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Mao S, Ren J, Xu Y, Lin J, Pan C, Meng Y, Xu N. Studies in the antiviral molecular mechanisms of 25-hydroxycholesterol: Disturbing cholesterol homeostasis and post-translational modification of proteins. Eur J Pharmacol 2022; 926:175033. [PMID: 35598845 PMCID: PMC9119167 DOI: 10.1016/j.ejphar.2022.175033] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 05/04/2022] [Accepted: 05/11/2022] [Indexed: 02/08/2023]
Abstract
Efficient antiviral drug discovery has been a pressing issue of global public health concern since the outbreak of coronavirus disease 2019. In recent years, numerous in vitro and in vivo studies have shown that 25-hydroxycholesterol (25HC), a reactive oxysterol catalyzed by cholesterol-25-hydroxylase, exerts broad-spectrum antiviral activity with high efficiency and low toxicity. 25HC restricts viral internalization and disturbs the maturity of viral proteins using multiple mechanisms. First, 25HC reduces lipid rafts and cholesterol in the cytomembrane by inhibiting sterol-regulatory element binding proteins-2, stimulating liver X receptor, and activating Acyl-coenzyme A: cholesterol acyl-transferase. Second, 25HC impairs endosomal pathways by restricting the function of oxysterol-binding protein or Niemann-pick protein C1, causing the virus to fail to release nucleic acid. Third, 25HC disturbs the prenylation of viral proteins by suppressing the sterol-regulatory element binding protein pathway and glycosylation by increasing the sensitivity of glycans to endoglycosidase. This paper reviews previous studies on the antiviral activity of 25HC in order to fully understand its role in innate immunity and how it may contribute to the development of urgently needed broad-spectrum antiviral drugs.
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Abstract
Cellular cholesterol levels are intricately controlled to maintain homeostasis. Here, we describe ways in which cellular cholesterol status can be manipulated for the study of cholesterol homeostasis, including sterol starvation (by culturing cells in lipoprotein-deficient serum and pretreating/treating with the cholesterol-lowering drug, statin) and sterol enrichment (using cholesterol complexed to cyclodextrin, and low-density lipoprotein). We also describe how to prepare lipoprotein-deficient serum and complex cholesterol to cyclodextrin.
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Affiliation(s)
- Winnie Luu
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Sciences Building D26, Sydney, NSW, 2052, Australia
| | - Ingrid C Gelissen
- Faculty of Pharmacy, The University of Sydney, Sydney, NSW, 2006, Australia
| | - Andrew J Brown
- School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Biological Sciences Building D26, Sydney, NSW, 2052, Australia.
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3
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Cheng C, Guo JY, Geng F, Wu X, Cheng X, Li Q, Guo D. Analysis of SCAP N-glycosylation and Trafficking in Human Cells. J Vis Exp 2016. [PMID: 27911384 DOI: 10.3791/54709] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.
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Affiliation(s)
- Chunming Cheng
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Jeffrey Yunhua Guo
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Feng Geng
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Xiaoning Wu
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Xiang Cheng
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Qiyue Li
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine
| | - Deliang Guo
- Department of Radiation Oncology, The Ohio State University Comprehensive Cancer Center and College of Medicine;
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Gupta V, Arora R, Gochhait S, Bairwa NK, Bamezai RNK. Gel-based nonradioactive single-strand conformational polymorphism and mutation detection: limitations and solutions. Methods Mol Biol 2014; 1105:365-380. [PMID: 24623242 DOI: 10.1007/978-1-62703-739-6_28] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/03/2023]
Abstract
Single-strand conformation polymorphism (SSCP) for screening mutations/single-nucleotide polymorphisms (SNPs) is a simple, cost-effective technique, saving an expensive exercise of sequencing each and every polymerase chain reaction product and assisting in choosing only the amplicons of interest with expected mutations. The principle of detection of small changes in DNA sequences is based on changes in single-strand DNA conformations. The changes in electrophoretic mobility that SSCP detects are sequence dependent. The limitations faced in SSCP range from routine polyacrylamide gel electrophoresis (PAGE) problems to the problems of resolving mutant DNA bands. Both these problems can be solved by controlling PAGE conditions and by varying physical and environmental conditions such as pH, temperature, voltage, gel type and percentage, addition of additives or denaturants, and others. Despite much upgrading of the technology for mutation detection, SSCP remains the method of choice to analyze mutations and SNPs in order to understand genomic variations, both spontaneous and induced, and the genetic basis of diseases.
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Affiliation(s)
- Vibhuti Gupta
- Human Genetics Section, School of Life Sciences, National Centre of Applied Human Genetics, Jawaharlal Nehru University, New Meharuli Road, New Delhi, 110067, India
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Jo Y, Debose-Boyd RA. Control of cholesterol synthesis through regulated ER-associated degradation of HMG CoA reductase. Crit Rev Biochem Mol Biol 2010; 45:185-98. [PMID: 20482385 DOI: 10.3109/10409238.2010.485605] [Citation(s) in RCA: 114] [Impact Index Per Article: 7.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Multiple mechanisms for feedback control of cholesterol synthesis converge on the rate-limiting enzyme in the pathway, 3-hydroxy-3-methylglutaryl coenzyme A reductase. This complex feedback regulatory system is mediated by sterol and nonsterol metabolites of mevalonate, the immediate product of reductase activity. One mechanism for feedback control of reductase involves rapid degradation of the enzyme from membranes of the endoplasmic reticulum (ER). This degradation results from the accumulation of sterols in ER membranes, which triggers binding of reductase to ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the recruitment of a membrane-associated ubiquitin ligase called gp78 that initiates ubiquitination of reductase. Ubiquitinated reductase then becomes extracted from ER membranes and is delivered to cytosolic 26S proteasomes through an unknown mechanism that is mediated by the gp78-associated ATPase Valosin-containing protein/p97 and appears to be augmented by nonsterol isoprenoids. Here, we will highlight several advances that have led to the current view of mechanisms for sterol-accelerated, ER-associated degradation of reductase. In addition, we will discuss potential mechanisms for other aspects of the pathway such as selection of reductase for gp78-mediated ubiquitination, extraction of the ubiquitinated enzyme from ER membranes, and the contribution of Insig-mediated degradation to overall regulation of reductase in whole animals.
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Affiliation(s)
- Youngah Jo
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA
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Abstract
Multiple epidemiologic studies have linked the development of renal cancer to obesity. In this chapter, we begin with a review of selected population studies, followed by recent mechanistic discoveries that further link lipid deregulation to the RCC development. The upregulation of leptin and downregulation of adiponectin pathways in obesity fit well with our molecular understanding of RCC pathogenesis. In addition, two forms of hereditary RCC involve proteins, Folliculin and TRC8, that are positioned to coordinately regulate lipid and protein biosynthesis. Both of these biosynthetic pathways have important downstream consequences on HIF-1/2alpha levels and angiogenesis, key aspects in the disease pathogenesis. The role of lipid biology and its interface with protein translation regulation represents a new dimension in RCC research with potential therapeutic implications.
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Affiliation(s)
- Harry A Drabkin
- Department of Medicine and Division of Hematology-Oncology, Medical University of South Carolina, Charleston, SC, USA
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7
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Chatterjee S, Szustakowski JD, Nanguneri NR, Mickanin C, Labow MA, Nohturfft A, Dev KK, Sivasankaran R. Identification of novel genes and pathways regulating SREBP transcriptional activity. PLoS One 2009; 4:e5197. [PMID: 19381295 PMCID: PMC2668173 DOI: 10.1371/journal.pone.0005197] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Accepted: 02/05/2009] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways. METHODOLOGY To identify novel regulators of SREBPs, we performed a genome-wide cDNA over-expression screen to identify proteins that might modulate the transcription of a luciferase gene driven from an SREBP-specific promoter. The results were verified through secondary biological assays and expression data were analyzed by a novel application of the Gene Set Enrichment Analysis (GSEA) method. CONCLUSIONS/SIGNIFICANCE We screened 10,000 different cDNAs and identified a number of genes and pathways that have previously not been implicated in SREBP control and cellular cholesterol homeostasis. These findings further our understanding of lipid biology and should lead to new insights into lipid associated disorders.
