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Speicherung, Mobilisierung und Umsatz von Neutrallipiden

English title Storage, Mobilization, and Turnover of Neutral Lipids
Applicant Schneiter Roger
Number 120650
Funding scheme Project funding (Div. I-III)
Research institution Division de Biochimie Département de Biologie Université de Fribourg
Institution of higher education University of Fribourg - FR
Main discipline Biochemistry
Start/End 01.07.2008 - 30.06.2011
Approved amount 510'636.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (12)

neutral lipids; triacylglycerol; steryl ester; lipid droplet; lipid particle; oil body; lipase; organelle biogenesis; protein sorting; detoxification; obesity; yeast (S. cerevisiae)

Lay Summary (English)

Lead
Lay summary
All organisms store excess of metabolic energy as neutral lipids or “fat”. Neutral lipids are composed of triacylglycerol (TAG) and, in eukaryotes, also contain steryl esters (STE). The fatty acids present in TAG and STE provide at least ten-times as much energy as that of an equal mass of hydrated carbohydrates or proteins. This energy is typically released by degradation through beta-oxidation of the long chain fatty acids present in TAG and STE, either in mitochondria (animal and plant cells) or in peroxisomes (yeasts). Because these neutral lipids are extremely hydrophobic, they are stored in intracellular lipid droplets (LDs), organelles that are dedicated to neutral lipid storage. Multicellular organisms have differentiated cell types such as adipocytes that are specialized for storage of neutral lipids in the form of giant cytosolic LDs, but have also developed pathways to secret neutral lipid in form of lipoproteins and milk lipid globules. Intracellular LDs are surrounded by an unusual lipid monolayer membrane and harbor only a limited number of different proteins, many of which are directly implicated in lipid metabolism. How precisely these LDs are formed and how their biogenesis is regulated, however, is poorly understood and will be addressed in the present research program. The second main question that will be addressed is how access, turnover, and degradation of this neutral lipid pool are governed. We and others have identified lipases that are required for the degradation of TAG and STE, and many of these lipases localize to LDs. How these lipases are activated and how they gain access to their insoluble substrates, however, remains to be defined. Understanding the regulation and turnover of neutral lipids is important, as it will help to define some of the problems associated with the metabolic syndrome, obesity, type 2 diabetes, and its associated cardiovascular problems, all of which are associated with an excess accumulation of neutral lipids and elevated levels of circulating free fatty acids, which ultimately results in an inflammatory response and insulin resistance.Our studies will be performed mainly in the unicellular eukaryote yeast, as a genetically tractable model, because it allows unprecedented experimental control over LD biogenesis and neutral lipid degradation. Employing a strain with which we can induce biogenesis as well as turnover of LDs, we will characterize the biogenetic- and degradative-intermediates morphologically by confocal as well as electron microscopy; determine the topology of the two TAG synthesizing enzymes, Lro1p and Dga1p, to define whether TAG synthesis is defined to the lumenal domain of the ER; and employ genetic screens to identify mutations that block LD biogenesis or turnover. We have recently identified protein kinases that are important for the mobilization of TAG, STE, and for the phosphorylation of a key lipase in STE turnover, Yeh2p. The role of these kinases in neutral lipid turnover will be characterized in more detail and their target will be defined. Neutral lipids in the ER have recently been suggested to act in retrotranslocation of misfolded proteins, a hypothesis that will be tested using yeast mutants that lack neutral lipids. Accumulation of neutral lipids in the ER may induce ER stress and insulin resistance in mammalian cells. A possible causative relation between TAG accumulation and ER stress will be investigated using yeast strains that accumulate TAG.We have recently characterized a novel neutral lipid modification, the reversible acetylation and deacetylation of sterols. We propose that this acetylation cycle acts to proofread the structure of sterols and possibly other hydrophobic compounds to ensure that only properly synthesized sterols are incorporated into cellular membranes, as acetylation acts as a signal for the secretion of the acetylated