EA2106

Protein isoprenylation

EA2106 Protein isoprenylation

Protein isoprenylation is a post-translational lipidation common to eukaryotes. This process engages CaaX-protein prenyl transferases (CaaX-PTases) that recognize a tetrapeptide (called CaaX-box) localized on the carboxy-terminus of target proteins and attach a prenyl moiety to the Cysteine (illustrated in A). The prenyl consists in a farnesyl or a geranylgeranyl that is specifically linked to the CaaX-box by two different PTases: farnesyltransferase (PFT) and geranylgeranyltransferase type I (PGGT-I).


These protein lipidations have a determinant role in protein subcellular localization, protein interactions or enzyme activity, and contribute to the functional diversity of the cellular proteome. In plants, the characterization of CaaX-PTases knockout mutants in Arabidopsis highlighted the importance of protein isoprenylation in developmental processes as well as in plant responses to biotic and abiotic stress (B, example of the PFT mutant). However, the identification and the molecular characterization of the prenylated proteins engaged in these physiological processes are poorly documented and require the development of specific strategies.

In vivo tagging of prenylated proteins is facilitated by the use of modified prenylazide isoprenoids. This new approach, called tagging-via-substrate, allows the detection (shown in C) and further, the identification of prenylated proteins. This approach combined with the in silico analysis of proteins harboring a CaaX-box are powerful tools to unravel the role of isoprenylation in specific physiological contexts.

The CaaX-box sequence is critical for PTase activity and determines which prenyl is attached. Thus, the identity of the PTases engaged for the prenylation of a specific CaaX-box may be assessed through in vitro prenylation assays using fluorescent prenyl substrates (exemplified in D). Remarkably, protein isoprenylation is commonly associated to subcellular localization. This is visualized by YFP imagery, using WT or CaaX-PTases deficient lines expressing the CaaX-protein or a SaaX-mutated version (E). In addition, the physiological role of the proteins is further studied by reverse genetics.

Studies performed by the EA2106 BBV research team has highlighted a role of prenylated proteins in hormonal regulations and cellular metabolism. For instance, the regulation of secondary metabolite biosynthesis in Catharanthus roseus or seed lipid storage in Arabidopsis thaliana by methyljasmonate or abscisic acid, respectively, engages isoprenylation events. The identification of proteins targeted by the CaaX-PTase enzymes will help to decipher the molecular mechanisms that govern these important medicinal and agronomical traits.

Contact: Nathalie Guivarc'h 

Publications:

Dutilleul, C., Ribeiro, I., Blanc, N., Nezames, C.D., Deng, X.W., Zglobicki, P., Palacio Barrera, A.M., Atehortùa, L, Courtois, M., Labas, V., Giglioli-Guivarc'h, N. & Ducos, E. (2015). ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis. Plant Cell Environment in press.

Courdavault, V., Clastre, M., Simkin, A. & Giglioli-Guivarc’h, N. (2013). Prenylated Proteins Are Required for Methyl-Jasmonate-Induced Monoterpenoid Indole Alkaloids Biosynthesis in Catharanthus roseus. In Isoprenoid Synth Plants Microorg SE  - 19, pp. 285–296. Edited by T. J. Bach & M. Rohmer. Springer New York.

Corbin, C., Decourtil, C., Marosevic, D., Bailly, M., Lopez, T., Renouard, S., Doussot, J., Dutilleul, C., Auguin, D., Giglioli-Guivarc'h, N., Lainé, E., Lamblin, F. & Hano, C. (2013). Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol-Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.). Plant Physiol Biochem 72, 96–111.

Courdavault, V., Burlat, V., St-Pierre, B. & Giglioli-Guivarc'h, N. (2009). Proteins prenylated by type I protein geranylgeranyltransferase act positively on the jasmonate signalling pathway triggering the biosynthesis of monoterpene indole alkaloids in Catharanthus roseus. Plant Cell Reports  28, 83-93.

Hedili, S., Courdavault, V., Giglioli-Guivarc’h, N. & Gantet, P. (2007). Regulation of the biosynthesis of the terpene moiety of Catharanthus roseus terpene indole alkaloids. Phytochemistry Review  6,341-51.

Courdavault, V., Burlat, V., St-Pierre, B. & Giglioli-Guivarc’h N.(2005) Characterisation of CaaX-prenyltransferases in Catharanthus roseus: relationships with the expression of genes involved in the early stages of monoterpenoid biosynthetic pathway. Plant Sci.  168, 1097–107.

Courdavault, V.,  Thiersault, M.,  Courtois, M.,  Gantet, P.,  Oudin, A.,  Doireau, P.,  St-Pierre, B. & Giglioli-Guivarc’h, N. (2005). CaaX-prenyltransferases are essential for expression of genes involved in the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells. Plant Mol. Biol. 57, 855–70.

Courdavault, V., Burlat, V., Gantet, P., St-Pierre, B. & Giglioli-Guivarc’h, N. (2005). Isolation of a cDNA encoding the α subunit of CaaX-prenyltransferases from Catharanthus roseus and the expression of the active recombinant protein farnesyltransferase. Cell. Mol. Biol. Lett. 10, 649-57.

 

 

EA2106 BBV, Biomolecules and Plant Biotechnology

For more 25 years, EA2106 Biomolecules and Plant Biotechnology (BBV) research programs aimed at understanding plant specialized metabolisms at physiological, biochemical, molecular and cellular biology levels, with a particular focus on the metabolism of Monoterpenoid Indole Alkaloid (MIA) in the Madagascar periwinkle and related plants. The Madagascar periwinkle (Catharanthus roseus) remains the main commercial source for the anticancer vincristine, vinblastine and several hemi-synthetic alkaloid pharmaceuticals. Despite numerous years spent on the study of this metabolism, the MIA biosynthetic pathway is still not entirely known as well as the modalities of its in planta regulation.  A large area of our research thus concern the study of isoprenoid biosynthesis with a focus on the terpenoid precursors of MIA and the global architecture of alkaloid metabolism in periwinkle including identification of critical enzymatic steps, organization at cellular and tissue levels of the metabolic pathway and its regulation.

In parallel of these fundamental approaches,  we have been developping for several years plant cell systems to produce specialized metabolites of interest. Most of these projects are in collaboration with industrial partners.  In addition, we have open our interest on specific yeast systems which offers also a larger panel of biotechnological applications. In particular, our attention have been focused on Candida species that have been, for some of them, described with a biotechnological potential. To this end, a large part of our activity consisted in development of molecular tools to permit the genetic manipulation of these yeast species for further biotechnological applications.

Our investigation topics have been enlarged to explore the physiological roles in planta of specialized metabolites in order to have a better understanding of their regulation. To this end we developed models of plant–pathogens interaction that led us to explore 1) the involvement of methyl jasmonate and cytokinin signaling and 2) the specific phenylpropanoid metabolism involved in plant defence mechanisms. These approaches are also favored because genomic data are now available and because some of these specialized metabolites could be isolated, purified and used for human health as pharmaceutical, cosmetic, insecticide or fungicide compounds or food complement.
 
Thus research areas of our team are divided in 6 domains which are enriched by our involvement in botanical expertise.

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