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Affiliation(s)
- Sandipan Chatterjee
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
| | - Joseph D. Szustakowski
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
| | - Nirmala R. Nanguneri
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
| | - Craig Mickanin
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
| | - Mark A. Labow
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
| | - Axel Nohturfft
- Division of Basic Medical Sciences, St. George's University of London, London, United Kingdom
| | - Kumlesh K. Dev
- Department of Anatomy, University College Cork, Cork, Ireland
- * E-mail: (KKD); (RS)
| | - Rajeev Sivasankaran
- Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts, United States of America
- * E-mail: (KKD); (RS)
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DeBose-Boyd RA. Feedback regulation of cholesterol synthesis: sterol-accelerated ubiquitination and degradation of HMG CoA reductase. Cell Res 2008; 18:609-21. [PMID: 18504457 DOI: 10.1038/cr.2008.61] [Citation(s) in RCA: 270] [Impact Index Per Article: 15.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase produces mevalonate, an important intermediate in the synthesis of cholesterol and essential nonsterol isoprenoids. The reductase is subject to an exorbitant amount of feedback control through multiple mechanisms that are mediated by sterol and nonsterol end-products of mevalonate metabolism. Here, I will discuss recent advances that shed light on one mechanism for control of reductase, which involves rapid degradation of the enzyme. Accumulation of certain sterols triggers binding of reductase to endoplasmic reticulum (ER) membrane proteins called Insig-1 and Insig-2. Reductase-Insig binding results in recruitment of a membrane-associated ubiquitin ligase called gp78, which initiates ubiquitination of reductase. This ubiquitination is an obligatory reaction for recognition and degradation of reductase from ER membranes by cytosolic 26S proteasomes. Thus, sterol-accelerated degradation of reductase represents an example of how a general cellular process (ER-associated degradation) is used to control an important metabolic pathway (cholesterol synthesis).
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Affiliation(s)
- Russell A DeBose-Boyd
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.
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9
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Kim SO, Ha SD, Lee S, Stanton S, Beutler B, Han J. Mutagenesis by retroviral insertion in chemical mutagen-generated quasi-haploid mammalian cells. Biotechniques 2007; 42:493-501. [PMID: 17489237 DOI: 10.2144/000112390] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Diploidy is a major obstacle to the mutagenic analysis of function in cultured mammalian cells. Here, we show that 6–8 rounds of chemical mutagenesis generates quasi-haploid cells that can be used as targets for insertional mutagenesis using a specially designed retroviral vector that permits rapid identification of disrupted genes in each cell that bears a phenotype of interest. The utility of combined chemical and insertional mutagenesis is illustrated by the identification of novel host genes that are required for macrophage sensitivity to anthrax lethal factor.
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Affiliation(s)
- Sung O Kim
- Department of Microbiology, University of Western Ontario, London, ON, Canada.
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Maguire JA, Reagan JW. Silencing of the mutant SCAP allele accounts for restoration of a normal phenotype in CT60 cells selected for NPC1 expression. J Lipid Res 2005; 46:1840-8. [PMID: 15995170 DOI: 10.1194/jlr.m500198-jlr200] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The sterol regulatory element binding protein (SREBP)/SREBP cleavage-activating protein (SCAP) complex regulates the transcription of numerous genes involved in cellular cholesterol metabolism. The CHO mutant, CT60, and its parental cell line, 25RA, possess a gain-of-function mutation in one allele of the SCAP gene that renders the cells resistant to sterol-mediated suppression of cholesterol synthesis and uptake. In addition, CT60 cells do not express a functional Niemann-Pick type C1 (NPC1) protein, which leads to lysosomal accumulation of free cholesterol. Correction of the NPC1 defect by expression of a yeast artificial chromosome (YAC) containing the NPC1 genetic interval restored normal mobilization of cholesterol from the lysosomal compartment. Unexpectedly, the YAC-containing cell lines have overall cellular cholesterol concentrations that are comparable to wild-type levels, despite the assumed presence of the SCAP mutation. This phenotypic change results from a reduction in endogenous sterol synthesis, LDL receptor message, and HMG-CoA reductase message. Genetic analysis of the SCAP gene revealed that the YAC-expressing CT60 cells have normal regulation of these sentinel cholesterogenic genes as a result of selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.
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Affiliation(s)
- Jean Ann Maguire
- Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
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11
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Wang Y, Castoreno AB, Stockinger W, Nohturfft A. Modulation of endosomal cholesteryl ester metabolism by membrane cholesterol. J Biol Chem 2005; 280:11876-86. [PMID: 15657032 PMCID: PMC1940112 DOI: 10.1074/jbc.m414676200] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Cells acquire cholesterol in part by endocytosis of cholesteryl ester containing lipoproteins. In endosomes and lysosomes cholesteryl ester is hydrolyzed by acidic cholesteryl ester hydrolase producing cholesterol and fatty acids. Under certain pathological conditions, however, such as in atherosclerosis, excessive levels of cholesteryl ester accumulate in lysosomes for reasons that are poorly understood. Here, we have studied endosomal and lysosomal cholesteryl ester metabolism in cultured mouse macrophages and with cell-free extracts. We show that net hydrolysis of cholesteryl ester is coupled to the transfer of cholesterol to membranes. When membrane cholesterol levels are low, absorption of cholesterol effectively drives cholesteryl ester hydrolysis. When cholesterol levels in acceptor membranes approach saturation or when cholesterol export is blocked, cholesterol is re-esterified in endosomes. These results reveal a new facet of cellular cholesterol homeostasis and provide a potential explanation for cholesteryl ester accumulation in lysosomes of atherosclerotic cells.
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Affiliation(s)
| | | | | | - Axel Nohturfft
- || To whom correspondence should be addressed: The Biological Laboratories, Dept. of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138. Tel.: 617-384-5846; Fax: 617-384-7423; E-mail:
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Higaki K, Almanzar-Paramio D, Sturley SL. Metazoan and microbial models of Niemann-Pick Type C disease. Biochim Biophys Acta Mol Cell Biol Lipids 2004; 1685:38-47. [PMID: 15465425 DOI: 10.1016/j.bbalip.2004.08.010] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
Niemann-Pick Type C (NP-C) disease compellingly provides insight into lipid transport and the association of this process with severe neuronal dysfunction. The two genes that define this syndrome, NPC1 and NPC2, are conserved throughout much of eukaryotic evolution, to the extent that the yeast and mammalian NPC1 genes are functionally interchangeable. We present here an evolutionary perspective of the genes defective in NP-C disease. We will describe how conservation of sequences and their biological roles in a variety of microbial and metazoan model systems may act as roadmaps to understanding this syndrome in humans.