compound. One part of the research program will thus further characterize this acetylation cycle with the aim of gaining a more refined understanding of the precise physiological function of this cycle. Therefore, we will characterize the function of two hitherto uncharacterized membrane proteins that are required for sterol acetylation; purify and identify proteins that bind acetylated sterols, identify additional substrates of the acetylation cycle by mass spectrometry; and examine the function of a conserved sterol deacetylase in mammalian cells.We expect that the results of this basic research program will significantly advance our understanding of the fundamental cellular processes that govern neutral lipid accumulation, degradation, and turnover, and hence the biogenesis and turnover of LDs. This is important to understand the pathophysiology of an overaccumulation of neutral lipids due to an excess of fatty acids, as is characteristic for obesity. A more detailed characterization of the sterol acetylation cycle, on the other hand, may prove more generally important to understand sterol transport and homeostasis in eukaryotic cells, which is aberrant in dyslipidemia. While the ultimate aim of course is to understand these processes in mammals, we think that at the current status, yeast is the organism of choice to perform such basic studies, as it offers all the genetic and biochemical tools that are essential to experimentally address these questions. Since basic cellular processes are mostly conserved between yeast and man, we think that the results we gain in yeast will also be relevant to understand the analogous processes in mammalian cells.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum
Hodge Christine A., Choudhary Vineet, Wolyniak Michael J., Scarcelli John J., Schneiter Roger, Cole Charles N. (2010), Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum, in JOURNAL OF CELL SCIENCE, 123(1), 141-151.
Integrating complex functions Coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins
Schneiter Roger, Cole Charles N. (2010), Integrating complex functions Coordination of nuclear pore complex assembly and membrane expansion of the nuclear envelope requires a family of integral membrane proteins, in NUCLEUS-AUSTIN, 1(5), 387-392.
Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control
Han Sumin, Lone Museer A., Schneiter Roger, Chang Amy (2010), Orm1 and Orm2 are conserved endoplasmic reticulum membrane proteins regulating lipid homeostasis and protein quality control, in PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 107(13), 5851-5856.
Ypk1, the yeast orthologue of the human serum- and glucocorticoid-induced kinase, is required for efficient uptake of fatty acids
Jacquier Nicolas, Schneiter Roger (2010), Ypk1, the yeast orthologue of the human serum- and glucocorticoid-induced kinase, is required for efficient uptake of fatty acids, in JOURNAL OF CELL SCIENCE, 123(13), 2218-2227.
Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells
Francois IE, Bink A, Vandercappellen J, Ayscough KR, Toulmay A, Schneiter R, van Gyseghem E, Van den Mooter G, Borgers M, Vandenbosch D, Coenye T, Cammue BP, Thevissen K (2009), Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells, in J Biol Chem, 284, 32680-32685.
Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation.
Schafer B., Quispe J., Choudhary V., Chipuk J. E., Ajero T. G., Du H., Schneiter R., Kuwana T. (2009), Mitochondrial outer membrane proteins assist Bid in Bax-mediated lipidic pore formation., in Mol. Biol. Cell., 20, 2276-2285.
Monitoring sterol uptake, acetylation, and export in yeast.
Choudhary V, Schneiter R (2009), Monitoring sterol uptake, acetylation, and export in yeast., in Methods Mol Biol, 580, 221-232.
Sterol acetylation and export from yeast and mammalian cells: Acetylation of sterols and steroids controls their export in yeast and mammalian cells
Schneiter R (2009), Sterol acetylation and export from yeast and mammalian cells: Acetylation of sterols and steroids controls their export in yeast and mammalian cells, VDM Verlag, Saarbrücken, Germany.
The Cdc42 Effectors Ste20, Cla4, and Skm1 Down-Regulate the Expression of Genes Involved in Sterol Uptake by a Mitogen-activated Protein Kinase-independent Pathway
Lin M, Unden H, Jacquier N, Schneiter R, Just U, Hofken T (2009), The Cdc42 Effectors Ste20, Cla4, and Skm1 Down-Regulate the Expression of Genes Involved in Sterol Uptake by a Mitogen-activated Protein Kinase-independent Pathway, in Mol Biol Cell, 20, 4826-4837.
Lipid signalling in disease
Wymann MP, Schneiter R (2008), Lipid signalling in disease, in Nat Rev Mol Cell Biol, 9, 162-167.

