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Dynamics and structure of lipid bodies
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Keywords :Lipid bodies, oleosome, dynamic, function, proteomic, crystallography, biophysics, low soluble proteins, protein interaction, lipid design, Arabidopsis thaliana, Brassica napus, Saccharomyces cerevisiae, Yarrowia lipolytica, Streptomyces.

Doctoral school affiliation : ED 435 ABIES

 


Contacts :

Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech
INRA Centre de Versailles-Grignon
Route de St-Cyr (RD10)
78026 Versailles Cedex France

tél : +33 (0)1 30 83 30 00 - fax : +33 (0)1 30 83 33 19

Group leader
Thierry Chardot

Senior scientist

Sabine d'Andréa
Associate Professor
AgroParisTech

Isabelle Bouchez-Mahiout
Engineer, ACP

Carine Deruyffelaere
Technician

Christelle Louis-Mondésir
Technician assitant AgroParisTech

 

Pierre Briozzo
Professor AgroParisTech

 

Yann Gohon
Associate Professor AgroParisTech

Franck Jagic
Technician

 

Michel Canonge
Technician

 

 


Summary :

Germination and seedling establishment require high energy input and the supply of carbon backbones for various biosynthetic pathways such as that of lipids. In effect, the plant organ exhibiting the highest levels of lipid accumulation is seeds. Storage lipids are accumulated in lipid bodies (LBs), also called lipid droplets or oil bodies. LBs structure is conserved among living organisms, with a core composed of neutral lipids surrounded by a monolayer of phospholipids, in which a highly variable number of proteins is embedded. These proteins in microbe or animal LBs are mostly comprised of enzymes, or proteins involved in subcellular transport. Seed LBs contain a limited set of proteins, with few enzymes. In particular, little is known about the dynamics of LBs during seed life cycle. To date, no structure has been determined to high resolution for a protein inserted in a phospholipid monolayer. To identify and characterize proteins contributing to the dynamic and the structure of LBs, we apply genetic, biochemical, molecular, and structural approaches. As models we use oleaginous plants (Arabidopsis thaliana, Brassica napus), and also yeasts (Saccharomyces cerevisae, Yarrowia lipolytica).

Our main goals are:
-to fill in the gaps between the knowledge of LB structure and function in plants and other organisms.
-to determine high resolution structures of selected plant oil body proteins.

In accordance with the principles of “Green Chemistry”, our research aims to modulate amounts and tailor the nature of lipids produced, as well as to facilitate storage lipid extraction.
Our expertise in the field of protein structure determination using crystallographic techniques is available to the scientific community, and we are known for our experience on bacterial kinases and plant oxidases.


Main Results :

Our efforts are focused on two investigative axes: « Structural and functional », and « Proteomic and dynamic ». Plants and microorganisms able to store lipids are used as models for our studies.

Structural and functional investigations
The three-dimensional structure determination of lipid body integral proteins is a challenge. Structural data on these very hydrophobic proteins are still scarce and contradictory. We are using original tools to solubilize and handle these proteins and to determine their structure, in isolation or inserted into lipid bodies. (Gohon 2008).
We have set up a simple and rapid protocol for the specific extraction of oleosins from A. thaliana seeds leading to the extensive identification of lipid body proteins (Andréa 2007b).
Using original amphiphilic polymers (Apols), we are able to maintain soluble S5 oleosin (with a low molecular weight and very hydrophobic) and caleosin (a calcium binding protein that is less hydrophobic). Original protein folds have been observed using synchrotron radiation circular dichroism (Gohon et al. submitted).
We are using similar approaches to study the structure of allergenic wheat proteins (bacterially expressed glutenins and gliadins).

We have expertise in the determination of protein post-translational modifications. We proved that A. thaliana caleosin was phosphorylated (Purkrtova 2007), and that its interfacial properties were modified upon calcium binding (Purkrtova 2008). Using structural proteomic approaches, the topology of proteins at the surface of lipid bodies has also been determined. (Vermachova).
We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote over accumulation of storage lipids in yeast oil bodies (Froissard 2009).


Proteomic, dynamic

Using proteomic approaches, we have identified more than 30 different proteins in Y. lipolytica lipid bodies, and proved that their composition was dictated by the carbon sources used for yeast growth (Athenstaed 2006). Yeast lipid bodies contain enzymes from triacylglycerol accumulation and mobilisation pathways, as well as proteins related to intracellular traffic, revealing the dynamics of the organelle. Glycerol-3 phosphate dehydrogenase is a protein found at the surface of lipid bodies. Its inactivation, in strains unable to perform ?-oxidation, redirected carbon fluxes and enabled an accumulation of up to 51 % neutral lipids in yeast (Beopoulos 2008, 2009).

