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Andrew J Brown - One of the best experts on this subject based on the ideXlab platform.
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the mammalian Cholesterol Synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form
Journal of Biological Chemistry, 2021Co-Authors: Hudson W Coates, Isabelle M Capellhattam, Andrew J BrownAbstract:Squalene monooxygenase (SM, also known as squalene epoxidase) is a rate-limiting enzyme of Cholesterol Synthesis that converts squalene to monooxidosqualene and is oncogenic in numerous cancer types. SM is subject to feedback regulation via Cholesterol-induced proteasomal degradation, which depends on its lipid-sensing N-terminal regulatory domain. We previously identified an endogenous truncated form of SM with a similar abundance to full-length SM, but whether this truncated form is functional or subject to the same regulatory mechanisms as full-length SM is not known. Here, we show that truncated SM differs from full-length SM in two major ways: it is Cholesterol resistant and adopts a peripheral rather than integral association with the endoplasmic reticulum membrane. However, truncated SM retains full SM activity and is therefore constitutively active. Truncation of SM occurs during its endoplasmic reticulum–associated degradation and requires the proteasome, which partially degrades the SM N-terminus and disrupts Cholesterol-sensing elements within the regulatory domain. Furthermore, truncation relies on a ubiquitin signal that is distinct from that required for Cholesterol-induced degradation. Using mutagenesis, we demonstrate that partial proteasomal degradation of SM depends on both an intrinsically disordered region near the truncation site and the stability of the adjacent catalytic domain, which escapes degradation. These findings uncover an additional layer of complexity in the post-translational regulation of Cholesterol Synthesis and establish SM as the first eukaryotic enzyme found to undergo proteasomal truncation.
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the mammalian Cholesterol Synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form
bioRxiv, 2020Co-Authors: Hudson W Coates, Andrew J BrownAbstract:Abstract Squalene monooxygenase (SM) is a rate-limiting enzyme of Cholesterol Synthesis that is oncogenic in a range of cancer types. SM is subject to feedback regulation via Cholesterol-induced degradation, which depends on its lipid-sensing N terminal regulatory domain. Here, we characterize an endogenous truncated form of SM and show that it is Cholesterol-resistant, and therefore constitutively active. Truncation of SM occurs during its endoplasmic reticulum-associated degradation and requires the proteasome, which partially degrades the SM N-terminus and eliminates Cholesterol-sensing elements within this region. Using mutagenesis studies, we demonstrate that partial degradation of SM depends on both an intrinsically disordered region near the truncation site and the stability of the adjacent catalytic domain. Finally, truncation converts SM from an integral to a peripheral ER membrane protein. These findings uncover an additional layer of complexity in the cellular control of Cholesterol Synthesis and establish SM as the first eukaryotic enzyme known to undergo proteasomal truncation.
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the degron architecture of squalene monooxygenase and how specific lipids calibrate levels of this key Cholesterol Synthesis enzyme
Advances in Experimental Medicine and Biology, 2020Co-Authors: Ngee Kiat Chua, Andrew J BrownAbstract:Cholesterol Synthesis is a fundamental process that contributes to cellular Cholesterol homeostasis. Cells execute transcriptional and post-translational mechanisms to control the abundance of enzymes of the Cholesterol Synthesis pathway, consequently affecting Cholesterol production. One such highly tuned enzyme is squalene monooxygenase (SM), which catalyzes a rate-limiting step in the pathway. A well-characterized mechanism is the Cholesterol-mediated degradation of SM. Notably, lipids (Cholesterol, plasmalogens, squalene, and unsaturated fatty acids) can act as cellular signals that either promote or reduce SM degradation. The N-terminal region of SM consists of the shortest known Cholesterol-responsive degron, characterized by atypical membrane anchoring structures, namely a re-entrant loop and an amphipathic helix. SM also undergoes non-canonical ubiquitination on serine, a relatively uncommon attachment site for ubiquitination. The structure of the catalytic domain of SM has been solved, providing insights into the catalytic mechanisms and modes of inhibition by well-known SM inhibitors, some of which have been effective in lowering Cholesterol levels in animal models. Certain human cancers have been linked to dysregulation of SM levels and activity, further emphasizing the relevance of SM in health and disease.