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Affiliation(s)
- Katsumi Higaki
- The Institute of Human Nutrition, Columbia University College of Physicians and Surgeons, 630 W168th St. New York, NY 10032, USA
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Abstract
The molecular mechanism of how hepatocytes maintain cholesterol homeostasis has become much more transparent with the discovery of sterol regulatory element binding proteins (SREBPs) in recent years. These membrane proteins are members of the basic helix-loop-helix-leucine zipper (bHLH-Zip) family of transcription factors. They activate the expression of at least 30 genes involved in the synthesis of cholesterol and lipids. SREBPs are synthesized as precursor proteins in the endoplasmic reticulum (ER), where they form a complex with another protein, SREBP cleavage activating protein (SCAP). The SCAP molecule contains a sterol sensory domain. In the presence of high cellular sterol concentrations SCAP confines SREBP to the ER. With low cellular concentrations, SCAP escorts SREBP to activation in the Golgi. There, SREBP undergoes two proteolytic cleavage steps to release the mature, biologically active transcription factor, nuclear SREBP (nSREBP). nSREBP translocates to the nucleus and binds to sterol response elements (SRE) in the promoter/enhancer regions of target genes. Additional transcription factors are required to activate transcription of these genes. Three different SREBPs are known, SREBPs-1a, -1c and -2. SREBP-1a and -1c are isoforms produced from a single gene by alternate splicing. SREBP-2 is encoded by a different gene and does not display any isoforms. It appears that SREBPs alone, in the sequence described above, can exert complete control over cholesterol synthesis, whereas many additional factors (hormones, cytokines, etc.) are required for complete control of lipid metabolism. Medicinal manipulation of the SREBP/SCAP system is expected to prove highly beneficial in the management of cholesterol-related disease.
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Affiliation(s)
- Lutz-W Weber
- Institute of Toxicology, GSF-National Research Center for Environment and Health, Munich, D-85758 Neuherberg, Germany.
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Sever N, Lee PCW, Song BL, Rawson RB, Debose-Boyd RA. Isolation of Mutant Cells Lacking Insig-1 through Selection with SR-12813, an Agent That Stimulates Degradation of 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase. J Biol Chem 2004; 279:43136-47. [PMID: 15247248 DOI: 10.1074/jbc.m406406200] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Insig-1 and Insig-2 are membrane proteins of the endoplasmic reticulum that regulate lipid metabolism by the following two actions: 1) sterol-induced binding to 3-hydroxy-3-methylglutaryl-coenzyme A reductase, an action that leads to ubiquitination and degradation of the enzyme; and 2) sterol-induced binding to SREBP cleavage-activating protein, an action that blocks the proteolytic processing of sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors that enhance the synthesis of cholesterol and fatty acids. Here we report the isolation of a new mutant line of Chinese hamster ovary cells, designated SRD-14, in which Insig-1 mRNA and protein are not produced due to a partial deletion of the INSIG-1 gene. The SRD-14 cells were produced by gamma-irradiation, followed by selection with the 1,1-bisphosphonate ester SR-12813, which mimics sterols in accelerating reductase degradation but does not block SREBP processing. SRD-14 cells fail to respond to sterols by promoting reductase ubiquitination and degradation. The rate at which sterols suppress SREBP processing is significantly slower in SRD-14 cells than wild type CHO-7 cells. Sterol regulation of reductase degradation and SREBP processing is restored when SRD-14 cells are transfected with expression plasmids encoding either Insig-1 or Insig-2. These results provide formal genetic proof for the essential role of Insig-1 in feedback control of lipid synthesis in cultured cells.
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Affiliation(s)
- Navdar Sever
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA
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15
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Malathi K, Higaki K, Tinkelenberg AH, Balderes DA, Almanzar-Paramio D, Wilcox LJ, Erdeniz N, Redican F, Padamsee M, Liu Y, Khan S, Alcantara F, Carstea ED, Morris JA, Sturley SL. Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution. ACTA ACUST UNITED AC 2004; 164:547-56. [PMID: 14970192 PMCID: PMC2171978 DOI: 10.1083/jcb.200310046] [Citation(s) in RCA: 109] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C–related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.
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Affiliation(s)
- Krishnamurthy Malathi
- Institute of Human Nutrition, Columbia University Medical Center, 630 W. 168 St., New York, NY 10032, USA
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Zhou RH, Yao M, Lee TS, Zhu Y, Martins-Green M, Shyy JYJ. Vascular endothelial growth factor activation of sterol regulatory element binding protein: a potential role in angiogenesis. Circ Res 2004; 95:471-8. [PMID: 15271857 DOI: 10.1161/01.res.0000139956.42923.4a] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
Abstract
By stimulating the migration and proliferation of endothelial cells (ECs), vascular endothelial growth factor (VEGF) is a potent angiogenic factor. However, the molecular mechanism involved in the VEGF-induced angiogenesis remains elusive. We hypothesized that sterol regulatory element binding proteins (SREBPs), transcription factors governing cellular lipid homeostasis, play an important role in regulating angiogenesis in response to VEGF. VEGF activated SREBP1 and SREBP2 in ECs, as demonstrated by the increased SREBPs, their cleavage products, and the upregulation of the targeted genes. VEGF-induced SREBP activation depended on SREBP cleavage-activating protein (SCAP), because knocking down SCAP by RNA interference (RNAi) inhibited SREBP activation in response to VEGF. SREBP activation was also blocked by 25-hydroxycholesterol (25-HC). To verify the functional implication of SREBPs in VEGF-induced angiogenesis, we tested the role of SREBPs in EC migration and proliferation. SCAP RNAi or 25-HC inhibited VEGF-induced pseudopodia extension and migration of ECs. Both treatments inhibited VEGF-induced EC proliferation, with cell growth arrested at the G(0)/G(1) phase and a concomitant decrease of the S phase. Blocking the PI3K-Akt pathway inhibited the VEGF-activated SREBPs, demonstrating that PI3K-Akt regulates SREBPs. Consistent with our in vitro data, SREBP1 was detected in newly developed microvasculatures in a rabbit skin partial-thickness wound-healing model. SREBP inhibition also markedly suppressed VEGF-induced angiogenesis in chick embryos. In summary, this study identifies SREBPs as the key molecules in regulating angiogenesis in response to VEGF.
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Affiliation(s)
- Rui-Hai Zhou
- Division of Biomedical Sciences, University of California, Riverside Riverside 92521-0121, USA
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17
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Ono K, Iwanaga Y, Hirayama M, Kawamura T, Sowa N, Hasegawa K. Contribution of caveolin-1 alpha and Akt to TNF-alpha-induced cell death. Am J Physiol Lung Cell Mol Physiol 2004; 287:L201-9. [PMID: 15020298 DOI: 10.1152/ajplung.00293.2003] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-alpha (TNF-alpha)-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1 alpha is required for TNF-alpha-induced cell death in L929 cells. The need for caveolin-1 alpha in TNF-alpha-induced cell death was confirmed by the restoration of sensitivity to TNF-alpha after ectopic reconstitution of caveolin-1 alpha/beta expression. This caveolin-1 alpha-mutated line was also resistant to H(2)O(2) and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1 alpha/beta were found to be much more sensitive to TNF-alpha than either parental cells transfected with caveolin-1 beta or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-alpha, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-alpha-treated L929 cells. The survival rates of caveolin-1 alpha-normal and caveolin-1 alpha-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-alpha. Similar results were obtained with HepG2 cells that stably expressed caveolin-1 alpha/beta or -beta and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1 alpha preferentially sensitizes L929 cells to TNF-alpha through the activation of a PI3K/Akt signaling pathway.
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Affiliation(s)
- Koh Ono
- Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555, Japan.