Compared to microbial or animal lipid bodies, (Athenstaedt 2006), LBs from A. thaliana or B. napus mature seed seem to be in a quiescent state (Jolivet 2004, 2009, d’Andréa 2007b, Popluechai submitted). Rapeseed LB proteins follow a sequential expression pattern, so it is likely that LBs undergo an ordered maturation scheme (Jolivet, unpublished).

 

Significant collaborations, contracts

Main collaborations: UMR INRA-INSA LISBP Toulouse, ESPCI ParisTech, ENSCP Paris, Institut Pasteur, Synchrotron Soleil, UMR AgroParisTech-INRA Micalis Grignon, UMR AgroCampus INRA APBV Rennes, UR INRA BIA Nantes, University Paris XI, Graz, Prague, Olomouc, Pittsburg; proteomic facility PAPPSO Le Moulon, CETIOM Pessac.

ANR Programs
-Genoplante GNP0036 Genobodies (2006-2008, 6 partners) “Structural and functional study of oil and protein storage bodies in A. thaliana and in B. napus: towards environmentally friendly oil and protein extraction process ?” . Coordination: T. chardot
-CP2D 08-CP2D-19 SOPOL (2009-2011, 5 partners) «Solubilization of seed oil bodies integral proteins by amphipols: structural studies for valorization » Coordination: T. Chardot
Participations
-GPLA-07-006C Genergy, (2008-2011). “Improvement of the oil yield of the rapeseed crop in the context of bio fuel production”. Coordination : N Nesi, INRA APBV Rennes.
-ANR 07-BIOE-008 Lipicaero (2008-2010) «Production microbienne de LIPIdes spécifiques à usage bioCarburant pour l’AEROnautique Approche intégrée de Physiologie au Procédé » Coordination C. Jouve, INSA LISBP, Toulouse
-ANR-08-ALIA-014-02 : Programme ALimentation et Industrie Alimentaires (ALIA), projet Predexpitope : « Prédiction in silico d’épitopes d’allergènes alimentaires et validation expérimentale sur les allergènes du blé. » Coordination S. Denery, INRA BIA Nantes.
-Prevalorisation programm « ArcoPress » (INRA Transfert). Coordination M. Miquel, IJPB Versailles
-Program CAER « Carburant pour l’Aéronautique », (2010-2013) Coordination C. Jouve, INSA LISBP, Toulouse.


Selected Publications :

 

Baud A, Ayme L, Gonnet F, Salard I, Gohon Y, Jolivet P, Brodolin K, Da Silva P, Giuliani A, Sclavi B, Chardot T, Mercere P, Roblin P, Daniel R (2017) SOLEIL shining on the solution-state structure of biomacromolecules by synchrotron X-ray footprinting at the Metrology beamline. Journal of Synchrotron Radiation 24: 576-585

Groeme R., Airouche S., Kope?ný D., Jaekel J., Savko M., Berjont N., Bussieres L., Le Mignon M., Jagic F., Zieglmayer P., Baron-Bodo V., Bordas-Le Floch V., Mascarell L., Briozzo P., Moingeon P. (2016) Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen. J. Biol. Chem. 291 : 13076-13087 

Haili N, Louap J, Canonge M, Jagic F, Louis-Mondesir C, Chardot T, Briozzo P (2016) Expression of Soluble Forms of Yeast Diacylglycerol Acyltransferase 2 That Integrate a Broad Range of Saturated Fatty Acids in Triacylglycerols. PLoS One 11: e0165431

Kopecný D., Koncitíková R., Popelká H., Briozzo P., Vigouroux A., Kopecná M, Zalabák D., Šebela M., Skopalová J., Frébort I., Moréra S. (2016) Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. FEBS J. 283: 361-377

Hluska T., Dobrev P.I., Tarkowská D., Frébortová J., Zalabák D., Kopecný D., Plíhal O., Kokáš F., Briozzo P., Zatloukal M., Motyka V., Galuszka P. 
(2016) Cytokinin metabolism in maize: Novel evidence of cytokinin abundance, interconversions and formation of a new trans-zeatin metabolic product with a weak anticytokinin activity. Plant Sci. 247: 127–137