Collaboration

Group / person Country
Types of collaboration
Peter Hunziker/Functional Genomic Center Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Paul Jenö/Biozentrum Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
André Schneider/Univ. Fribourg/Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
Matthias Wymann/DKBW Universität Basel Switzerland (Europe)
- Publication
Amy Chang United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Associated projects

Number Title Start Funding scheme
110450 Lipid homeostasis in yeast 01.07.2006 SNSF Professorships
134742 Lipid Acetylation, Storage, Export and Degradation 01.07.2011 Project funding (Div. I-III)
125232 Membrane topology of lipid synthesis, transport, and turnover and their role in the physiology of starvation and aging 01.08.2009 Sinergia
134742 Lipid Acetylation, Storage, Export and Degradation 01.07.2011 Project funding (Div. I-III)

Abstract

All organisms store excess of metabolic energy as neutral lipids or “fat”. Neutral lipids are composed of triacylglycerol (TAG) and, in eukaryotes, also contain steryl esters (STE). The fatty acids present in TAG and STE provide at least ten-times as much energy as that of an equal mass of hydrated carbohydrates or proteins. This energy is typically released by degradation through beta-oxidation of the long chain fatty acids present in TAG and STE, either in mitochondria (animal and plant cells) or in peroxisomes (yeasts). Because these neutral lipids are extremely hydrophobic, they are stored in intracellular lipid droplets (LDs), organelles that are dedicated to neutral lipid storage. Multicellular organisms have differentiated cell types such as adipocytes that are specialized for storage of neutral lipids in the form of giant cytosolic LDs, but have also developed pathways to secret neutral lipid in form of lipoproteins and milk lipid globules. Intracellular LDs are surrounded by an unusual lipid monolayer membrane and harbor only a limited number of different proteins, many of which are directly implicated in lipid metabolism. How precisely these LDs are formed and how their biogenesis is regulated, however, is poorly understood and will be addressed in the present research program. The second main question that will be addressed is how access, turnover, and degradation of this neutral lipid pool are governed. We and others have identified lipases that are required for the degradation of TAG and STE, and many of these lipases localize to LDs. How these lipases are activated and how they gain access to their insoluble substrates, however, remains to be defined. Understanding the regulation and turnover of neutral lipids is important, as it will help to define some of the problems associated with the metabolic syndrome, obesity, type 2 diabetes, and its associated cardiovascular problems, all of which are associated with an excess accumulation of neutral lipids and elevated levels of circulating free fatty acids, which ultimately results in an inflammatory response and insulin resistance.Our studies will be performed mainly in the unicellular eukaryote yeast, as a genetically tractable model, because it allows unprecedented experimental control over LD biogenesis and neutral lipid degradation. Employing a strain with which we can induce biogenesis as well as turnover of LDs, we will characterize the biogenetic- and degradative-intermediates morphologically by confocal as well as electron microscopy; determine the topology of the two TAG synthesizing enzymes, Lro1p and Dga1p, to define whether TAG synthesis is defined to the lumenal domain of the ER; and employ genetic screens to identify mutations that block LD biogenesis or turnover. We have recently identified protein kinases that are important for the mobilization of TAG, STE, and for the phosphorylation of a key lipase in STE turnover, Yeh2p. The role of these kinases in neutral lipid turnover will be characterized in more detail and their target will be defined. Neutral lipids in the ER have recently been suggested to act in retrotranslocation of misfolded proteins, a hypothesis that will be tested using yeast mutants that lack neutral lipids. Accumulation of neutral lipids in the ER may induce ER stress and insulin resistance in mammalian cells. A possible causative relation between TAG accumulation and ER stress will be investigated using yeast strains that accumulate TAG.We have recently characterized a novel neutral lipid modification, the reversible acetylation and deacetylation of sterols. We propose that this acetylation cycle acts to proofread the structure of sterols and possibly other hydrophobic compounds to ensure that only properly synthesized sterols are incorporated into cellular membranes, as acetylation acts as a signal for the secretion of the acetylated compound. One part of the research program will thus further characterize this acetylation cycle with the aim of gaining a more refined understanding of the precise physiological function of this cycle. Therefore, we will characterize the function of two hitherto uncharacterized membrane proteins that are required for sterol acetylation; purify and identify proteins that bind acetylated sterols, identify additional substrates of the acetylation cycle by mass spectrometry; and examine the function of a conserved sterol deacetylase in mammalian cells.We expect that the results of this basic research program will significantly advance our understanding of the fundamental cellular processes that govern neutral lipid accumulation, degradation, and turnover, and hence the biogenesis and turnover of LDs. This is important to understand the pathophysiology of an overaccumulation of neutral lipids due to an excess of fatty acids, as is characteristic for obesity. A more detailed characterization of the sterol acetylation cycle, on the other hand, may prove more generally important to understand sterol transport and homeostasis in eukaryotic cells, which is aberrant in dyslipidemia. While the ultimate aim of course is to understand these processes in mammals, we think that at the current status, yeast is the organism of choice to perform such basic studies, as it offers all the genetic and biochemical tools that are essential to experimentally address these questions. Since basic cellular processes are mostly conserved between yeast and man, we think that the results we gain in yeast will also be relevant to understand the analogous processes in mammalian cells.
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