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squalene monooxygenase a journey to the heart of Cholesterol Synthesis
Progress in Lipid Research, 2020Co-Authors: Ngee Kiat Chua, Hudson W Coates, Andrew J BrownAbstract:Squalene monooxygenase (SM) is a vital sterol Synthesis enzyme across eukaryotic life. In yeast, it is a therapeutic target for treating certain fungal infections, and in mammals it is a rate-limiting enzyme that represents a key control point in the Cholesterol Synthesis pathway. SM introduces an oxygen atom to squalene, which becomes the signature oxygen of the hydroxyl group in Cholesterol. Our knowledge of SM has advanced tremendously since its initial cloning and characterization. Early research developed mammalian SM inhibitors to target SM for Cholesterol-lowering purposes. The substrate squalene has gained considerable interest for its health benefits and in nanomedicine for delivery of drugs. More recently, SM has been implicated as a key dysregulated component in certain cancers. In this review, we summarize our present knowledge of SM, focusing on the regulation of SM and the gene encoding it, SQLE. Furthermore, we offer insights into the role of SM across different organisms and its significance in human health and disease.
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the Cholesterol Synthesis enzyme lanosterol 14α demethylase is post translationally regulated by the e3 ubiquitin ligase march6
Biochemical Journal, 2020Co-Authors: Nicola A Scott, Laura J Sharpe, Isabelle M Capellhattam, Samuel J Gullo, Andrew J BrownAbstract:: Cholesterol Synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of Cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in Cholesterol Synthesis, also control levels of LDM and the terminal Cholesterol Synthesis enzyme, 24-dehydroCholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of Cholesterol Synthesis.
Joseph L. Goldstein - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol induced conformational changes in the sterol sensing domain of the scap protein suggest feedback mechanism to control Cholesterol Synthesis
Journal of Biological Chemistry, 2017Co-Authors: Yansong Gao, Michael S. Brown, Joseph L. Goldstein, Yulian Zhou, Arun RadhakrishnanAbstract:Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases Cholesterol Synthesis. Previously, we provided evidence that Cholesterol inhibition is initiated when Cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added Cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of Cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When Cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that Cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the Cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of Cholesterol Synthesis. These insights suggest a new structural model for Cholesterol-mediated regulation of Scap activity.
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Cholesterol induced conformational changes in the sterol sensing domain of the scap protein suggest feedback mechanism to control Cholesterol Synthesis
Journal of Biological Chemistry, 2017Co-Authors: Yulian Zhou, Michael S. Brown, Joseph L. Goldstein, Arun RadhakrishnanAbstract:Abstract Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases Cholesterol Synthesis. Previously, we provided evidence that Cholesterol inhibition is initiated when Cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added Cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of Cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When Cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that Cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the Cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of Cholesterol Synthesis. These insights suggest a new structural model for Cholesterol-mediated regulation of Scap activity.
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sterol regulated ubiquitination and degradation of insig 1 creates a convergent mechanism for feedback control of Cholesterol Synthesis and uptake
Cell Metabolism, 2006Co-Authors: Yi Gong, Michael S. Brown, Joseph L. Goldstein, Jin YeAbstract:Summary This paper describes a convergent mechanism for the feedback control of Cholesterol Synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for Cholesterol Synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for Synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and Cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.