Groeme R., Airouche S., Kopecný D., Jaekel J., Savko M., Berjont N., Bussieres L., Le Mignon M., Jagic F., Zieglmayer P., Baron-Bodo V., Bordas-Le Floch V., Mascarell L., Briozzo P., Moingeon P. 
(2016) Structural and Functional Characterization of the Major Allergen Amb a 11 from Short Ragweed Pollen. J. Biol. Chem

D'Andréa S 
(2015) Lipid droplet mobilization: The different ways to loosen the purse strings. Biochimie. DOI: 10.1016/j.biochi.2015.07.010

Aymé L, Jolivet P, Nicaud JM, Chardot T 
(2015) Molecular Characterization of the Elaeis guineensis Medium-Chain Fatty Acid Diacylglycerol Acyltransferase DGAT1-1 by Heterologous Expression in Yarrowia lipolytica. PLoS One 10: e0143113

Boulard C, Bardet M, Chardot T, Dubreucq B, Gromova M, Guillermo A, Miquel M, Nesi N, Yen-Nicolay S, Jolivet P
 (2015) The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes. Planta 242: 53-68

Bouchez I, Pouteaux M, Canonge M, Genet M, Chardot T, Guillot A, Froissard M 
(2015) The Rab7-like protein Ypt7p is involved in Saccharomyces cerevisiae lipid droplet dynamics. Biolopen 4: 764-775

Deruyffelaere C, Bouchez I, Morin H, Guillot A, Miquel M, Froissard M, Chardot T, D'Andréa S
 (2015) Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis. Plant Cell Physiol. 56: 1374-1387

Dulermo T, Coze F, Virolle MJ, Méchin V, Baumberger S, Froissard M (2015) 
Bioconversion of agricultural lignocellulosic residues into branched-chain fatty acids using Streptomyces lividans. OCL - Oléagineux, Corps Gras, Lipides 23 :2-8

Froissard M, Canonge M, Pouteaux M, Cintrat B, Mohand-Oumoussa S, Guillouet SE, Chardot T, Jacques N, Casaregola S 
(2015) Lipids containing medium-chain fatty acids are specific to post-whole genome duplication Saccharomycotina yeasts. BMC Evol Biol 15: 97

Kopecny D, Koncitikova R, Popelka H, Briozzo P, Vigouroux A, Kopecna M, Zalabak D, Sebela M, Skopalova J, Frebort I, Morera S
 (2015) Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. FEBS J J 283: 361-377

Purkrtova Z, Chardot T, Froissard M 
(2015) N-terminus of seed caleosins is essential for lipid droplet sorting but not for lipid accumulation. Arch Biochem Biophys 579: 47-54

Gallardo, K. Jolivet, P., Vernoud, V. Canonge, M. Larré, C. Chardot, T.
 , (2015) Storage cells - oil and protein bodies, in Molecular Cell Biology of the Growth and Differentiation of Plant Cells, Ray Rose editor, CRC Press

D'Andrea S (2015) Lipid droplet mobilization: The different ways to loosen the purse strings. Biochimie. DOI: 10.1016/j.biochi.2015.07.010

Kopecny D, Koncitikova R, Popelka H, Briozzo P, Vigouroux A, Kopecna M, Zalabak D, Sebela M, Skopalova J, Frebort I, Morera S (2015) Kinetic and structural investigation of the cytokinin oxidase/dehydrogenase active site. FEBS J 10.1111/febs.13581

Aymé L, Baud S, Dubreucq B, Joffre F, Chardot T (2014) Function and Localization of the Arabidopsis thaliana Diacylglycerol Acyltransferase DGAT2 Expressed in Yeast. PLoS One 9: e92237

Miquel M, Trigui G, d'Andréa S, Kelemen Z, Baud S, Berger A, Deruyffelaere C, Trubuil A, Lepiniec L, Dubreucq B
 (2014) Specialization of oleosins in oil body dynamics during seed development in Arabidopsis seeds. Plant Physiol. 164: 1866-1878

Purkrtova Z, Chardot T, Froissard M (2015) N-terminus of seed caleosins is essential for lipid droplet sorting but not for lipid accumulation. Arch Biochem Biophys 579: 47-54

Deruyffelaere C, Bouchez I, Morin H, Guillot A, Miquel M, Froissard M, Chardot T, D'Andrea S (2015) Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis. Plant Cell Physiol. 56: 1374-1387

Boulard C, Bardet M, Chardot T, Dubreucq B, Gromova M, Guillermo A, Miquel M, Nesi N, Yen-Nicolay S, Jolivet P (2015) The structural organization of seed oil bodies could explain the contrasted oil extractability observed in two rapeseed genotypes. Planta 242: 53-68

