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Lipid Storage and Export

English title Lipid Storage and Export
Applicant Schneiter Roger
Number 153416
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 01.10.2014 - 30.11.2017
Approved amount 582'213.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (8)

Saccharomyces cerevisiae; endoplasmic reticulum; sterol-binding proteins; CAP/SCP proteins; lipid droplets; cholesterol; topology of lipid synthesis; lipid homeostasis

Lay Summary (German)

Lead
Fett dient als wichtiger Energiespeicher und wird innerhalb von Zellen in sogenannten Lipidtröpfchen aufbewahrt. Wie genau und wo diese Fetttröpfchen entstehen und wie deren Biogenese reguliert wird, ist jedoch nur ungenügend verstanden. In diesem Forschungprojekt wird die molekulare Grundlage der Biogenese dieser Lipidtröpchen im Modellorganimus Hefe mittels zellbiologischer Methoden untersucht.
Lay summary

Membrane bilden molekulare Barrieren aus, welche der Zelle ermöglichen, unterschiedliche Funktionen voneinander zu trennen und sich auch gegenüber der Umgebung abzugrenzen. Diese Membrane sind komplexe makromolekulare Strukturen und bestehen aus Proteinen und Lipiden, mit einem gewichtsmässig äquivalenten Anteil. Die Kontrolle und Regulation der Zusammensetzung zellulärer Membrane erfolgt dynamisch und ist notwendig, um die Funktionalität der Membran und damit die Lebensfähigkeit der Zelle zu ermöglichen.

In diesem Forschungsvorhaben werden zwei fundamentale Aspekte dieser homöostatischen Kontrolle untersucht. Einerseits wird die Biogenese von sogenannten Lipidtröpfchen untersucht. Diese kugelförmigen intrazellulären Strukturen dienen der temporären Speicherung von Fett, welches bei Bedarf wieder abgebaut werden kann und damit der Zelle Energie liefert. Fett ist ein wesentlich besserer Energiespeicher als Zucker. Die zentrale Frage, welche behandelt wird, ist wie und wo sich diese Strukturen genau ausbilden und wie der Prozess reguliert wird. In einem zweiten Teil wird die Struktur und Funktion einer Klasse von Proteinen untersucht, den sogenannten CAP/SCP Proteinen. CAP/SCP Proteine kommen im Menschen (33 Gene), in Pflanzen, Pilzen aber auch in einzelnen Bakterien vor, ihre Funktion wird allerdings nur ungenügend verstanden. Wir konnten kürzlich zeigen, dass die entsprechenden Proteine der Hefe Cholesterol binden und dass das proteingebundene Lipid dann aus den Zellen ausgeschieden/sekretiert wird. Wir vermuten daher, dass diese Proteine generell dazu dienen, Lipide zu binden und diese dann aus der Zelle auszuscheiden. Diese Hypothese soll anhand von Struktur/Funktionsstudien von CAP/SCP Proteinen aus verschiedenen Organismen untersucht werden.

