1. GBPs: Immunity to intracellular pathogens Juliana Ueda Thais Herrero Claudia Polli GBP

2. IFN-γ : Central in host resistance to infection WT IFN KO

3. IFN-γ regulates the expression of more than 1.200 genes Products: Only a fraction: Mediators of host immune responses Remainder: little is known Mainly produced by: GTPases GTPases: GTP-binding proteins NRAMP1: natural-resistance associated macrophage protein 1

4. Cellular functions of GTPases Two conformations: Bound to GTP: active Hydrolisis GTP GDP and GMP Bound t GDP: inactive GEF (guanine exchange factor) GDP GTP Active GTPase Cellular functions: Activation of cell-surface receptors to modulation of membrane-fusion events Membrane tafficking Cell signaling and migration Translation and protein translocation Nuclear transport

5. Families of GTPases 4 major families: - Mx family (type I and II IFN antiviral activity ) - Very large inducible GTPases (type II IFN ???) - p47 immunity-related GTPases (IRGs) antimicrobial activity - Guanylate-binding proteins (GBPs) ??? Type I IFN and IFN-γ * Mice: IRGs against intracellular pathogens Human: lack IRGs GBPs

6. 11 mGBPs 7 hGBPs GBPs: Guanylate-binding proteins

7. Induced by: type I IFN, IL-1β, LPS, IFN-γ 65-KDa GTP-binding domain(G domain) Isoprenylation: Addition of either a C15 farnesyl or C20 geranygeranyl lipid to the C-terminus of the GBPs Isoprenylation is important in targeting proteins to intracellular membranes and/or to facilitate protein/protein interactions GBPs are predominantly cytosolic and have, at most, a relatively small portion of the total amount associated with membranes. leucine serine

8. Functions of GBPs Regulation of vasculogenesis by proinflammatory cytokines Mutant GTPase active site These are strinking phenomena but seem unlike to constitute the adaptative function of hGBP because the G domain was not required

9. Functions of GBPs Regulation of MMP1 production MMP1: Matrix metalloprotease 1 is required for the breakdown of extracellular colagen, enabling endothelial cells to form vessels in vivo

10. Functions of GBPs Since GBPs are induced by both type I and type II IFNs, it seemed logical to examine whether they are involved in host defense

11. Functions of GBPs Antiviral activity HeLa cells hGBP-1 SVS, EMCV m.o.i 1 mGBP-2 S52N: single point mutation GTP binding region SVS, EMCV m.o.i 0.1

12. Functions of GBPs Antiviral activity The basis of antiviral effects is unknown. The antiproliferative activity might help to limit the cell-to-cell spread of vírus. Control Clone 1GBP-2 Clone 2 GBP-2 SN52

13. Functions of GBPs Response to Protozoan infections MEFs

14. Functions of GBPs Response to Bacterial infections

15. Functions of GBPs Antibacterial activity Thais Claudia

16. NADPH oxidase complex is a cluster of proteins that donate an electron from NADPH to molecular oxygen (O 2 ) to produce superoxide anion (O 2 - ). This initiates the respiratory burst, a key step in immune defense against bacterial and fungal pathogens Neutrophil NADPH oxidase http://www.clinsci.org

17. http://www.caymanchem.com Gp91 phox large glycosylated protein p22 phox smaller adapter protein FAD:cofactor Soluble components: p47 phox P67 phox p40 phox PKC-mediated phosphorylation p47 phox Translocation to the membrane 47 phox binding to p22 phox Electrons from NADPH are donated to O 2 to produce superoxide O 2 - Protons dissociated from NADPH proton channels interact with O 2 - to produce H 2 O 2 Phagosome acidification Also engulfed with the bacterium will be membrane proteins, including gp91/p22 and FAD

18. Second antimicrobial pathway… Nonoxidative mechanisms: http://www.fbs.osaka-u.ac.jp

19. Microtubule-associated protein light-chain 3 (LC3) Antioxid Redox Signal. 2009 August; 11(8): 1975–1988.