GBPs: Immunity to intracellular pathogens

Juliana UedaThais HerreroClaudia Polli

GBP

IFN-γ : Central in host resistance to infection

WT

IFN KO

IFN-γ regulates the expressionof more than 1.200 genes

Products:Only a fraction:Mediators of host immune responsesRemainder: little is known

Mainly produced by:

GTPases GTPases: GTP-binding proteins

NRAMP1: natural-resistance associated macrophage protein 1

Cellular functions of GTPases

Two conformations:

• Bound to GTP: active Hydrolisis GTP GDP and GMP

• Bound t GDP: inactiveGEF (guanine exchange factor)GDP GTPActive GTPase

Cellular functions:Activation of cell-surface receptorsto modulation of membrane-fusion eventsMembrane taffickingCell signaling and migrationTranslation and protein translocationNuclear transport

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

11 mGBPs

7 hGBPs

GBPs: Guanylate-binding proteins

GBPs: Guanylate-binding proteins

Induced by: type I IFN, IL-1β, LPS, IFN-γ65-KDa

GTP-binding domain(G domain)

Isoprenylation:Addition of either a C15 farnesyl orC20 geranygeranyl lipid to the C-terminus of the GBPs

Isoprenylation is important in targeting proteinsto intracellular membranes and/or to facilitate protein/protein interactions

GBPs are predominantly cytosolic and have,at most, a relatively small portion of the totalamount associated with membranes.

leucine

serine

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

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

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

Functions of GBPs

Antiviral activity

HeLa cellshGBP-1SVS, EMCV m.o.i 1

mGBP-2S52N: single point mutation GTP binding regionSVS, EMCV m.o.i 0.1

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

Functions of GBPs

Response to Protozoan infections

MEFs

Functions of GBPs

Response to Bacterial infections

Functions of GBPs

Antibacterial activity

Thais

Claudia

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

Neutrophil

NADPH oxidase

http://www.clinsci.org

http://www.caymanchem.com

Gp91phox large glycosylated proteinp22phoxsmaller adapter proteinFAD:cofactor

Soluble components:p47phox

P67phox

p40phox

PKC-mediated phosphorylation p47phox

Translocation to the membrane47phox binding to p22phox

Electrons from NADPH are donated to O2 to produce superoxide O2

-

Protons dissociated from NADPH proton channels interact with O2

- to produce H2O2

Phagosome acidification

Also engulfed with the bacterium will be membrane proteins, including gp91/p22 and FAD

Second antimicrobial pathway…Nonoxidative mechanisms:

http://www.fbs.osaka-u.ac.jp

Microtubule-associated protein light-chain 3 (LC3)

Antioxid Redox Signal. 2009 August; 11(8): 1975–1988.