Direct link to Lay Summary Last update: 23.06.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
A Ligand-Binding Assay to Measure the Affinity and Specificity of Sterol-Binding Proteins In Vitro.
Darwiche R, Schneiter R (2017), A Ligand-Binding Assay to Measure the Affinity and Specificity of Sterol-Binding Proteins In Vitro., in Barredo Jose-Luis and Herraiz Ignacio (ed.), Springer, New York, 361-368.
Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.
Debelyy MO, Waridel P, Quadroni M, Schneiter R, Conzelmann A (2017), Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins., in PLoS One, 12(10), 0186840-0186840.
Crystal Structure of Borrelia turicatae protein, BTA121, a differentially regulated  gene in the tick-mammalian transmission cycle of relapsing fever spirochetes.
Luo Z, Kelleher AJ, Darwiche R, Hudspeth EM, Shittu OK, Krishnavajhala A, Schneiter R, Lopez JE, Asojo OA (2017), Crystal Structure of Borrelia turicatae protein, BTA121, a differentially regulated  gene in the tick-mammalian transmission cycle of relapsing fever spirochetes., in Sci Rep, 7(1), 15310-15310.
Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches' Broom Disease of Cacao.
Baroni RM, Luo Z, Darwiche R, Hudspeth EM, Schneiter R, Pereira GAG, Mondego JMC, Asojo OA (2017), Crystal Structure of MpPR-1i, a SCP/TAPS protein from Moniliophthora perniciosa, the fungus that causes Witches' Broom Disease of Cacao., in Sci Rep, 7(1), 7818-7818.
Plant pathogenesis-related proteins of the cacao fungal pathogen Moniliophthora perniciosa differ in their lipid-binding specificities.
Darwiche R, El Atab O, Baroni RM, Teixeira PJPL, Mondego JMC, Pereira GAG, Schneiter R (2017), Plant pathogenesis-related proteins of the cacao fungal pathogen Moniliophthora perniciosa differ in their lipid-binding specificities., in Plant J.
Sphingolipid accumulation causes mitochondrial dysregulation and cell death.
Knupp J, Martinez-Montañés F, Van Den Bergh F, Cottier S, Schneiter R, Beard D, Chang A (2017), Sphingolipid accumulation causes mitochondrial dysregulation and cell death., in Cell Death Differ, 24(12), 2044-2053.
The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein.
Darwiche R, Mène-Saffrané L, Gfeller D, Asojo OA, Schneiter R (2017), The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein., in J Biol Chem, 292(20), 8304-8314.
The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein.
Gamir J, Darwiche R, Van't Hof P, Choudhary V, Stumpe M, Schneiter R, Mauch F (2017), The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein., in Plant J, 89(3), 502-509.
Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether.
Martínez-Montañés F, Lone MA, Hsu FF, Schneiter R (2016), Accumulation of long-chain bases in yeast promotes their conversion to a long-chain base vinyl ether., in J Lipid Res, 57(11), 2040-2050.
Chemogenetic E-MAP in Saccharomyces cerevisiae for Identification of Membrane Transporters Operating Lipid Flip Flop.
Vazquez HM, Vionnet C, Roubaty C, Mallela SK, Schneiter R, Conzelmann A (2016), Chemogenetic E-MAP in Saccharomyces cerevisiae for Identification of Membrane Transporters Operating Lipid Flip Flop., in PLoS Genet, 12(7), e1006160-e1006160.
Cholesterol-binding by the yeast CAP faimily member, Pry1, requires the presence of an aliphatic side chain on cholesterol.
Darwiche R, Schneiter R (2016), Cholesterol-binding by the yeast CAP faimily member, Pry1, requires the presence of an aliphatic side chain on cholesterol., in J Steroids Hormon Sci, 7(172), 2-2.
Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry.
Martínez-Montañés F, Schneiter R (2016), Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry., in J Lipid Res, 57(5), 906-15.
Mature lipid droplets are accessible to ER luminal proteins.
Mishra S, Khaddaj R, Cottier S, Stradalova V, Jacob C, Schneiter R (2016), Mature lipid droplets are accessible to ER luminal proteins., in J Cell Sci, 129(20), 3803-3815.
Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein.
Darwiche R, Kelleher A, Hudspeth EM, Schneiter R, Asojo OA (2016), Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein., in Sci Rep, 6, 28838-28838.
Tools for the analysis of metabolic flux through the sphingolipid pathway.
Martínez-Montañés F, Schneiter R (2016), Tools for the analysis of metabolic flux through the sphingolipid pathway., in Biochimie, 130, 76-80.
Valproate Induces the Unfolded Protein Response by Increasing Ceramide Levels.
Jadhav S, Russo S, Cottier S, Schneiter R, Cowart A, Greenberg ML (2016), Valproate Induces the Unfolded Protein Response by Increasing Ceramide Levels., in J Biol Chem, 291(42), 22253-22261.
Expression of perilipin 5 promotes lipid droplet formation in yeast.
Mishra S, Schneiter R (2015), Expression of perilipin 5 promotes lipid droplet formation in yeast., in Commun Integr Biol, 8(6), e1071728-e1071728.
Match-making for posaconazole through systems thinking.
Fugi MA, Kaiser M, Tanner M, Schneiter R, Maser P, Guan XL (2015), Match-making for posaconazole through systems thinking., in Trends Parasitol, 31(2), 46-51.
Yeast integral membrane proteins Apq12, Brl1, and Brr6 form a complex important for regulation of membrane homeostasis and nuclear pore complex biogenesis.
Lone MA, Atkinson AE, Hodge CA, Cottier S, Martínez-Montañés F, Maithel S, Mène-Saffrané L, Cole CN, Schneiter R (2015), Yeast integral membrane proteins Apq12, Brl1, and Brr6 form a complex important for regulation of membrane homeostasis and nuclear pore complex biogenesis., in Eukaryot Cell, 14(12), 1217-1227.
Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs.
Kelleher A, Darwiche R, Rezende WC, Farias LP, Leite LC, Schneiter R, Asojo OA (2014), Schistosoma mansoni venom allergen-like protein 4 (SmVAL4) is a novel lipid-binding SCP/TAPS protein that lacks the prototypical CAP motifs., in Acta Crystallogr D Biol Crystallogr, 70(8), 2186-96.
The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate.
Choudhary V, Darwiche R, Gfeller D, Zoete V, Michielin O, Schneiter R (2014), The caveolin-binding motif of the pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is required for in vivo export of cholesteryl acetate., in J Lipid Res, 55(5), 883-94.
TORC1 Regulates Pah1 Phosphatidate Phosphatase Activity via the Nem1/Spo7 Protein Phosphatase Complex.
Dubots E, Cottier S, Peli-Gulli MP, Jaquenoud M, Bontron S, Schneiter R, De Virgilio C (2014), TORC1 Regulates Pah1 Phosphatidate Phosphatase Activity via the Nem1/Spo7 Protein Phosphatase Complex., in PLoS One, 9(8), 104194-104194.
The function of yeast CAP family proteins in lipid export, mating and pathogen defense.
Darwiche R, El Atab O, Cottier S, Schneiter R, The function of yeast CAP family proteins in lipid export, mating and pathogen defense., in FEBS Lett.