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Sever N, Song BL, Yabe D, Goldstein JL, Brown MS, DeBose-Boyd RA. Insig-dependent ubiquitination and degradation of mammalian 3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols and geranylgeraniol. J Biol Chem 2003; 278:52479-90. [PMID: 14563840 DOI: 10.1074/jbc.m310053200] [Citation(s) in RCA: 233] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The endoplasmic reticulum enzyme 3-hydroxy-3-methylglutaryl-CoA reductase produces mevalonate, which is converted to sterols and to other products, including geranylgeraniol groups attached to proteins. The enzyme is known to be ubiquitinated and rapidly degraded when sterols and nonsterol end products of mevalonate metabolism accumulate in cells. Here, we use RNA interference to show that sterol-accelerated ubiquitination of reductase requires Insig-1 and Insig-2, membrane-bound proteins of the endoplasmic reticulum that were shown previously to accelerate degradation of reductase when overexpressed by transfection. Alanine substitution experiments reveal that binding of reductase to Insigs and subsequent ubiquitination require the tetrapeptide sequence YIYF in the second membrane-spanning helix of reductase. The YIYF peptide is also found in the sterol-sensing domain of SCAP, another protein that binds to Insigs in a sterol-stimulated fashion. When lysine 248 of reductase is substituted with arginine, Insig binding persists, but the reductase is no longer ubiquitinated and degradation is markedly slowed. Lysine 248 is predicted to lie immediately adjacent to a membrane-spanning helix, suggesting that a membrane-bound ubiquitin transferase is responsible. Finally, we show that Insig-dependent, sterol-stimulated degradation of reductase is further accelerated when cells are also supplied with the 20-carbon isoprenoid geranylgeraniol, but not the 15-carbon farnesol, raising the possibility that the nonsterol potentiator of reductase regulation is a geranylgeranylated protein.
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Affiliation(s)
- Navdar Sever
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA
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19
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Abstract
Animal cells coordinate lipid homeostasis by end-product feedback regulation of transcription. The control occurs through the proteolytic release of transcriptionally active sterol regulatory element binding proteins (SREBPs) from intracellular membranes. This feedback system has unexpected features that are found in all cells. Here, we consider recently discovered components of the regulatory machinery that govern SREBP processing, as well as studies in Drosophila that indicate an ancient role for the SREBP pathway in integrating membrane composition and lipid biosynthesis.
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Affiliation(s)
- Robert B Rawson
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.
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20
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Abstract
The mammalian cell continuously adjusts its sterol content by regulating levels of key sterol synthetic enzymes and levels of LDL receptors that mediate uptake of cholesterol-laden particles. Control is brought about by sterol-regulated transcription of relevant genes and by regulated degradation of the committed step enzyme HMG-CoA reductase (HMGR). Current work has revealed that proteolysis is at the heart of each of these mechanistically distinct axes. Transcriptional control is effected by regulated cleavage of the membrane-bound transcription factor sterol regulatory element binding protein (SREBP), and HMGR degradation is brought about by ubiquitin-mediated degradation. In each case, ongoing cell biological processes are being harnessed to bring about regulation. The secretory pathway plays a central role in allowing sterol-mediated control of transcription. The constitutively active endoplasmic reticulum (ER) quality control apparatus is employed to bring about regulated destruction of HMGR. This review describes the methods and results of various studies to understand the mechanisms and molecules involved in these distinct but interrelated aspects of sterol regulation and the intriguing similarities that appear to exist at the levels of protein sequence and cell biology.
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Affiliation(s)
- Randolph Y Hampton
- Section of Cell and Developmental Biology, Division of Biology, University of California, San Diego, La Jolla 92093-0347, USA.
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21
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Salek L, Lutucuta S, Ballantyne CM, Gotto AM, Marian AJ. Effects of SREBF-1a and SCAP polymorphisms on plasma levels of lipids, severity, progression and regression of coronary atherosclerosis and response to therapy with fluvastatin. J Mol Med (Berl) 2002; 80:737-44. [PMID: 12436350 PMCID: PMC2896566 DOI: 10.1007/s00109-002-0381-z] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2002] [Accepted: 07/25/2002] [Indexed: 10/27/2022]
Abstract
Sterol regulatory elements binding factor-1a (SREBF-1a) and SREBF cleavage activating protein (SCAP) regulate lipids homeostasis. Polymorphisms in SREBF-1a and SCAP could affect plasma levels of lipids and risk of atherosclerosis. We determined association of SREBF-1a -36del/G and SCAP 2386A/G genotypes with plasma levels of lipids, severity and progression/regression of coronary atherosclerosis, and response to treatment with fluvastatin in a well-characterized Lipoprotein Coronary Atherosclerosis Study population. Plasma lipids and quantitative indices of coronary atherosclerosis were obtained at baseline and 2.5 years following randomization to fluvastatin or placebo in 372 subjects. Fluvastatin reduced plasma levels of total cholesterol by 16%, LDL-C by 25%, and ApoB by 16% and increased plasma levels of HDL-C by 9% and apoA-1 by 7%. Distributions of SREBF-1a SCAP genotypes were 60 GG, 172 del-G and 140 del-del and 88 GG, 188 GA and 96 AA, respectively. There were no significant differences in baseline plasma levels of lipids or indices of severity of atherosclerosis among the genotypes of each gene. There was a strong graded genotype-treatment interaction between SREBF-1a genotypes and change in apoA-I levels in response to fluvastatin (16.5% increase in GG, 10.5% in del/G, and 0.4% in del/del groups). Modest interactions between SREBF-1a genotypes and changes in HDL-C, and apoC-III levels in response to fluvastatin were also present. No genotype-treatment interaction for progression or regression of coronary atherosclerosis was detected. There were no significant interactions between SCAP genotypes and response to therapy. Thus we detected a strong graded interaction between SREBF-1a -36del/G genotypes and response of plasma apoA-I to treatment with fluvastatin.
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Affiliation(s)
- Lorraine Salek
- Section of Cardiology, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
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22
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Johnson RL, Zhou L, Bailey EC. Distinct consequences of sterol sensor mutations in Drosophila and mouse patched homologs. Dev Biol 2002; 242:224-35. [PMID: 11820817 DOI: 10.1006/dbio.2001.0524] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
The membrane protein Patched (Ptc) is a critical regulator of Hedgehog signaling. Ptc is among a family of proteins that contain a sterol sensor motif. The function of this domain is poorly understood, but some proteins that contain sterol sensors are involved in cholesterol homeostasis. In the SREBP cleavage-activating protein (SCAP), sterols inhibit the protein's activity through this domain. Mutations in two highly conserved residues in the SCAP sterol sensor have been identified that confer resistance to sterol regulation. We introduced the analogous mutations in the sterol sensor motif of fly Ptc and mouse Ptc1 and examined their effect on protein activity. In contrast to SCAP, the sterol sensor mutations had different affects on Drosophila Ptc; Ptc Y442C retained function, while Ptc D584N conferred dominant negative activity. In the wing imaginal disc, Ptc D584N overexpression induced Hedgehog targets by stabilizing Cubitus interruptus and inducing decapentaplegic. However, Ptc D584N did not induce collier, a gene that requires high levels of Hedgehog signaling. In mouse Ptc1, the Y438C and D585N mutations did not stimulate signaling in Shh-responsive cell lines but did complement murine ptc1(-/-) cells. The results suggest that mutations in sterol sensor motifs alter function differently between sterol sensor family members.
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Affiliation(s)
- Ronald L Johnson
- Departments of Cell Biology and Neurobiology, University of Alabama at Birmingham, 35294-0005, USA.
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23
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Seegmiller AC, Dobrosotskaya I, Goldstein JL, Ho YK, Brown MS, Rawson RB. The SREBP pathway in Drosophila: regulation by palmitate, not sterols. Dev Cell 2002; 2:229-38. [PMID: 11832248 DOI: 10.1016/s1534-5807(01)00119-8] [Citation(s) in RCA: 143] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. Here, we show that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the two proteases that process SREBP at sites 1 and 2 to release the nuclear fragment. In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis.
