The Biotechnology of Isoprenoids - All Things Considered? Tobacco BY-2 Cells as a Model System

Transcription

1 The Biotechnology of Isoprenoids - All Things Considered? Tobacco BY-2 Cells as a Model System Thomas J. Bach Co-workers workers: Hemmerlin A. 1, Hartmann M. 1, Hartmann, M.-A. 1, Heintz D. 1, Simonovik B. 1, Gas- Pascual E. 1, Schaller H. 1, Tritsch D. 2, Crowell D.N. 3 and Rohmer M. 2 1 Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, F Strasbourg; 2 Université de Strasbourg/CNRS, Institut de Chimie UMR 7177, F Strasbourg; 3 Idaho State University, Pocatello,, ID Collaborations: Prof. Albert Boronat Prof. Herbert Waldmann Universitat de Barcelona Dr. Reinhard Reents Prof. Mee-Len Chye Max-Planck-Institut für University of Hong Kong Molekulare Physiologie Prof. Joe Noel & Dr. Florence Pojer Dortmund Salk Institute Dr. Pierrette Bouvier-Navé Navé,, IBMP, Dépt. Réseaux métaboliques Dr. Alain Van Dorsselaer, Institut Pluridisciplinaire Hubert Curien, Strasbourg

2 CYTOSOL AACT + HMGS Acetyl-CoA HMG-CoA F244 HMGL Geranylgeranylated proteins PGGT-1 IPA Cytokinins Sesquiterpenoids Dolichol Chaetomellic acid Farnesylated proteins GGPP PFT DMAPP Squalene SQE HMGR GGTi-2133? 3 x IDI FDS FPP 2 x SQS Squalestatin MVA Terbinafine Squalene epoxide Ro CAS Cycloartenol IPP Mevinolin FPP Cytochrome a 3 Ubiquinones MITOCHONDRIA Sterols Brassinosteroids, Triterpenoids

3 Structure of Brassica juncea HMGS1 and inhibitor (F-244) Eukaryotic specific extra-domain F. Pojer et al., PNAS 2006

4 F-244 tail and substrates bind in different cavities F-244 Acetyl-CoA F. Pojer et al., PNAS 2006

5 G1 HMGR activity peaks > Mevinolin- induced cell cycle arrests MVA requirement MVA-derived signal G2 cf. Hemmerlin et al. Lipids (2004)

6 Differences in cell cycle phases of mevinolin-treated TBY-2 cells as compared to control cells. Note the onset of clear differences at around 8 h following treatment. Mevinolin-control (%) Time (hours) G0/G1 S G2/M (Hemmerlin & Bach, Plant J 1998)

7 CYTOSOL AACT + HMGS Acetyl-CoA HMG-CoA F244 HMGL Geranylgeranylated proteins IPA Cytokinins Sesquiterpenoids Dolichol Chaetomellic acid Farnesylated proteins endogenous Farnesol? GGPP PFT DMAPP Squalene SQE? Sterols HMGR 3 x IDI FDS FPP SQS Squalestatin Brassinosteroids 2 x MVA Terbinafine Squalene epoxide IPP Ro Mevinolin Farnesol, a regulator molecule? A model for compounds that could pile up under certain conditions? exogenous Farnesol FPP Cytochrome a 3 Ubiquinones MITOCHONDRIA

8 Farnesol-induced cell death Farnesol Cell death (%) Cell death (%) Cell dilution Farnesol (µm) x 17x 9x 5x Hemmerlin & Bach, Plant Physiol. (2000)

9 Stimulation by farnesol of apparent HMGR activity Western blot Northern blot Hemmerlin & Bach, Plant Physiol. (2000)

10 Effect of different concentrations of farnesol on the viability of TBY-2 cells in the absence or presence of 1 µm mevinolin (24 h of incubation). Percentage Cellules of vivantes living TBY-2 (%) cells Concentration µm Farnesol en farnésol (!M) J.-F. Feldtrauer & A. Hemmerlin, unpublished Farnésol Farnesol Mevinoline + Farnésol Farnesol + Mevinolin

11 Chemical structures of Fol* and GGol* HO HN NO 2 N O N HO HN NO 2 N O N

12 Hemmerlin et al. ABB, 2006

13 Putative phosphorylation site N-terminal, membrane-spanning amino acid sequences of plant HMGR isozymes. Note the apparent existence of two families that are either containing a proline residue in the arginine-rich part or a serine.. An important exception is Arabidopsis

14 Labeling of actin by talin-gfp after bombardment of BY-2 cells with pysc14 (N.H. Chuaʼs s lab, Rockefeller University); Z-stack, 3-D reconstitution (R. Merret, diploma thesis 2007)

15 Localization of HMGR isozymes: BY-2 cells, transiently transformed with A) TMD1-GFP; B) TDM2-GFP. Note that TDM1-GFP localizes mainly to network-like structures (ER?), while TDM2 forms major aggregates that seem to be rather associated with filaments.