Collaboration

Group / person Country
Types of collaboration
Oluwatoyin A. Asojo/Baylor College United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
David Gfeller/Swiss Institute of Bioinformatics Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
A. Puoti/University of Fribourg Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Felix Mauch/University Fribourg Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Molecular and Cellular Biology of Helminth Parasites XI - Hydra, Greece. September 2017. Poster The role of CAP proteins in sterol transport 03.09.2017 Hydra, Greece Schneiter Roger; Darwiche Rabih;
28th International Conference on Yeast Genetics and Molecular Biology (ICYGMB) Talk given at a conference Lipid Droplets and the ER membrane – A Mysterious Connection 27.08.2017 Prag, Czech Republic Schneiter Roger;
FASEB Lipid Droplet Meeting Snowmass Colorado, USA. June 2016 Poster Mature lipid droplets are accessible to endoplasmic reticulum luminal proteins​ 24.07.2017 Snowmass Colorado, United States of America Khaddaj Rasha;
EMBO| EMBL Symposium: Molecular and cell Biology of of membranes, Heidelberg, Germany Poster EMBO| EMBL Symposium: Molecular and cell Biology of of membranes, Heidelberg, Germany 21.05.2017 Heidelberg, Germany Schneiter Roger;
13th Yeast Lipid Conference- Paris, France. May 2017. Talk given at a conference Lipid droplet dynamic 17.05.2017 Paris, France Darwiche Rabih; Khaddaj Rasha; Schneiter Roger; El Atab Ola;
LS2 Annual Meeting-Lausanne, Switzerland. February 2016. Poster Lipid binding by the CAP family member, Pry1 15.02.2016 Lausanne, Switzerland Khaddaj Rasha; Darwiche Rabih;
12th Yeast Lipid Conference- Ghent, Belgium. May 2015. Talk given at a conference Lipid droplets and the ER membrane 20.05.2015 Ghent, Belgium Martinez Fernando; Schneiter Roger; Darwiche Rabih; Khaddaj Rasha;
LS2 Annual Meeting- Zurich, Switzerland. January 2015 Poster Keeping the ER membrane clean: Lipid acetyla- tion and export 29.01.2015 Zürich, Switzerland Darwiche Rabih; Khaddaj Rasha;
Jacques Monod Conference on Molecular basis for membrane remodelling and organization, Roscoff, France, November 2014 Poster Function and functioning of Pry proteins in yeast cells 15.11.2014 Roscoff, France Schneiter Roger;
EMBO Meeting: The Endoplasmic Reticulum (ER) as a hub for organelle communication, Girona, Spain. October 2014 Talk given at a conference Keeping the ER membrane clean: Lipid acetylation and export 26.10.2014 Girona, Spain Schneiter Roger;