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Affiliation(s)
- Adam C Seegmiller
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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24
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Goldstein JL, Rawson RB, Brown MS. Mutant mammalian cells as tools to delineate the sterol regulatory element-binding protein pathway for feedback regulation of lipid synthesis. Arch Biochem Biophys 2002; 397:139-48. [PMID: 11795864 DOI: 10.1006/abbi.2001.2615] [Citation(s) in RCA: 192] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The tools of somatic cell genetics have been instrumental in unraveling the pathway by which sterol regulatory element-binding proteins (SREBPs) control lipid metabolism in animal cells. SREBPs are membrane-bound transcription factors that enhance the synthesis and uptake of cholesterol and fatty acids. The activities of the SREBPs are controlled by the cholesterol content of cells through feedback inhibition of proteolytic processing. When cells are replete with sterols, SREBPs remain bound to membranes of the endoplasmic reticulum (ER) and are therefore inactive. When cells are depleted of sterols, the SREBPs move to the Golgi complex where two proteases release the active portions of the SREBPs, which then enter the nucleus and activate transcription of target genes. This processing requires three membrane proteins-a sterol-sensing escort protein (SCAP) that transports SREBPs from the ER to the Golgi and two Golgi-located proteases (S1P and S2P) that release SREBPs from membranes. The existence of all three proteins was revealed through analysis of mutant mammalian cells in tissue culture. Their cDNAs and genes were isolated by genetic complementation or by expression cloning. The somatic cell genetic approach described in this article should prove useful for unraveling other complex biochemical pathways in animal cells.
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Affiliation(s)
- Joseph L Goldstein
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA.
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25
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Martín V, Carrillo G, Torroja C, Guerrero I. The sterol-sensing domain of Patched protein seems to control Smoothened activity through Patched vesicular trafficking. Curr Biol 2001; 11:601-7. [PMID: 11369205 DOI: 10.1016/s0960-9822(01)00178-6] [Citation(s) in RCA: 139] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The Hedgehog (Hh) family of signaling molecules function as organizers in many morphogenetic processes. Hh signaling requires cholesterol in both signal-generating and -receiving cells, and it requires the tumor suppressor Patched (Ptc) in receiving cells in which it plays a negative role. Ptc both blocks the Hh pathway and limits the spread of Hh. Sequence analysis suggests that it has 12 transmembrane segments, 5 of which are homologous to a conserved region that has been identified in several proteins involved in cholesterol homeostasis and has been designated the sterol-sensing domain (SSD). In the present study, we show that a Ptc mutant with a single amino acid substitution in the SSD induces target gene activation in a ligand-independent manner. This mutant Ptc(SSD) protein shows dominant-negative activity in blocking Hh signaling by preventing the downregulation of Smoothened (Smo), a positive effector of the Hh pathway. Despite its dominant-negative activity, the mutant Ptc protein functioned like the wild-type protein in sequestering and internalizing Hh. In addition, we show that Ptc(SSD) preferentially accumulates in endosomes of the endocytic compartment. All these results suggest a role of the SSD of Ptc in mediating the vesicular trafficking of Ptc to regulate Smo activity.
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Affiliation(s)
- V Martín
- Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, E-28049, Madrid, Spain
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26
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Matsuda M, Korn BS, Hammer RE, Moon YA, Komuro R, Horton JD, Goldstein JL, Brown MS, Shimomura I. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver induced by cholesterol deprivation and insulin elevation. Genes Dev 2001; 15:1206-16. [PMID: 11358865 PMCID: PMC313801 DOI: 10.1101/gad.891301] [Citation(s) in RCA: 274] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
In liver, the synthesis of cholesterol and fatty acids increases in response to cholesterol deprivation and insulin elevation, respectively. This regulatory mechanism underlies the adaptation to cholesterol synthesis inhibitors (statins) and high calorie diets (insulin). In nonhepatic cells, lipid synthesis is controlled by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose active domains are released proteolytically to enter the nucleus and activate genes involved in the synthesis and uptake of cholesterol and fatty acids. SCAP (SREBP cleavage-activating protein) is a sterol-regulated escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of cleavage in the Golgi. Here, we produced a conditional deficiency of SCAP in mouse liver by genomic recombination mediated by inducible Cre recombinase. SCAP-deficient mice showed an 80% reduction in basal rates of cholesterol and fatty acid synthesis in liver, owing to decreases in mRNAs encoding multiple biosynthetic enzymes. Moreover, these mRNAs failed to increase normally in response to cholesterol deprivation produced by a cholesterol synthesis inhibitor and to insulin elevation produced by a fasting-refeeding protocol. These data provide in vivo evidence that SCAP and the SREBPs are required for hepatic lipid synthesis under basal and adaptive conditions.
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Affiliation(s)
- M Matsuda
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9046, USA
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27
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Wang X, Han J. Elucidating tumor necrosis factor signaling pathway using a functional gene identification approach. Immunol Res 2000; 21:55-61. [PMID: 10852102 DOI: 10.1385/ir:21:2-3:55] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Functional identification of genes is an efficient way to study many biological processes in lower eukaryotes. However, an effective approach in mammalian cells is still under development. We designed a functional gene identification procedure and applied it in a study of tumor necrosis factor (TNF)-induced cell killing. This procedure employed a specially designed retroviral vector that allows random truncation of genes, efficiently selecting clones in which a gene was disrupted and quickly identifying disrupted genes. We have identified several novel genes by a preliminary test of this approach and confirmed by reconstitution that the genes we identified are required for TNF cytotoxicity in L929 cells. Because of the efficient identification of these components in TNF-induced cell killing, we have already been able to outline the killing pathway of TNF in L929 cells. Application of this method could be widespread because it can be used in studying any cellular responses if a specific selection assay can be set up.
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Affiliation(s)
- X Wang
- Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA
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28
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Bailey EC, Scott MP, Johnson RL. Hedgehog signaling in animal development and human disease. ERNST SCHERING RESEARCH FOUNDATION WORKSHOP 2000:211-35. [PMID: 10943312 DOI: 10.1007/978-3-662-04264-9_12] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Affiliation(s)
- E C Bailey
- Department of Cell Biology, University of Alabama at Birmingham 35294-0005, USA
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29
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Davies JP, Ioannou YA. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein. J Biol Chem 2000; 275:24367-74. [PMID: 10821832 DOI: 10.1074/jbc.m002184200] [Citation(s) in RCA: 229] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
The Niemann-Pick C1 (NPC1) protein is predicted to be a polytopic glycoprotein, and it contains a region with extensive homology to the sterol-sensing domains (SSD) of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-R) and sterol regulatory element binding protein cleavage-activating protein (SCAP). To aid the functional characterization of NPC1, a model of NPC1 topology was evaluated by expression of epitope-tagged NPC1 proteins and investigation of epitope accessibility in selectively permeabilized cells. These results were further confirmed by expression of NPC1 and identification of glycosylated domains that are located in the lumen of the endoplasmic reticulum. Our data indicate that this glycoprotein contains 13 transmembrane domains, 3 large and 4 small luminal loops, 6 small cytoplasmic loops, and a cytoplasmic tail. Furthermore, our data show that the putative SSD of NPC1 is oriented in the same manner as those of HMG-R and SCAP, providing strong evidence that this domain is functionally important.
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Affiliation(s)
- J P Davies
- Department of Human Genetics, Mount Sinai School of Medicine, New York, New York 10029, USA
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30
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Thewke D, Kramer M, Sinensky MS. Transcriptional homeostatic control of membrane lipid composition. Biochem Biophys Res Commun 2000; 273:1-4. [PMID: 10873553 DOI: 10.1006/bbrc.2000.2826] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Plasma membranes have a structural property, commonly referred to as membrane fluidity, that is compositionally regulated. The two main features of plasma membrane lipid composition that determine membrane fluidity are the ratio of cholesterol to phospholipids and the ratio of saturated to unsaturated fatty acids that are incorporated into the phospholipids. These ratios are determined, at least in part, by regulation of membrane lipid biosynthesis-particularly that of cholesterol and oleate. It now appears that cholesterol and oleate biosynthesis are feedback regulated by a common transcriptional mechanism which is governed by the maturation of the SREBP transcription factors. In this article, we briefly review our current understanding of transcriptional regulation of plasma membrane lipid biosynthesis by sterols and oleate. We also discuss studies related to the mechanism by which the physical state of membrane lipids signals the transcriptional regulatory machinery to control the rates of synthesis of these structural components of the lipid bilayer.