Bouchez I, Pouteaux M, Canonge M, Genet M, Chardot T, Guillot A, Froissard M (2015) The Rab7-like protein Ypt7p is involved in Saccharomyces cerevisiae lipid droplet dynamics. Biolopen 4: 764-775

Ayme L, Nicaud J-M, Jolivet P, Chardot T (2015) Molecular characterization of the Elaeis guineensis medium-chain fatty acid diacylglycerol acyltransferase DGAT1-1 by heterologous expression in Yarrowia lipolytica. PLoS One 10: e0143113 (pdfCommuniqué de presse INRA

Vermachova M, Purkrtova Z, Jolivet P, Chardot T, Kodicek M (2014) Combining chymotrypsin/trypsin digestion to identify hydrophobic proteins from oil bodies. In Plant Proteomics. Methods and Protocols. Se49

Ayme L, Baud S, Dubreucq B, Joffre F, Chardot T (2014) Function and Localization of the Arabidopsis thaliana Diacylglycerol Acyltransferase DGAT2 Expressed in Yeast. PLoS One 9: e92237 (pdf) Communiqué de press INRA

Le Marechal P, Decottignies P, Marchand CH, Degrouard J, Jaillard D, Dulermo T, Froissard M, Smirnov A, Chapuis V, Virolle MJ (2013) Comparative Proteomic Analysis of Streptomyces lividans Wild-Type and ppk Mutant Strains Reveals the Importance of Storage Lipids for Antibiotic Biosynthesis. Appl Environ Microbiol 79: 5907-5917 (pdf

Haïli N, Arnal N, Quadrado M, Amiar S, Tcherkez G, Dahan J, Briozzo P, Colas des Francs Small C, Vrielynck N, Mireau H (2013) The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria. Nucleic Acids Res 41:6650-6663 (pdf)

Jamme F, Vindigni JD, Méchin V, Cherifi T, Chardot TFroissard, M. (2013) Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae. PLOS ONE. DOI: 10.1371/journal.pone.0074421 (pdfActualité Centre INRA Versailles-Grignon 

Jolivet P, Acevedo F, Boulard Cd'Andrea S, Faure JD, Kohli, A, Nesi, N, Valot, B, Chardot, T. (2013) Crop seed oil bodies: From challenges in protein identification to an emerging picture of the oil body proteome. Proteomics 13: 1836-1849.

Jolivet PDeruyffelaere CBoulard C, Quinsac A, Savoire R, Nesi N, Chardot T (2013) Deciphering the structural organization of the oil bodies in the Brassica napus seed as a mean to improve the oil extraction yield. Ind Crop Prod 44: 549-557

Kubienova L, Kopecny DTylichova MBriozzo P, Skopalova J, Sebela M, Navratil M, Tache R, Luhova L, Barroso JB, Petrivalsky M (2013) Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum. Biochimie 95: 889-902

Snegaroff JBouchez ISmaali Mel A, Pecquet C, Raison-Peyron N, Jolivet PLauriere M (2013) Barley gamma3-hordein: glycosylation at an atypical site, disulfide bridge analysis, and reactivity with IgE from patients allergic to wheat. Biochim Biophys Acta Proteins and Proteomics 1834: 395-403

Vindigni JD, Wien F, Giuliani A, Erpapazoglu Z, Tache RJagic FChardot TGohon YFroissard M (2013) Fold of an oleosin biologically targeted into cellular oil bodies. Biochim Biophys Acta Biomembranes 1828: 1881-1888

Acevedo F, Rubilar M, Shene C, Navarrete P, Romero F, Rabert C, Jolivet P, Valot B, Chardot T (2012) Seed oil bodies from Gevuina avellana and Madia sativa. J Agric Food Chem 60: 6994-7004

Beopoulos A, Haddouche R, Kabran P, Dulermo T, Chardot T, Nicaud JM (2012) Identification and characterization of DGA2, an acyltransferase of the DGAT1 acyl-CoA:diacylglycerol acyltransferase family in the oleaginous yeast Yarrowia lipolytica. New insights into the storage lipid metabolism of oleaginous yeasts. Appl Microbiol Biotechnol 93: 1523-1537

Mameri HBouchez I, Pecquet C, Raison-Peyron N, Choudat D, Chabane H, Kerre S, Denery-Papini S, Gohon YBriozzo PLauriere MSnegaroff J (2012a) A Recombinant omega-Gliadin-like D-Type Glutenin and an alpha-Gliadin from Wheat (Triticum aestivum): Two Immunoglobulin E Binding Proteins, Useful for the Diagnosis of Wheat-Dependent Allergies. J Agric Food Chem 60: 8059-8068