Awards

Title Year
SNF Postdoctoral Fellowship 2017

Associated projects

Number Title Start Funding scheme
134742 Lipid Acetylation, Storage, Export and Degradation 01.07.2011 Project funding (Div. I-III)
173003 Lipid Storage, Binding, and Export 01.12.2017 Project funding (Div. I-III)

Abstract

Lipid homeostasis is essential to ensure proper membrane function and cell viability. In this proposal, we address two basic aspects of this homeostatic regulation. In the first part, the topology of lipid storage and the biogenesis of lipid droplets are being addressed. In the second part, we propose to perform a detailed structural and functional analysis of a novel class of secreted sterol-binding proteins, the Pry (pathogen related in yeast) family. Both projects build on our established expertise and they constitute a rational continuation of our previously published work in this field. Lipid droplets constitute a globular intracellular compartment that is dedicated to the storage of fat/neutral lipids. The droplet is composed of a core of neutral lipids, particularly triacylglycerols and steryl esters and covered by a monolayer membrane onto which a small number of proteins associate. Many of these proteins function in lipid metabolism, for example as acyltransferases or lipases. We have previously shown that some of these lipid droplet associated proteins are integral membrane proteins and that they can move between the membrane of the endoplasmic reticulum (ER) and the surface of lipid droplets. These as well as other observations indicate that lipid droplets are closely associated with the ER membrane. The nature of this association, however, has not yet been resolved and is currently debated. Here, we will test if lipid droplets are accessible from within the ER lumen by targeting soluble lipid droplet-associated proteins into the luminal space of the ER. Preliminary results indicate that this indeed is the case, thus challenging some of the current models of lipid droplet biogenesis. To complement these studies, we will artificially generate ER-luminal lipid droplets by targeting heterologous lipid droplet-scaffolding proteins inside the ER luminal space. We have previously shown that cytosolic expression of such proteins in yeast results in the induction of lipid droplets and preliminary results indicate that lipid droplet induction is also observed if these proteins are targeted into the ER lumen. Thus having an experimental system to form cytosolic as well as ER luminal lipid droplets will allows us to compare their properties with naturally formed lipid droplets and thus allow us to define their origin and the nature of their association with the ER. Pry proteins belong to a large protein superfamily known as CAP/SCP (sperm coating proteins). These proteins are implicated in a multitude of physiologically important processes in all kingdoms of life, including but not limited to immune defense in animals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity and prostate and brain cancer. CAP proteins are mostly secreted glycoproteins and are stable in the extracellular environments over a wide range of conditions. Their mode of action, however, has remained elusive. We could recently show that the yeast Pry proteins bind sterols and related small hydrophobic compounds in vivo and in vitro. This sterol-binding activity is confined to the CAP domain of Pry1, and conserved in a human CAP family member, CRISP2, suggesting that CAP family members exert their various functions through binding sterols or related small hydrophobic compounds. Aim of this part of the project is to characterize the ligand binding activity of Pry1 at the structural level and to define its ligand-specificity. The results of this work will provide the foundation to understand the mode of action of this important class of proteins; not only in fungi but also in plants and vertebrates.
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