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activation of Cholesterol Synthesis in preference to fatty acid Synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element binding protein 2
Journal of Clinical Investigation, 1998Co-Authors: Jay D Horton, Michael S. Brown, Joseph L. Goldstein, Iichiro Shimomura, Robert E Hammer, Hitoshi ShimanoAbstract:We produced transgenic mice that express a dominant-posi- tive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The en- coded protein lacks the membrane-binding and COOH-ter- minal regulatory domains, and it is therefore not susceptible to negative regulation by Cholesterol. Livers from the trans- genic mice showed increases in mRNAs encoding multi- ple enzymes of Cholesterol bioSynthesis, the LDL recep- tor, and fatty acid bioSynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the trans- genic livers showed a 28-fold increase in the rate of choles- terol Synthesis and a lesser fourfold increase in fatty acid Synthesis, as measured by intraperitoneal injection of ( 3 H)water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid Synthesis more than Cholesterol Synthesis. In adi- pose tissue of the SREBP-2 transgenics, the mRNAs for Cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of Cholesterol Synthesis, as opposed to fatty acid Synthesis, in liver and adipose tissue of mice. ( J. Clin. Invest. 1998. 101: 2331-2339.) Key words: Cholesterollow density lipoprotein • sterol regulatory element binding proteinsfatty acids • transgenic mice
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elevated levels of srebp 2 and Cholesterol Synthesis in livers of mice homozygous for a targeted disruption of the srebp 1 gene
Journal of Clinical Investigation, 1997Co-Authors: Hitoshi Shimano, Michael S. Brown, Joseph L. Goldstein, Iichiro Shimomura, Robert E Hammer, Joachim Herz, Jay D HortonAbstract:The Synthesis of Cholesterol and its uptake from plasma LDL are regulated by two membrane-bound transcription factors, designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). Here, we used the technique of homologous recombination to generate mice with disruptions in the gene encoding the two isoforms of SREBP-1, termed SREBP-1a and SREBP-1c. Heterozygous gene-disrupted mice were phenotypically normal, but 50- 85% of the homozygous (-/-) mice died in utero at embryonic day 11. The surviving -/- mice appeared normal at birth and throughout life. Their livers expressed no functional SREBP-1. There was a 1.5-fold upregulation of SREBP-2 at the level of mRNA and a two- to threefold increase in the amount of mature SREBP-2 in liver nuclei. Previous studies showed that SREBP-2 is much more potent than SREBP-1c, the predominant hepatic isoform of SREBP-1, in activating transcription of genes encoding enzymes of Cholesterol Synthesis. Consistent with this observation, the SREBP-1 -/- animals manifested elevated levels of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, farnesyl diphosphate synthase, and squalene synthase. Cholesterol Synthesis, as measured by the incorporation of [3H]water, was elevated threefold in livers of the -/- mice, and hepatic Cholesterol content was increased by 50%. Fatty acid Synthesis was decreased in livers of the -/- mice. The amount of white adipose tissue was not significantly decreased, and the levels of mRNAs for lipogenic enzymes, adipocyte lipid binding protein, lipoprotein lipase, and leptin were normal in the -/- mice. We conclude from these studies that SREBP-2 can replace SREBP-1 in regulating Cholesterol Synthesis in livers of mice and that the higher potency of SREBP-2 relative to SREBP-1c leads to excessive hepatic Cholesterol Synthesis in these animals.
Michael S. Brown - One of the best experts on this subject based on the ideXlab platform.
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Cholesterol induced conformational changes in the sterol sensing domain of the scap protein suggest feedback mechanism to control Cholesterol Synthesis
Journal of Biological Chemistry, 2017Co-Authors: Yansong Gao, Michael S. Brown, Joseph L. Goldstein, Yulian Zhou, Arun RadhakrishnanAbstract:Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases Cholesterol Synthesis. Previously, we provided evidence that Cholesterol inhibition is initiated when Cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added Cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of Cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When Cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that Cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the Cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of Cholesterol Synthesis. These insights suggest a new structural model for Cholesterol-mediated regulation of Scap activity.
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Cholesterol induced conformational changes in the sterol sensing domain of the scap protein suggest feedback mechanism to control Cholesterol Synthesis
Journal of Biological Chemistry, 2017Co-Authors: Yulian Zhou, Michael S. Brown, Joseph L. Goldstein, Arun RadhakrishnanAbstract:Abstract Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases Cholesterol Synthesis. Previously, we provided evidence that Cholesterol inhibition is initiated when Cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added Cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of Cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When Cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that Cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the Cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of Cholesterol Synthesis. These insights suggest a new structural model for Cholesterol-mediated regulation of Scap activity.