16 A BY-2 cell co-transformed with TDM1 P->S >S-GFP and TMD2-RFP. Note the nearly perfect match of intracellular localization in the overlay image.

17 BY-2 cells transformed with mouse talin-gfp and TMD2-RFP. A) The image was taken after treatment with Cytochalasin D (5 µm, ~ 6 h), which disrupts actin-based network organization and leads to separation of TMD2 protein bodies and their final diffusion into the peripheric cytoplasm. B) Treatment with 5 µm calmidazolium for 15 min leads also to diffusion.

18 Reactions in the phytosterol pathway (Pierre Benveniste, 2002) HO SMT1 C4-demethylase HO 4α -methyl-ergosta- 8,24-dien-3 β -ol Cyclopropyl- sterol isomerase HO cycloartenol cycloeucalenol obtusifoliol C14-demethylase HO 4α -methyl-ergosta- 8,14,24-trien-3β-ol -ol FACKEL (C14-reductase) HO HO 24-ethylidene lophenol Δ8- Δ7-isomerase SMT2 HO 24-methylene lophenol C4-demethylase STE (C5-desaturase) DWF5 ( Δ 7-reductase) HO C4-demethylase STE (C5-desaturase) DWF5 (Δ7-reductase)( isofucosterol HO 24-methylene cholesterol DIM HO sitosterol DIM HO campesterol

19 Growth curve of BY-2 cells, relation to sterol content and enzyme activities. Effects of squalestatin on HMGR, FDS, SQS and SMTs A B C nmol.h -1.mg -1 protein µg.mg -1 protein log fresh weight SQS HMGR SMT-1 SMT-2 4-demethylsterols Days D E Relative HMGR activity HMGR SQS FDS EF1-α HMGR SQS FDS Sq concentration (nm) Sq concentration (nm) Sq concentration (nm)

20 Terbinafine induces squalene accumulation at the expense of sterols, and is labeled with 14 C-acetate A B mg.g -1 dwt cpm.g -1 dwt x squalene 4,4-dimethylsterols 4α-methylsterols 4-desmethylsterols Tb concentration (µm) squalene 4,4-dimethylsterols 4α-methylsterols 4-desmethylsterols Tb concentration (µm) mg.g -1 dwt h Control squalene 4,4-dimethylsterols 4α-desmethylsterols h + Tb Wentzinger et al., Plant Physiol (2002)

21 Lipid particles in the cytosol of TBY-2 cells treated with Terbinafine (Tb). 3-d-old TBY-2 cells were treated for 24 h with Tb 30 µm, then observed in optical microscopy after staining by Sudan IV. Lipid particles containing triglycerides plus squalene appear as spherical droplets. Bar = 20 µm.

22 Mevinolin-induced growth inhibition of tobacco BY-2 cells can be overcome by MVA, but also by deoxyxylulose (DX), the dephosphorylated first product in the MEP pathway 100 Growth (% of control) / DX 0.5 mm / DX 1 mm DX 2 mm MVA 1 mm MVA 2 mm / / MV 5!M MV 5!M MV 5!M MV 5!M MV 5!M Hemmerlin et al. JBC (2003)

23 CYTOSOL Geranylgeranylated proteins AACT + HMGS HMGR Acetyl-CoA HMG-CoA F244 Mevinolin HMGL Pyruvate + Glyceraldehyde 3-P DXS 1-Deoxy Deoxy-D-xylulose -xylulose 5-P DXRI Oxoclomazone Fosmidomycin 2-C-Methyl- -Methyl-D-erythritol-erythritol 4-P BY-2 PLASTIDS Thiamine IPA Cytokinins Sesquiterpenoids Dolichol Chaetomellic acid endogenous Farnesol? GGPP PFT DMAPP Squalene SQE? Sterols 3 x IDI FDS FPP SQS Squalestatin Brassinosteroids 2 x MVA Terbinafine Squalene epoxide IPP? FPP Cytochromes a, a 3 Ubiquinones MITOCHONDRIA 3 x IPP IDI DMAPP IPP GGPP GPP Diterpenoids (GAs)? (α-tocopherol,, Phylloquinone) Phytoene Carotenoids? Isoprene? (Monoterpenoids?) Plastoquinone ABA? The plastidial MEP pathway in tobacco BY-2 cells : branching

24 MEP pathway for isoprenoid biosynthesis: hypothetical biogenetic scheme for the incorporation of [2-13 C, 4-2 H]deoxyxylulose (1)( ) into the isoprene units of plastoquinone and cis-phytoene, the plastid isoprenoids of a Tobacco Bright Yellow-2 cell culture. D. Tritsch et al., FEBS Lett 2010

