GBPs: Immunity to intracellular pathogens

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

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: GTP-binding proteins GTPases 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. GBPs: Guanylate-binding proteins 7 hGBPs 11 mGBPs

7. GBPs: Guanylate-binding proteins Isoprenylation: Addition of either a C15 farnesyl or C20 geranygeranyl lipid to the C-terminus of the GBPs Induced by: type I IFN, IL-1β, LPS, IFN-γ 65-KDa leucine serine 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. GTP-binding domain(G domain)

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. Clone 1GBP-2 Clone 2 GBP-2 Control 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 Neutrophil http://www.clinsci.org NADPH oxidase complex is a cluster of proteins that donate an electron from NADPH to molecular oxygen (O2) to produce superoxide anion (O2-). This initiates the respiratory burst, a key step in immune defense against bacterial and fungal pathogens

17. Also engulfed with the bacterium will be membrane proteins, including gp91/p22 and FAD Gp91phox large glycosylated protein p22phoxsmaller adapter protein FAD:cofactor Electrons from NADPH are donated to O2 to produce superoxide O2- Soluble components: p47phox P67phox p40phox PKC-mediated phosphorylation p47phox Translocation to the membrane 47phox binding to p22phox Protons dissociated from NADPH proton channels interact with O2- to produce H2O2 Phagosome acidification http://www.caymanchem.com

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

19. Microtubule-associated protein light-chain 3 (LC3)