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sterol regulated ubiquitination and degradation of insig 1 creates a convergent mechanism for feedback control of Cholesterol Synthesis and uptake
Cell Metabolism, 2006Co-Authors: Yi Gong, Michael S. Brown, Joseph L. Goldstein, Jin YeAbstract:Summary This paper describes a convergent mechanism for the feedback control of Cholesterol Synthesis and uptake mediated by SREBPs, membrane bound transcription factors. Endoplasmic reticulum (ER) bound SREBPs form complexes with Scap, a polytopic ER protein. In sterol-overloaded cells, Scap/SREBP binds to Insig-1, which retains the complex in the ER. Upon sterol deprivation, the Scap/SREBP complex dissociates from Insig-1, which is then ubiquitinated on lysines 156 and 158 and degraded in proteasomes. Scap/SREBP moves to the Golgi, where SREBP is processed to liberate a nuclear fragment that activates genes for Cholesterol Synthesis and uptake and the gene for Insig-1. Ubiquitination is not necessary for release of Scap/SREBP from Insig-1, but it establishes a requirement for Synthesis of new Insig-1 for feedback inhibition. When the new Insig-1 and Cholesterol converge on Scap, Scap/SREBP binds to Insig-1, preventing ubiquitination. The Insig-1/Scap/SREBP complex accumulates in the ER, ready for liberation when the cell is again sterol deprived.
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activation of Cholesterol Synthesis in preference to fatty acid Synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element binding protein 2
Journal of Clinical Investigation, 1998Co-Authors: Jay D Horton, Michael S. Brown, Joseph L. Goldstein, Iichiro Shimomura, Robert E Hammer, Hitoshi ShimanoAbstract:We produced transgenic mice that express a dominant-posi- tive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The en- coded protein lacks the membrane-binding and COOH-ter- minal regulatory domains, and it is therefore not susceptible to negative regulation by Cholesterol. Livers from the trans- genic mice showed increases in mRNAs encoding multi- ple enzymes of Cholesterol bioSynthesis, the LDL recep- tor, and fatty acid bioSynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the trans- genic livers showed a 28-fold increase in the rate of choles- terol Synthesis and a lesser fourfold increase in fatty acid Synthesis, as measured by intraperitoneal injection of ( 3 H)water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid Synthesis more than Cholesterol Synthesis. In adi- pose tissue of the SREBP-2 transgenics, the mRNAs for Cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of Cholesterol Synthesis, as opposed to fatty acid Synthesis, in liver and adipose tissue of mice. ( J. Clin. Invest. 1998. 101: 2331-2339.) Key words: Cholesterollow density lipoprotein • sterol regulatory element binding proteinsfatty acids • transgenic mice
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elevated levels of srebp 2 and Cholesterol Synthesis in livers of mice homozygous for a targeted disruption of the srebp 1 gene
Journal of Clinical Investigation, 1997Co-Authors: Hitoshi Shimano, Michael S. Brown, Joseph L. Goldstein, Iichiro Shimomura, Robert E Hammer, Joachim Herz, Jay D HortonAbstract:The Synthesis of Cholesterol and its uptake from plasma LDL are regulated by two membrane-bound transcription factors, designated sterol regulatory element binding protein-1 and -2 (SREBP-1 and SREBP-2). Here, we used the technique of homologous recombination to generate mice with disruptions in the gene encoding the two isoforms of SREBP-1, termed SREBP-1a and SREBP-1c. Heterozygous gene-disrupted mice were phenotypically normal, but 50- 85% of the homozygous (-/-) mice died in utero at embryonic day 11. The surviving -/- mice appeared normal at birth and throughout life. Their livers expressed no functional SREBP-1. There was a 1.5-fold upregulation of SREBP-2 at the level of mRNA and a two- to threefold increase in the amount of mature SREBP-2 in liver nuclei. Previous studies showed that SREBP-2 is much more potent than SREBP-1c, the predominant hepatic isoform of SREBP-1, in activating transcription of genes encoding enzymes of Cholesterol Synthesis. Consistent with this observation, the SREBP-1 -/- animals manifested elevated levels of mRNAs for 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase, farnesyl diphosphate synthase, and squalene synthase. Cholesterol Synthesis, as measured by the incorporation of [3H]water, was elevated threefold in livers of the -/- mice, and hepatic Cholesterol content was increased by 50%. Fatty acid Synthesis was decreased in livers of the -/- mice. The amount of white adipose tissue was not significantly decreased, and the levels of mRNAs for lipogenic enzymes, adipocyte lipid binding protein, lipoprotein lipase, and leptin were normal in the -/- mice. We conclude from these studies that SREBP-2 can replace SREBP-1 in regulating Cholesterol Synthesis in livers of mice and that the higher potency of SREBP-2 relative to SREBP-1c leads to excessive hepatic Cholesterol Synthesis in these animals.