25 Specific targeting of prenylated GFP fusion proteins GFP GFP GFP GFP S C V I L S C V I M S V I X The white bar corresponds to 10 µm E. Gerber, thèse de doctorat, Strasbourg 2005

26 In vivo inhibition of protein isoprenylation in BY-2 cells and selective chemical complementation E. Gerber, A. Hemmerlin et al., Plant Cell 2009

27 A Control 30 µm Oxoclomazone 100 µm 5 µm Mevinolin 30 µm OC + 5 µm Mevinolin Gerber, Hemmerlin et al., Plant Cell (2009) Data: Michael Hartmann

28 Miniaturization of the assay, adapted for multiwell plates for the Zeiss fluorescence microscope Volume down-scaled from 3 ml to 250 µl (100 µl)? Treatment and observation take place in special glass-bottom plates (96 wells)? Field of view > 1 mm 2, with 100 to 200 countable cells depending on the dilution Untreated control Zoom 40 µm oxoclomazone

29 Doubly transformed BY-2 cell line showing different intracellular localisation of fluorescence marker fusion proteins (M. Hartmann & E. Gas-Pascual, unpublished) The existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector (Zuo et al., 2000) driving the independent expression of a RFP protein, C-terminally fused to a NLS (nuclear localisation signal). With this new strategy, the total number of viable cells versus the number of inhibited cells after various treatments can be quantified.

30 CONTROL Fosmidomycin G R G R G DXR amirna -Est G +Est G R G R G G Mevinolin Oxoclomazone -Est +Est HMGR amirna

31 Cloning of transgenic BY-2 cells with new features: inducible nuclear staining (SV40-NLS-mRFP) and with strongly reduced heterogeneity

32 Principal steps of image processing and analysis Image analysis is performed with ImageJ software. Multi-channel images are first separated into red and green channels. After adjusting the threshold of the red channel, the signals (from the nuclei) are converted to a mask, which is superposed on the green channel image. After adjusting the threshold for the latter, automatic particle counting is performed. Mask (nuclei) Red Channel Original image Green Channel thresholding create mask apply mask apply mask Results Overlay thresholding particle analysis M. Hartmann et al., Poster TERPNET 2009, Tokyo

33 CYTOSOL AACT + HMGS HMGR Geranylgeranylated proteins Acetyl-CoA HMG-CoA F244 Mevinolin HMGL Pyruvate + Glyceraldehyde 3-P Oxoclomazone DXS 1-Deoxy Deoxy-D-xylulose -xylulose 5-P DXRI Fosmidomycin 2-C-Methyl- -Methyl-D-erythritol-erythritol 4-P PLASTIDS Thiamine IPA Cytokinins Sesquiterpenoids Dolichol Chaetomellic acid Farnesylated proteins endogenous Farnesol? GGPP PFT DMAPP Squalene SQE Sterols 3 x IDI FDS FPP SQS Squalestatin Squalene epoxide CAS??? Brassinosteroids 2 x MVA Terbinafine IPP? FPP? Cytochrome a 3 Ubiquinones MITOCHONDRIA 3 x IPP IDI DMAPP IPP GGPP GPP Isoprene Monoterpenoids Plastoquinone Diterpenoids (GAs) (Chlorophylls, α-tocopherol,, Phylloquinone) Carotenoids ABA To export, or not to export intermediates (whatsoever): That is the question! Is on the way

34 Deficiency in vital compounds Accumulation of toxic compounds & intermediates? Specific function of isozymes; deregulation Aim: Overproduction of (Secondary) Metabolites of High Value Vitamines, Antioxidants Anticancerigenic Compounds Nutritional Additives Hypercholesterolemic Agents Retro-inhibition & retroactivation of enzymes in metabolic pathways Reorientation of metabolic fluxes Modification of hormonal balances What needs to be considered

35

36 2D-PAGE of proteins from mevinolin-treated BY-2 cells Data: B. Simonovik, unpublished

37 Cytosolic Isoprenoid Biosynthesis - Regulatory Interactions Glycolysis...!?!! Acetyl-CoA AACT HMGS HMG-CoA HMGR MVA Toxic products? Limiting metabolic channels or action of inhibitors Feedback control by de novo synthesized (end) products Direct feedback on enzyme Regulation at gene level, by phosphorylation, etc. MVAK IPP DMAPP Dolichols, polyprenols Squalene Sterols, triterpenoids, brassinosteroids... (Final) products (on the way to cell membranes?) FPP Ubiquinone side chain Farnesylated proteins Lyase Farnesol Recycling of toxic farnesol by double phosphorylation

38 Differences in cell cycle phase distribution of squalestatin-treated and control TBY-2 cells Squalestatin - Control (%) Time (hours) G0/G1 S G2/M A. Hemmerlin, unpublished