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Affiliation(s)
- D Thewke
- Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, Johnson City, Tennessee 37614-0581, USA
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31
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Lagace TA, Storey MK, Ridgway ND. Regulation of phosphatidylcholine metabolism in Chinese hamster ovary cells by the sterol regulatory element-binding protein (SREBP)/SREBP cleavage-activating protein pathway. J Biol Chem 2000; 275:14367-74. [PMID: 10799518 DOI: 10.1074/jbc.275.19.14367] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Sterol regulation-defective (SRD) 4 cells expressing a mutant sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP D443N) and Chinese hamster ovary (CHO) cells stably expressing SCAP (CHO-SCAP) and SCAP D443N (CHO-SCAP-D443N) have increased cholesterol and fatty acid synthesis because of constitutive processing of SREBPs. We assessed whether constitutive activation of SREBPs also influenced the CDP-choline pathway for phosphatidylcholine (PtdCho) biosynthesis. Relative to control CHO 7 cells, SRD 4 cells displayed increased PtdCho synthesis and degradation as indicated by a 4-6-fold increase in [(3)H]choline incorporation into PtdCho and 10-15-fold increase in intracellular [(3)H]glycerophosphocholine. [(3)H]Phosphocholine levels in SRD 4 cells were reduced by over 10-fold, suggesting enhanced activity of CTP:phosphocholine cytidylyltransferase alpha (CCTalpha). CHO-SCAP and CHO-SCAP D443N cells displayed modest increases in [(3)H]choline incorporation into PtdCho (2-fold) and only a 2-fold reduction in [(3)H]phosphocholine. Elevated PtdCho metabolism in SRD 4, compared with SCAP-overexpressing cells, was correlated with fatty acid synthesis. Inhibition of fatty acid synthesis by cerulenin resulted in almost complete normalization of PtdCho synthesis and choline metabolite profiles in SRD 4 cells, indicating that fatty acids or a fatty acid-derived metabolite was responsible for up-regulation of PtdCho synthesis. In contrast to apparent activation in vivo, CCTalpha protein, mRNA, and in vitro activity were reduced in SRD 4 cells and unchanged in SCAP transfected cells. Unlike control and SCAP transfected cells, CCTalpha in SRD 4 cells was localized by immunofluorescence to the nuclear envelope, suggesting that residual enzyme activity in these cells was in an active membrane-associated form. Translocation of CCTalpha to the nuclear envelope was reproduced by treatment of CHO 7 cells with exogenous oleate. We conclude that the SREBP/SCAP pathway regulates PtdCho synthesis via post-transcriptional activation of nuclear CCTalpha by fatty acids or a fatty acid-derived signal.
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Affiliation(s)
- T A Lagace
- Atlantic Research Center and the Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
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32
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Abstract
Oxygenated derivatives of cholesterol (oxysterols) present a remarkably diverse profile of biological activities, including effects on sphingolipid metabolism, platelet aggregation, apoptosis, and protein prenylation. The most notable oxysterol activities center around the regulation of cholesterol homeostasis, which appears to be controlled in part by a complex series of interactions of oxysterol ligands with various receptors, such as the oxysterol binding protein, the cellular nucleic acid binding protein, the sterol regulatory element binding protein, the LXR nuclear orphan receptors, and the low-density lipoprotein receptor. Identification of the endogenous oxysterol ligands and elucidation of their enzymatic origins are topics of active investigation. Except for 24, 25-epoxysterols, most oxysterols arise from cholesterol by autoxidation or by specific microsomal or mitochondrial oxidations, usually involving cytochrome P-450 species. Oxysterols are variously metabolized to esters, bile acids, steroid hormones, cholesterol, or other sterols through pathways that may differ according to the type of cell and mode of experimentation (in vitro, in vivo, cell culture). Reliable measurements of oxysterol levels and activities are hampered by low physiological concentrations (approximately 0.01-0.1 microM plasma) relative to cholesterol (approximately 5,000 microM) and by the susceptibility of cholesterol to autoxidation, which produces artifactual oxysterols that may also have potent activities. Reports describing the occurrence and levels of oxysterols in plasma, low-density lipoproteins, various tissues, and food products include many unrealistic data resulting from inattention to autoxidation and to limitations of the analytical methodology. Because of the widespread lack of appreciation for the technical difficulties involved in oxysterol research, a rigorous evaluation of the chromatographic and spectroscopic methods used in the isolation, characterization, and quantitation of oxysterols has been included. This review comprises a detailed and critical assessment of current knowledge regarding the formation, occurrence, metabolism, regulatory properties, and other activities of oxysterols in mammalian systems.
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Affiliation(s)
- G J Schroepfer
- Departments of Biochemistry, Rice University, Houston, Texas, USA.
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33
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DeBose-Boyd RA, Brown MS, Li WP, Nohturfft A, Goldstein JL, Espenshade PJ. Transport-dependent proteolysis of SREBP: relocation of site-1 protease from Golgi to ER obviates the need for SREBP transport to Golgi. Cell 1999; 99:703-12. [PMID: 10619424 DOI: 10.1016/s0092-8674(00)81668-2] [Citation(s) in RCA: 260] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Cholesterol homeostasis in animal cells is achieved by regulated cleavage of membrane-bound transcription factors, designated SREBPs. Proteolytic release of the active domains of SREBPs from membranes requires a sterol-sensing protein, SCAP, which forms a complex with SREBPs. In sterol-depleted cells, SCAP escorts SREBPs from ER to Golgi, where SREBPs are cleaved by Site-1 protease (S1P). Sterols block this transport and abolish cleavage. Relocating active S1P from Golgi to ER by treating cells with brefeldin A or by fusing the ER retention signal KDEL to S1P obviates the SCAP requirement and renders cleavage insensitive to sterols. Transport-dependent proteolysis may be a common mechanism to regulate the processing of membrane proteins.
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Affiliation(s)
- R A DeBose-Boyd
- Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75390-9046, USA
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34
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Rawson RB, DeBose-Boyd R, Goldstein JL, Brown MS. Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein. J Biol Chem 1999; 274:28549-56. [PMID: 10497220 DOI: 10.1074/jbc.274.40.28549] [Citation(s) in RCA: 145] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
We describe a line of mutant Chinese hamster ovary cells, designated SRD-13A, that cannot cleave sterol regulatory element-binding proteins (SREBPs) at site 1, due to mutations in the gene encoding SREBP cleavage-activating protein (SCAP). The SRD-13A cells were obtained by two rounds of gamma-irradiation followed first by selection for a deficiency of low density lipoprotein receptors and second for cholesterol auxotrophy. In the SRD-13A cells, the only detectable SCAP allele encodes a truncated nonfunctional protein. In the absence of SCAP, the site 1 protease fails to cleave SREBPs, and their transcriptionally active NH(2)-terminal fragments cannot enter the nucleus. As a result, the cells manifest a marked reduction in the synthesis of cholesterol and its uptake from low density lipoproteins. The SRD-13A cells grow only when cholesterol is added to the culture medium. SREBP cleavage is restored and the cholesterol requirement is abolished when SRD-13A cells are transfected with expression vectors encoding SCAP. These results provide formal proof that SCAP is essential for the cleavage of SREBPs at site 1.