Laura J Sharpe - One of the best experts on this subject based on the ideXlab platform.
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the Cholesterol Synthesis enzyme lanosterol 14α demethylase is post translationally regulated by the e3 ubiquitin ligase march6
Biochemical Journal, 2020Co-Authors: Nicola A Scott, Laura J Sharpe, Isabelle M Capellhattam, Samuel J Gullo, Andrew J BrownAbstract:: Cholesterol Synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of Cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in Cholesterol Synthesis, also control levels of LDM and the terminal Cholesterol Synthesis enzyme, 24-dehydroCholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of Cholesterol Synthesis.
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Phosphorylation regulates activity of 7-dehydroCholesterol reductase (DHCR7), a terminal enzyme of Cholesterol Synthesis.
The Journal of steroid biochemistry and molecular biology, 2016Co-Authors: Anika V Prabhu, Laura J Sharpe, Winnie Luu, Andrew J BrownAbstract:Cholesterol is essential for survival, but too much or too little can cause disease. Thus, Cholesterol levels must be kept within close margins. 7-dehydroCholesterol reductase (DHCR7) is a terminal enzyme of Cholesterol Synthesis, and is essential for embryonic development. Largely, DHCR7 research is associated with the developmental disease Smith-Lemli-Opitz syndrome, which is caused by mutations in the DHCR7 gene. However, little is known about what regulates DHCR7 activity. Here we provide evidence that phosphorylation plays a role in controlling DHCR7 activity, which may provide a means to divert flux from Cholesterol Synthesis to vitamin D production. DHCR7 activity was significantly decreased when we used pharmacological inhibitors against two important kinases, AMP-activated protein kinase and protein kinase A. Moreover, mutating a known phosphorylated residue, S14, also decreased DHCR7 activity. Thus, we demonstrate that phosphorylation modulates DHCR7 activity in cells, and contributes to the overall Synthesis of Cholesterol, and probably vitamin D.
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a march6 and idol e3 ubiquitin ligase circuit uncouples Cholesterol Synthesis from lipoprotein uptake in hepatocytes
Molecular and Cellular Biology, 2015Co-Authors: Anke Loregger, Laura J Sharpe, Andrew J Brown, Emma Claire Laura Cook, Jessica K Nelson, Martina Moeton, Susanna Engberg, Madina Karimova, Gilles Lambert, Noam ZelcerAbstract:Cholesterol Synthesis and lipoprotein uptake are tightly coordinated to ensure that the cellular level of Cholesterol is adequately maintained. Hepatic dysregulation of these processes is associated with pathological conditions, most notably cardiovascular disease. Using a genetic approach, we have recently identified the E3 ubiquitin ligase MARCH6 as a regulator of Cholesterol bioSynthesis, owing to its ability to promote degradation of the rate-limiting enzymes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) and squalene epoxidase (SQLE). Here, we present evidence for MARCH6 playing a multifaceted role in the control of Cholesterol homeostasis in hepatocytes. We identify MARCH6 as an endogenous inhibitor of the sterol regulatory element binding protein (SREBP) transcriptional program. Accordingly, loss of MARCH6 increases expression of SREBP-regulated genes involved in Cholesterol bioSynthesis and lipoprotein uptake. Unexpectedly, this is associated with a decrease in cellular lipoprotein uptake, induced by enhanced lysosomal degradation of the low-density lipoprotein receptor (LDLR). Finally, we provide evidence that induction of the E3 ubiquitin ligase IDOL represents the molecular mechanism underlying this MARCH6-induced phenotype. Our study thus highlights a MARCH6-dependent mechanism to direct cellular Cholesterol accretion that relies on uncoupling of Cholesterol Synthesis from lipoprotein uptake.
