acid-binding protein (fabp), and peroxisome proliferator-activated receptor gamma (pparγ) in the liver. In addition, 6.7 µg/L BPAF significantly down-regulated the expression levels of antioxidant-related genes [superoxide dismutase (sod), glutathione peroxidase (gpx), and catalase (cat)], and complement system-related genes [complement component 5 (c5), complement component 7a (c7a), mannan-binding lectin serine peptidase 1 (masp1), and tumor necrosis factor (tnf)] were significantly up-regulated in the 73.4 and 367.0 µg/L groups, which implies the effect of BPAF on the immune system in the liver. In the hypothalamic-pituitary-ovarian axis (HPG) results, the transcription levels of estrogen receptor α (erα), estrogen receptor ß (erß), androgen receptor (arα), gonadotropin-releasing hormone 2 (gnrh2), cytochrome P450 19b (cyp19b), aromatase (cyp19a), and luteinizing hormone receptor (lhr) in the brain and ovary, and vitellogenin (vtg) and choriogenin (chg) in the liver of 367.0 µg/L BPAF group showed a downward trend. In addition, exposure to 367.0 µg/L BPAF for 120 d inhibited the spawning behavior of marine medaka. Our results showed that long-term BPAF treatment influenced growth (body weight and condition factors), lipid metabolism, and ovarian maturation, and significantly altered the immune response and the transcriptional expression levels of HPG axis-related genes.

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.

SREBP and central nervous system disorders: genetic overlaps revealed by in silico analysis.

BACKGROUND: The central nervous system (CNS) is enriched in lipids; despite this, studies exploring the functional roles of lipids in the brain are still limited. Sterol regulatory element binding protein (SREBP) signaling is a transcriptomic pathway that predominantly participates in the maintenance of lipid homeostasis; however, its involvement in the CNS dysfunction is not well-established. In this study, we aimed to characterize and pinpoint specific genes of the SREBP pathway which may be implicated in neurodegenerative, neurological, and neuropsychiatric diseases. METHODS: In silico bioinformatic analysis was performed using the open-source databases DisGeNET and MSigDB. Protein-protein interaction data were visualized and analyzed using STRING, after which GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were conducted via DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: Several common genes were identified between the SREBP pathway and CNS disorders. In GO enrichment analysis, the most enriched biological processes included lipid, cholesterol, and steroid biosynthetic processes; the most enriched molecular functions were transcription factor-related; and the most enriched subcellular compartments revealed that the genes involved in CNS disorders were mainly associated with the enzyme complexes of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). In KEGG enrichment analysis, the most enriched pathway was the AMP-activated protein kinase (AMPK) signaling pathway, and the top-ranked genes significantly enriched under this pathway were ACACA, ACACB, FASN, HMGCR, MTOR, PPARGC1A, PRKAA1, SCD, SIRT1, and SREBF1. CONCLUSIONS: The findings of this study strengthen the evidence linking the involvement of lipid homeostasis in CNS functions. We suggest herein the roles of downstream ACC and FASN enzymes and upstream AMPK signaling in the SREBP pathway as mechanisms underlying neurodegenerative, neurological, and neuropsychiatric CNS disorders.

YAP regulates an SGK1/mTORC1/SREBP-dependent lipogenic program to support proliferation and tissue growth.

The coordinated regulation of growth control and metabolic pathways is required to meet the energetic and biosynthetic demands associated with proliferation. Emerging evidence suggests that the Hippo pathway effector Yes-associated protein 1 (YAP) reprograms cellular metabolism to meet the anabolic demands of growth, although the mechanisms involved are poorly understood. Here, we demonstrate that YAP co-opts the sterol regulatory element-binding protein (SREBP)-dependent lipogenic program to facilitate proliferation and tissue growth. Mechanistically, YAP stimulates de novo lipogenesis via mechanistic target of rapamcyin (mTOR) complex 1 (mTORC1) signaling and subsequent activation of SREBP. Importantly, YAP-dependent regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1) is required to activate mTORC1/SREBP and stimulate de novo lipogenesis. We also find that the SREBP target genes fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) are conditionally required to support YAP-dependent proliferation and tissue growth. These studies reveal that de novo lipogenesis is a metabolic vulnerability that can be targeted to disrupt YAP-dependent proliferation and tissue growth.

The effect of simvastatin on gene expression of low-density lipoprotein receptor, sterol regulatory element-binding proteins, stearoyl-CoA desaturase 1 mRNA in rat hepatic tissues.