Mameri H, Denery-Papini S, Pietri M, Tranquet O, Larre C, Drouet M, Paty E, Jonathan AM, Beaudouin E, Moneret-Vautrin DA, Moreau T, Briozzo P, Gaudin JC (2012b) Molecular and immunological characterization of wheat Serpin (Tri a 33). Mol Nutr Food Res 56: 1874-1883

Mameri HSnegaroff JGohon Y, Pecquet C, Choudat D, Raison-Peyron N, Denery-Papini S, Wien F, Briozzo P (2012c) Immunoglobulin-E Reactivity and Structural Analysis of Wheat Low-Molecular-Weight Glutenin Subunits and Their Repetitive and Nonrepetitive Halves. J Agric Food Chem 60: 7538-7547

Gohon YVindigni JD, Pallier A, Wien F, Celia H, Giuliani A, Tribet C, Chardot TBriozzo P (2011) High water solubility and fold in amphipols of proteins with large hydrophobic regions: oleosins and caleosin from seed lipid bodies. Biochim Biophys Acta Biomembranes 1808: 706-716

Kopecny DTylichova MSnegaroff J, Popelkova H, Sebela M (2011) Carboxylate and aromatic active-site residues are determinants of high-affinity binding of omega-aminoaldehydes to plant aminoaldehyde dehydrogenases. FEBS J 278: 3130-3139

Jolivet PBoulard C, Bellamy A, Valot B, d'Andrea S, Zivy M, Nesi N, Chardot T (2011) Oil body proteins sequentially accumulate throughout seed development in Brassica napus. J Plant Physiol 168: 2015-2020

Popluechai S, Froissard MJolivet P, Breviario D, Gatehouse AM, O'Donnell AG, Chardot T, Kohli A (2011) Jatropha curcas oil body proteome and oleosins: L-form JcOle3 as a potential phylogenetic marker. Plant Physiol Biochem 49: 352-356

Vermachova MPurkrtova Z, Santrucek J, Jolivet PChardot T, Kodicek M (2011) New protein isoforms identified within Arabidopsis thaliana seed oil bodies combining chymotrypsin/trypsin digestion and peptide fragmentation analysis. Proteomics 11: 3430-3434

Bouchez-Mahiout I, Boulenc E, Snegaroff J, Choudat D, Pecquet C, Raison-Peyron N, Vigan M, Chabane H, Dron-Gonzalves M, Branlard G, Tanis-Plant S, Lauriere M (2010a) Immunoblotting analysis of wheat allergens: control of side reactions through wheat polypeptides naturally present in dried cow milk. Food and Agr Immunol 21: 237 - 251

Bouchez-Mahiout I, Pecquet C, Kerre S, Snegaroff J, Raison-Peyron N, Lauriere M (2010b) High molecular weight entities in industrial wheat protein hydrolysates are immunoreactive with IgE from allergic patients. J Agric Food Chem 58: 4207-4215

Bouchez-Mahiout ISnegaroff JTylichova M, Pecquet C, Branlard G, Lauriere M (2010c) Low molecular weight glutenins in wheat-dependant, exercise-induced anaphylaxis: allergenicity and antigenic relationships with omega 5-gliadins. Int Arch Allergy Imm 153: 35-45

Kopecny DBriozzo P, Popelkova H, Sebela M, Koncitikova R, Spichal L, Nisler J, Madzak C, Frebort I, Laloue M, Houba-Herin N (2010) Phenyl- and benzylurea cytokinins as competitive inhibitors of cytokinin oxidase/dehydrogenase: a structural study. Biochimie 92: 1052-1062

Pedrono F, Blanchard H, Kloareg M, D'Andrea S, Daval S, Rioux V, Legrand P (2010) The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues. J Lipid Res 51: 472-479

Stranska J, Tylichova MKopecny DSnegaroff J, Sebela M (2010) Biochemical characterization of pea ornithine-delta-aminotransferase: Substrate specificity and inhibition by di- and polyamines. Biochimie 92: 940-948

Tylichova MKopecny D, Morera S, Briozzo P, Lenobel R, Snegaroff J, Sebela M (2010) Structural and functional characterization of plant aminoaldehyde dehydrogenase from Pisum sativum with a broad specificity for natural and synthetic aminoaldehydes. J Mol Biol 396: 870-882

 

 

 

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