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the sterol based transcriptional control of human 7 dehydroCholesterol reductase dhcr7 evidence of a cooperative regulatory program in Cholesterol Synthesis
Biochimica et Biophysica Acta, 2014Co-Authors: Anika V Prabhu, Laura J Sharpe, Andrew J BrownAbstract:Abstract The enzyme 7-dehydroCholesterol reductase (DHCR7) catalyzes the final step of Cholesterol Synthesis via the Kandutsch–Russell pathway, and is crucial in maintaining cellular Cholesterol levels. Its absence leads to the devastating fetal developmental disorder Smith–Lemli–Opitz Syndrome (SLOS). How this enzyme is regulated has implications in controlling not only Cholesterol Synthesis, but also the Synthesis of Vitamin D — another product of 7-dehydroCholesterol. In this study, we look specifically at how DHCR7 is regulated by the sterol regulatory element–binding protein-2 (SREBP-2) transcription factor. Sterol regulation has previously been studied in the rat DHCR7 promoter, but we have found that its regulatory elements are not all conserved in humans. Rather, the human promoter contains two binding sites for SREBP-2 (at − 155 and − 55) and a binding site for the nuclear factor-Y (NF-Y) cofactor (at − 136). The − 155 site is a particularly responsive sterol regulatory element (SRE) which is well conserved in mammals, and was possibly overlooked in the rat promoter study. The exact location of the weaker − 55 site (close to the known rat SRE) may have shifted during evolution. Furthermore, we established that the two SREs that bind SREBP-2 work in cooperation to synergistically activate DHCR7. We have previously characterized the SREs in DHCR24, the final enzyme in the alternate Bloch pathway of Cholesterol Synthesis. Here, comparison of the sterol regulation of these terminal enzymes demonstrates the unique cooperative system that helps to control Cholesterol Synthesis.
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controlling Cholesterol Synthesis beyond 3 hydroxy 3 methylglutaryl coa reductase hmgcr
Journal of Biological Chemistry, 2013Co-Authors: Laura J Sharpe, Andrew J BrownAbstract:3-Hydroxy-3-methylglutaryl-CoA reductase (HMGCR) is the target of the statins, important drugs that lower blood Cholesterol levels and treat cardiovascular disease. Consequently, the regulation of HMGCR has been investigated in detail. However, this enzyme acts very early in the Cholesterol Synthesis pathway, with ∼20 subsequent enzymes needed to produce Cholesterol. How they are regulated is largely unexplored territory, but there is growing evidence that enzymes beyond HMGCR serve as flux-controlling points. Here, we introduce some of the known regulatory mechanisms affecting enzymes beyond HMGCR and highlight the need to further investigate their control.
Ingiald Hafstrom - One of the best experts on this subject based on the ideXlab platform.
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circulating proprotein convertase subtilisin kexin type 9 has a diurnal rhythm synchronous with Cholesterol Synthesis and is reduced by fasting in humans
Arteriosclerosis Thrombosis and Vascular Biology, 2010Co-Authors: Lena Persson, Guoqing Cao, Lars Stahle, Beatrice G Sjoberg, Jason S Troutt, Robert J Konrad, Cecilia Galman, Hakan Wallen, Mats Eriksson, Ingiald HafstromAbstract:Objective— To gain insight into the function of proprotein convertase subtilisin kexin type 9 (PCSK9) in humans by establishing whether circulating levels are influenced by diurnal, dietary, and hormonal changes. Methods and Results— We monitored circulating PCSK9 in a set of dynamic human experiments and could show that serum PCSK9 levels display a diurnal rhythm that closely parallels that of Cholesterol Synthesis, measured as serum lathosterol. In contrast to these marked diurnal changes in Cholesterol metabolism, serum low-density lipoprotein (LDL) Cholesterol levels remained stable during the diurnal cycle. Depletion of liver Cholesterol by treatment with the bile acid–binding resin, cholestyramine, abolished the diurnal rhythms of both PCSK9 and lathosterol. Fasting (>18 hours) strongly reduced circulating PCSK9 and lathosterol levels, whereas serum LDL levels remained unchanged. Growth hormone, known to be increased during fasting in humans, reduced circulating PCSK9 in parallel to LDL Cholesterol levels. Conclusion— Throughout the day, and in response to fasting and Cholesterol depletion, circulating PCSK9 displays marked variation, presumably related to oscillations in hepatic Cholesterol that modify its activity in parallel with Cholesterol Synthesis. In addition to this sterol-mediated regulation, additional effects on LDL receptors may be mediated by hormones directly influencing PCSK9.