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Affiliation(s)
- R B Rawson
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
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35
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Brown MS, Goldstein JL. A proteolytic pathway that controls the cholesterol content of membranes, cells, and blood. Proc Natl Acad Sci U S A 1999; 96:11041-8. [PMID: 10500120 PMCID: PMC34238 DOI: 10.1073/pnas.96.20.11041] [Citation(s) in RCA: 1016] [Impact Index Per Article: 39.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The integrity of cell membranes is maintained by a balance between the amount of cholesterol and the amounts of unsaturated and saturated fatty acids in phospholipids. This balance is maintained by membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) that activate genes encoding enzymes of cholesterol and fatty acid biosynthesis. To enhance transcription, the active NH(2)-terminal domains of SREBPs are released from endoplasmic reticulum membranes by two sequential cleavages. The first is catalyzed by Site-1 protease (S1P), a membrane-bound subtilisin-related serine protease that cleaves the hydrophilic loop of SREBP that projects into the endoplasmic reticulum lumen. The second cleavage, at Site-2, requires the action of S2P, a hydrophobic protein that appears to be a zinc metalloprotease. This cleavage is unusual because it occurs within a membrane-spanning domain of SREBP. Sterols block SREBP processing by inhibiting S1P. This response is mediated by SREBP cleavage-activating protein (SCAP), a regulatory protein that activates S1P and also serves as a sterol sensor, losing its activity when sterols overaccumulate in cells. These regulated proteolytic cleavage reactions are ultimately responsible for controlling the level of cholesterol in membranes, cells, and blood.
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Affiliation(s)
- M S Brown
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA.
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Nohturfft A, DeBose-Boyd RA, Scheek S, Goldstein JL, Brown MS. Sterols regulate cycling of SREBP cleavage-activating protein (SCAP) between endoplasmic reticulum and Golgi. Proc Natl Acad Sci U S A 1999; 96:11235-40. [PMID: 10500160 PMCID: PMC18017 DOI: 10.1073/pnas.96.20.11235] [Citation(s) in RCA: 192] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
The proteolytic cleavage of sterol regulatory element-binding proteins (SREBPs) is regulated by SREBP cleavage-activating protein (SCAP), which forms complexes with SREBPs in membranes of the endoplasmic reticulum (ER). In sterol-depleted cells, SCAP facilitates cleavage of SREBPs by Site-1 protease, thereby initiating release of active NH(2)-terminal fragments from the ER membrane so that they can enter the nucleus and activate gene expression. In sterol-overloaded cells, the activity of SCAP is blocked, SREBPs remain bound to membranes, and transcription of sterol-regulated genes declines. Here, we provide evidence that sterols act by inhibiting the cycling of SCAP between the ER and Golgi. We use glycosidases, glycosidase inhibitors, and a glycosylation-defective mutant cell line to demonstrate that the N-linked carbohydrates of SCAP are modified by Golgi enzymes in sterol-depleted cells. After modification, SCAP returns to the ER, as indicated by experiments that show that the Golgi-modified forms of SCAP cofractionate with ER membranes on density gradients. In sterol-overloaded cells, the Golgi modifications of SCAP do not occur, apparently because SCAP fails to leave the ER. Golgi modifications of SCAP are restored when sterol-overloaded cells are treated with brefeldin A, which causes Golgi enzymes to translocate to the ER. These studies suggest that sterols regulate the cleavage of SREBPs by modulating the ability of SCAP to transport SREBPs to a post-ER compartment that houses active Site-1 protease.
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Affiliation(s)
- A Nohturfft
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA
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Nohturfft A, Brown MS, Goldstein JL. Sterols regulate processing of carbohydrate chains of wild-type SREBP cleavage-activating protein (SCAP), but not sterol-resistant mutants Y298C or D443N. Proc Natl Acad Sci U S A 1998; 95:12848-53. [PMID: 9789003 PMCID: PMC23627 DOI: 10.1073/pnas.95.22.12848] [Citation(s) in RCA: 108] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/16/1998] [Indexed: 11/18/2022] Open
Abstract
SREBP cleavage activating protein (SCAP), a membrane-bound glycoprotein, regulates the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which are membrane-bound transcription factors that control lipid synthesis in animal cells. SCAP-stimulated proteolysis releases active fragments of SREBPs from membranes of the endoplasmic reticulum and allows them to enter the nucleus where they activate transcription. Sterols such as 25-hydroxycholesterol inactivate SCAP, suppressing SREBP proteolysis and turning off cholesterol synthesis. We here report the isolation of Chinese hamster ovary cells with a point mutation in SCAP (Y298C) that renders the protein resistant to inhibition by 25-hydroxycholesterol. Like the previously described D443N mutation, the Y298C mutation occurs within the putative sterol-sensing domain, which is part of the polytopic membrane attachment region of SCAP. Cells that express SCAP(Y298C) continued to process SREBPs in the presence of 25-hydroxycholesterol and hence they resisted killing by this sterol. In wild-type Chinese hamster ovary cells the N-linked carbohydrate chains of SCAP were mostly in the endoglycosidase H-sensitive form when cells were grown in medium containing 25-hydroxycholesterol. In contrast, when cells were grown in sterol-depleted medium, these chains were converted to an endoglycosidase H-resistant form. 25-Hydroxycholesterol had virtually no effect in cells expressing SCAP(D443N) or SCAP(Y298C). The relation between this regulated carbohydrate processing to the SCAP-regulated proteolysis of SREBP remains to be explored.
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Affiliation(s)
- A Nohturfft
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA
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Rawson RB, Cheng D, Brown MS, Goldstein JL. Isolation of cholesterol-requiring mutant Chinese hamster ovary cells with defects in cleavage of sterol regulatory element-binding proteins at site 1. J Biol Chem 1998; 273:28261-9. [PMID: 9774448 DOI: 10.1074/jbc.273.43.28261] [Citation(s) in RCA: 82] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The synthesis and uptake of cholesterol requires transcription factors designated sterol regulatory element-binding proteins (SREBPs). SREBPs are bound to membranes in a hairpin orientation with their transcriptionally active NH2-terminal segments facing the cytosol. The NH2-terminal segments are released from membranes by two-step proteolysis initiated by site 1 protease (S1P), which cleaves in the luminal loop between two membrane-spanning segments. Next, site 2 protease (S2P) releases the NH2-terminal fragment of SREBP. The S2P gene was recently isolated by complementation cloning using Chinese hamster ovary cells that require cholesterol for growth, due to a mutation in the S2P gene. A similar approach cannot be used for S1P because all previous cholesterol auxotrophs manifest defects in S2P, which is encoded by a single copy gene. To circumvent this problem, in the current studies we transfected Chinese hamster ovary cells with the S2P cDNA, assuring multiple copies. We mutagenized the cells, selected for cholesterol auxotrophy, and identified two mutant cell lines (SRD-12A and -12B) that fail to cleave SREBPs at site 1. Complementation analysis demonstrated that the defects in both cell lines are recessive and noncomplementing, indicating a mutation in the same gene. These cells should now be useful for expression cloning of the sterol-regulated S1P gene.
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Affiliation(s)
- R B Rawson
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
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Sakai J, Rawson RB, Espenshade PJ, Cheng D, Seegmiller AC, Goldstein JL, Brown MS. Molecular identification of the sterol-regulated luminal protease that cleaves SREBPs and controls lipid composition of animal cells. Mol Cell 1998; 2:505-14. [PMID: 9809072 DOI: 10.1016/s1097-2765(00)80150-1] [Citation(s) in RCA: 303] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The lipid composition of animal cells is controlled by SREBPs, transcription factors released from membranes by sterol-regulated proteolysis. Release is initiated by Site-1 protease (S1P), which cleaves SREBPs in the ER luminal loop between two membrane-spanning regions. To clone S1P, we prepared pCMV-PLAP-BP2, which encodes a fusion protein that contains placental alkaline phosphatase (PLAP) in the ER lumen flanked by cleavage sites for signal peptidase and S1P. In sterol-deprived cells, cleavage by both proteases leads to PLAP secretion. PLAP is not secreted by SRD-12B cells, cholesterol auxotrophs that lack S1P. We transfected SRD-12B cells with pCMV-PLAP-BP2 plus pools of CHO cDNAs and identified a cDNA that restores Site-1 cleavage and PLAP secretion. The cDNA encodes S1P, an intraluminal 1052-amino-acid membrane-bound subtilisin-like protease. We propose that S1P is the sterol-regulated protease that controls lipid metabolism in animal cells.
