Antioxidant reductive systems in plants : The poplar glutaredoxins C1 and C4.

1. Nter GSH C27 Cter Antioxidant reductive systems in plants : The poplar glutaredoxins C1 and C4. Why genetic engineering and protein structure are a necessary complement to Phylogenomics. Jean-Pierre Jacquot UMR 1136 INRA UHP Interaction Arbres Microorganismes IFR 110 Génomique, Ecophysiologie et Ecologie fonctionnelles Université Henri Poincaré, Nancy I, France Décembre 2006 Ecole Phylogénomique Carry Le Rouet

2. Nter GSH C27 Cter What is a glutaredoxin? Small oxidoreductases cousin to thioredoxins Generally small molecular weight (ca 12 kDa, 100 to 120 amino acids in the processed form) High degree of secondary structures. Central pleated b sheet surrounded by a helices. Often thermostable Active site generally of the CXXC form (most well known YCPYC) thioredoxin is generally WCGPC and PDI WCGHC Reduced via glutathione Function : reducers of disulfide bridges, glutathionylation/ de, electron donors to peroxidases, domain of APS reductase

3. Thioredoxin pathway NTR Trx h,o S SH Target SH Inactive form protein S SH SH NADPH Grx SH SH Target HS Active form protein HS SH SH SH GSH 2 Nter GR GSH/Glutaredoxin pathway GSSG GSH C27 Cter The cytosolic and mitochondrial pathways leading to reduction of disulfide bonds

4. ROS S-SG SH Protein GSSG Monothiol pathway GSH S-SG Protein S NADPH Grx GR GSH SH S S Protein S S- S- HS GR NADP+ Dithiol pathway Grx GSSG SH Protein SH S-S HS Grx SH Protein HS Nter S Grx S GSH C27 Cter Catalytic mechanisms of glutaredoxins

5. Nter GSH C27 Cter yeast Grx 4 CGFS yeast Grx 3 At Grx S14 human Grx3 At Grx S1 CCx[C/S/G] At Grx S2 At GrxS3 At Grx S15 At GrxC12 At Grx C7 At Grx C8 yeast Grx 5 At Grx C13 At Grx C14 E. coli Grx4 At Grx C9 E. coli Grx 3 At Grx C10 human Grx1 human Grx2 yeast Grx2 E. coli Grx1 At Grx C4 yeast Grx1 At Grx C2 At GrxC1 At Grx C3 Cxx[C/S] At Grx C5 There are active site variants and subgroups of glutaredoxin in all known genomes Rouhier et al. Cell Mol Life Sci. 2004 Jun;61(11):1266-77. 0.1

6. Nter GSH C27 Cter CxxC8 CxxC7 CCx[C/S/G] CxxC11 CxxC12 CxxS10 CxxS4 CxxS8 CxxS7 CxxC13 CxxS6 CxxS5 CxxC14 CxxS3 CxxS2 CxxS9 CxxS1 CxxS11 CxxC6 CxxC1 CxxC10 CxxC2 CxxS12 CxxS13 CxxC5 CxxC9 Cxx[C/S] CxxC4 CxxC3 CxxS17 CxxS15 CxxS16 CxxS14 CGFS There are 31 glutaredoxingenes in A. thaliana, and a similar number in poplar Rouhier et al J Exp Bot. 2006;57(8):1685-96.

7. Nter GSH C27 Cter Characterization of poplar glutaredoxins of the Cxx[C/S] group Glutaredoxins C4 and C1

8. Nter GSH C27 Cter Glutaredoxins C1 and C4 are present in all sequenced plants. They display extensive homologies

9. Nter GSH C27 Cter Glutaredoxin C4 has a CPYC active site What are its targets? What functions can it have? What is its 3D structure? What do we know about the molecular contacts with established targets of known 3D structure?

10. Nter GSH C27 Cter Plant glutaredoxin C4 targets Rouhier et al., 2005, Antiox & Red. Signal., 2005 Jul-Aug;7(7-8):919-29.

11. NADPH,H+ ROOH ROOH NTR ROH ROH NADP 2 GSH NADP SH Grx SH SH SH Prx Prx GR SH SH GSSG NADPH,H+ S S Nter SOH SOH Trx Grx Prx Prx S S SH SH SH Trx GSH SH C27 Cter In vitro reduction systems Rouhier et al. J Biol Chem. 2002 Apr 19;277(16):13609-14.

12. C76 Nter C51 Cter Nter Nter GSH GSH C27 C27 Cter Cter X ray structures Poplar glutaredoxin C4 Poplar type II peroxiredoxin Echalier et al. Biochemistry. 2005 Feb 15;44(6):1755-67 Corbier et al. unpublished

13. Nter GSH C27 Cter 3D structure of the Haemophilus influenzae Prx Grx hybrid enzyme Kim et al, J Biol Chem. 2003 Mar 21;278(12):10790-8.

14. Nter GSH C27 Cter NMR structure of poplar glutaredoxin C4 Noguera et al. J Mol Biol. 2005 Oct 28;353(3):629-41.

15. Glutaredoxin C1 has a CGYC active site What is its 3D structure? What are its targets? What functions can it have? What do we know about the molecular contacts with established targets of known 3D structure?

16. When expressed in E.coli cells, poplar glutaredoxin C1 is distributed into two fractions of approximately equal importance One with a monomeric apoprotein (ca 12 kda) One with a dimeric holoprotein (apparent mass 24 kDa) The dimeric holoprotein faction is reddish brown suggesting the presence of an ironsulfur centre Chemical analyses indicate that a dimer contains one 2Fe-2S center

17. Nter GSH C27 Cter Spectral properties of poplar glutaredoxin C1 holo Rouhier et al. unpublished

18. Nter GSH C27 Cter X ray 3D structure of poplar Grx C1 Rouhier et al. unpublished

19. Nter GSH C27 Cter NMR structure of poplar Grx C1 Feng et al. Biochemistry. 2006 Jul 4;45(26):7998-8008.

20. Both NMR spectroscopy and X ray crystallography indicate that the iron sulfur centre of poplar glutaredoxin C1 bridges two identical subunits The ligands are the active site catalytic cysteine and external molecules of reduced glutathione The backup cysteine or other conserved cysteines in various C1 glutaredoxins are not involved in the binding of the ISC

21. GSH GSSG ROS sensor? Aconitase GRX Cytosol C1 CGYC CYTOSOL Mitochondrial CGFS GRX SDH ROS sensor? MITOCHONDRIA Is glutaredoxin C1 involved in the ISC assembly of cytosolic iron sulfur containing enzymes?

22. Conclusion Glutaredoxins are likely to play multiple functions in plant cells Donors for peroxide reduction (interaction with type II peroxiredoxin) Electron donors to APS reductase Possibly implicated in iron sulfur assembly processes (yeast Grx 5 is involved in these pathways) The implication of certain Grx isoforms in the ISC assembly could be related to their own capacity to assemble ISC centers. There are reports that some Grx are also involved/required for heme assembly

23. Collaborations X ray crystallography A Aubry, C Corbier, Nancy, France T Hase, M Kusunoki, Osaka, Japan NMR spectroscopy JM Lancelin, I Krimm, Lyon, France B Xia, Beijing, China RAMAN, EPR spectroscopy M Johnson, Georgia, USA