The study aimed to assess the effect of simvastatin on gene expression of LDLR, SREBPs, and SCD1 in rat hepatic tissues fed with high-fat diets (HFD) and its association with some biochemical parameters. Thirty-two male Wister albino rats were divided into four equal groups (three test and one control groups). The biochemical parameters were determined by using spectrophotometer techniques and the Elisa method. Low-density lipoprotein receptor, sterol regulatory element-binding proteins, stearoyl-CoA desaturase1, Beta-actin were analysed by real-time quantitative polymerase chain reaction (RT-PCR) method. At the end of study, the livers of the rats were separated and changes of hepatic tissue were determined. LDLR, SREBP2, and SCD1 expression increased significantly when compared G1 versus G4 and G2 versus G4. The expression of LDLR, SREBP2, and SCD1 also increased significantly when compared G2 versus G3, G1versus G3 and G1 versus G3 and G2 versus G3. The serum level of cholesterol, triglyceride, glucose, LDL, and HDL increased significantly when compared G1 versus G3. LDL showed significantly decreased when compared G1 versus G2. Cholesterol, glucose and HDL and triglyceride levels were increased significantly when compared G1 versus G4 and G2. Treatment of rats with HFD and simvastatin 20 mg/kg, triglyceride and LDL were almost the same as a control group and LDLR expression increased 98% in liver tissue. Gene expressions may be up-regulated in liver tissue and they showed different effects on biochemical parameters.

The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage.

In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

LH Induces De Novo Cholesterol Biosynthesis via SREBP Activation in Granulosa Cells During Ovulation in Female Mice.

In the liver, the sterol response element binding protein (SREBP) and the SREBP cleavage-activated protein (SCAP) complex upregulate cholesterol biosynthesis by gene induction of de novo cholesterol synthetic enzymes (Hmgcr, Cyp51, and Dhcr7). Insulin induced gene 1 (INSIG1) negatively regulates cholesterol biosynthesis by the inhibition of de novo cholesterol biosynthetic gene expression. In the ovary, cholesterol is de novo synthesized; however, the roles of SREBP and its regulators (SCAP and INSIG1) are not well understood. In this study, when immature mice were treated with gonadotropins (eCG followed by hCG), eCG induced and hCG maintained the expression of SREBP-1a, -2, and SCAP granulosa cells, whereas INSIG1 expression was dramatically downregulated after hCG injection. Downregulation of INSIG1 led to generate the SREBPs active form and translocate the SREBPs active form to nuclei. Inhibition of generation of the SREBPs active form by fatostatin or Scap siRNA in both in vivo and in vitro significantly decreased the expressions of de novo cholesterol biosynthetic enzymes, cholesterol accumulation, and progesterone (P4) production compared with the control group. Fatostatin treatment inhibited the ovulation and increased the formation of abnormal corpus luteum which trapped the matured oocyte in the corpus luteum; however, the phenomenon was abolished by P4 administration. The results showed that decreasing INSIG1 level after hCG stimulation activated SREBP-induced de novo cholesterol biosynthesis in granulosa cells of preovulatory follicles, which is essential for P4 production and the rupture of matured oocyte during ovulation process.

mTOR Driven Gene Transcription Is Required for Cholesterol Production in Neurons of the Developing Cerebral Cortex.

Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders (ASD) to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy, and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood. Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1, and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels, pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.

Discovery of a Vitamin D Receptor-Silent Vitamin D Derivative That Impairs Sterol Regulatory Element-Binding Protein In Vivo.

Vitamin D3 metabolites inhibit the expression of lipogenic genes by impairing sterol regulatory element-binding protein (SREBP), a master transcription factor of lipogenesis, independent of their canonical activity through a vitamin D receptor (VDR). Herein, we designed and synthesized a series of vitamin D derivatives to search for a drug-like small molecule that suppresses the SREBP-induced lipogenesis without affecting the VDR-controlled calcium homeostasis in vivo. Evaluation of the derivatives in cultured cells and mice led to the discovery of VDR-silent SREBP inhibitors and to the development of KK-052 (50), the first vitamin D-based SREBP inhibitor that has been demonstrated to mitigate hepatic lipid accumulation without calcemic action in mice. KK-052 maintained the ability of 25-hydroxyvitamin D3 to induce the degradation of SREBP but lacked in the VDR-mediated activity. KK-052 serves as a valuable compound for interrogating SREBP/SCAP in vivo and may represent an unprecedented translational opportunity of synthetic vitamin D analogues.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation.

Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/ß-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Enterohepatic Transcription Factor CREB3L3 Protects Atherosclerosis via SREBP Competitive Inhibition.

BACKGROUND & AIMS: cAMP responsive element-binding protein 3 like 3 (CREB3L3) is a membrane-bound transcription factor involved in the maintenance of lipid metabolism in the liver and small intestine. CREB3L3 controls hepatic triglyceride and glucose metabolism by activating plasma fibroblast growth factor 21 (FGF21) and lipoprotein lipase. In this study, we intended to clarify its effect on atherosclerosis. METHODS: CREB3L3-deficifient, liver-specific CREB3L3 knockout, intestine-specific CREB3L3 knockout, both liver- and intestine-specific CREB3L3 knockout, and liver CREB3L3 transgenic mice were crossed with LDLR-/- mice. These mice were fed with a Western diet to develop atherosclerosis. RESULTS: CREB3L3 ablation in LDLR-/- mice exacerbated hyperlipidemia with accumulation of remnant APOB-containing lipoprotein. This led to the development of enhanced aortic atheroma formation, the extent of which was additive between liver- and intestine-specific deletion. Conversely, hepatic nuclear CREB3L3 overexpression markedly suppressed atherosclerosis with amelioration of hyperlipidemia. CREB3L3 directly up-regulates anti-atherogenic FGF21 and APOA4. In contrast, it antagonizes hepatic sterol regulatory element-binding protein (SREBP)-mediated lipogenic and cholesterogenic genes and regulates intestinal liver X receptor-regulated genes involved in the transport of cholesterol. CREB3L3 deficiency results in the accumulation of nuclear SREBP proteins. Because both transcriptional factors share the cleavage system for nuclear transactivation, full-length CREB3L3 and SREBPs in the endoplasmic reticulum (ER) functionally inhibit each other. CREB3L3 promotes the formation of the SREBP-insulin induced gene 1 complex to suppress SREBPs for ER-Golgi transport, resulting in ER retention and inhibition of proteolytic activation at the Golgi and vice versa. CONCLUSIONS: CREB3L3 has multi-potent protective effects against atherosclerosis owing to new mechanistic interaction between CREB3L3 and SREBPs under atherogenic conditions.

Dipyridamole Inhibits Lipogenic Gene Expression by Retaining SCAP-SREBP in the Endoplasmic Reticulum.

Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.

The novel rexinoid MSU-42011 is effective for the treatment of preclinical Kras-driven lung cancer.

Effective drugs are needed for lung cancer, as this disease remains the leading cause of cancer-related deaths. Rexinoids are promising drug candidates for cancer therapy because of their ability to modulate genes involved in inflammation, cell proliferation or differentiation, and apoptosis through activation of the retinoid X receptor (RXR). The only currently FDA-approved rexinoid, bexarotene, is ineffective as a single agent for treating epithelial cancers and induces hypertriglyceridemia. Here, we used a previously validated screening paradigm to evaluate 23 novel rexinoids for biomarkers related to efficacy and safety. These biomarkers include suppression of inducible nitric oxide synthase (iNOS) and induction of sterol regulatory element-binding protein (SREBP). Because of its potent iNOS suppression, low SREBP induction, and activation of RXR, MSU-42011 was selected as our lead compound. We next used MSU-42011 to treat established tumors in a clinically relevant Kras-driven mouse model of lung cancer. KRAS is one of the most common driver mutations in human lung cancer and correlates with aggressive disease progression and poor patient prognosis. Ultrasound imaging was used to detect and monitor tumor development and growth over time in the lungs of the A/J mice. MSU-42011 markedly decreased the tumor number, size, and histopathology of lung tumors compared to the control and bexarotene groups. Histological sections of lung tumors in mice treated with MSU-42011 exhibited reduced cell density and fewer actively proliferating cells compared to the control and bexarotene-treated tumors. Although bexarotene significantly (p < 0.01) elevated plasma triglycerides and cholesterol, treatment with MSU-42011 did not increase these biomarkers, demonstrating a more favorable toxicity profile in vivo. The combination of MSU-42011 and carboplatin and paclitaxel reduced macrophages in the lung and increased activation markers of CD8+T cells compared to the control groups. Our results validate our screening paradigm for in vitro testing of novel rexinoids and demonstrate the potential for MSU-42011 to be developed for the treatment of KRAS-driven lung cancer.