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Affiliation(s)
- J Sakai
- Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75235, USA
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40
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Nohturfft A, Brown MS, Goldstein JL. Topology of SREBP cleavage-activating protein, a polytopic membrane protein with a sterol-sensing domain. J Biol Chem 1998; 273:17243-50. [PMID: 9642295 DOI: 10.1074/jbc.273.27.17243] [Citation(s) in RCA: 151] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The NH2-terminal fragments of sterol regulatory element-binding proteins (SREBPs) are released from endoplasmic reticulum membranes by proteases whose activities depend upon SREBP cleavage-activating protein (SCAP), a polytopic endoplasmic reticulum membrane protein. The activity of SCAP is inhibited by sterols, which appear to interact with the polytopic membrane domain of SCAP. Here, we use protease protection and N-linked glycosylation site-mapping techniques to define the topology of the eight membrane-spanning domains of SCAP. The data indicate that the NH2 terminus and COOH terminus of SCAP face the cytosol. The long intralumenal loops after membrane-spanning segments 1 and 7 are glycosylated, confirming their lumenal location. The region comprising membrane-spanning segments 2-6 shows sequence resemblance to putative sterol-sensing domains in three other proteins: 3-hydroxy-3-methylglutaryl CoA reductase (HMG-CoA reductase), the Niemann-Pick C1 protein, and the morphogen receptor Patched. The orientation of the eight membrane-spanning segments in SCAP is consistent with the model proposed for HMG-CoA reductase (Olender, E. H., and Simoni, R. D. (1992) J. Biol. Chem. 267, 4223-4235). The membrane-spanning domains of SCAP and HMG-CoA reductase confer sterol sensitivity upon the functional activities of the two molecules. The common membrane topology of the two proteins is consistent with the notion that sterols regulate both proteins by a common mechanism.
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Affiliation(s)
- A Nohturfft
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
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41
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Ross SL, Martin F, Simonet L, Jacobsen F, Deshpande R, Vassar R, Bennett B, Luo Y, Wooden S, Hu S, Citron M, Burgess TL. Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells. J Biol Chem 1998; 273:15309-12. [PMID: 9624107 DOI: 10.1074/jbc.273.25.15309] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.
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Affiliation(s)
- S L Ross
- Department of Mammalian Cell Molecular Biology, Amgen Inc., Thousand Oaks, California 91320-1789, USA
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Abstract
Hydroxymethylglutaryl-coenzyme A reductase degradation occurs in the endoplasmic reticulum, and is regulated by the mevalonate pathway. In order to discover the molecules that mediate the degradation process and its control, we conducted a genetic analysis of the degradation of the yeast Hmg2p isozyme of hydroxymethylglutaryl-coenzyme A reductase. Hmg2p degradation occurs by the action of HRD genes that direct Hmg2p to the ubiquitin-proteasome pathway. Regulation of HRD-dependent Hmg2p degradation appears to occur by the action of a separate set of CRD genes.
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Affiliation(s)
- R Y Hampton
- UCSD Department of Biology 0347, La Jolla 92093, USA.
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Osborne TF, Rosenfeld JM. 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. Curr Opin Lipidol 1998; 9:137-40. [PMID: 9559271 DOI: 10.1097/00041433-199804000-00010] [Citation(s) in RCA: 39] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
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.
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Affiliation(s)
- T F Osborne
- Department of Molecular Biology and Biochemistry, University of California at Irvine 92697-3900, USA
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44
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Sakai J, Nohturfft A, Goldstein JL, Brown MS. Cleavage of sterol regulatory element-binding proteins (SREBPs) at site-1 requires interaction with SREBP cleavage-activating protein. Evidence from in vivo competition studies. J Biol Chem 1998; 273:5785-93. [PMID: 9488713 DOI: 10.1074/jbc.273.10.5785] [Citation(s) in RCA: 181] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that promote lipid synthesis in animal cells. They are embedded in the membranes of the endoplasmic reticulum (ER) in a helical hairpin orientation and are released from the ER by a two-step proteolytic process. Proteolysis begins when the SREBPs are cleaved at Site-1, which is located at a leucine residue in the middle of the hydrophobic loop in the lumen of the ER. Sterols suppress Site-1 cleavage, apparently by interacting with a polytopic membrane protein designated SREBP cleavage-activating protein (SCAP). SREBPs and SCAP are joined together in ER membranes through interaction of their cytoplasmic COOH-terminal domains. Here we use an in vivo competition assay in transfected cells to show that the SREBP.SCAP complex is essential for Site-1 cleavage. Overexpression of the truncated COOH-terminal domains of either SREBP-2 or SCAP disrupted the complex between full-length SREBP-2 and SCAP as measured by co-immunoprecipitation. This resulted in a complete inhibition of Site-1 cleavage that was restored by concomitant overexpression of full-length SCAP. The transfected COOH-terminal domains also inhibited the transcription of a reporter gene driven by an SRE-containing promoter, and this, too, was restored by overexpression of full-length SCAP. We interpret these data to indicate that the SREBP.SCAP complex directs the Site-1 protease to its target in the lumenal domain of SREBP and that disruption of this complex inactivates the Site-1 cleavage reaction.
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Affiliation(s)
- J Sakai
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
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45
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Scheek S, Brown MS, Goldstein JL. Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1. Proc Natl Acad Sci U S A 1997; 94:11179-83. [PMID: 9326582 PMCID: PMC23408 DOI: 10.1073/pnas.94.21.11179] [Citation(s) in RCA: 96] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.
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Affiliation(s)
- S Scheek
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-9046, USA
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Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997; 89:331-40. [PMID: 9150132 DOI: 10.1016/s0092-8674(00)80213-5] [Citation(s) in RCA: 2870] [Impact Index Per Article: 102.5] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Affiliation(s)
- M S Brown
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA
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47
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Hua X, Nohturfft A, Goldstein JL, Brown MS. Sterol resistance in CHO cells traced to point mutation in SREBP cleavage-activating protein. Cell 1996; 87:415-26. [PMID: 8898195 DOI: 10.1016/s0092-8674(00)81362-8] [Citation(s) in RCA: 393] [Impact Index Per Article: 13.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
Through expression cloning we have isolated a cDNA-encoding SREBP cleavage-activating protein (SCAP), which regulates cholesterol metabolism by stimulating cleavage of transcription factors SREBP-1 and -2, thereby releasing them from membranes. The cDNA was isolated from Chinese hamster ovary cells with a dominant mutation that renders them resistant to sterol-mediated suppression of cholesterol synthesis and uptake. Sterol resistance was traced to a G-->A transition at codon 443 of SCAP, changing aspartic acid to asparagine. The D443N mutation enhances the cleavage-stimulating ability of SCAP and renders it resistant to inhibition by sterols. SCAP has multiple membrane-spanning regions, five of which resemble the sterol-sensing domain of HMG CoA reductase, an endoplasmic reticulum enzyme whose degradation is accelerated by sterols. SCAP appears to be a central regulator of cholesterol metabolism in animal cells.
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Affiliation(s)
- X Hua
- Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA
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