Institute of Molecular and Cellular Biology FACULTY OF BIOLOGICAL SCIENCES

Regulation of NOD Signalling by Optineurin

John Short, Summer Placement Student;

Dr Andrew Macdonald

Introduction

NOD Signalling

Bacterium Peptidoglycan

Invasion, Endocytosis

NOD Receptors

Cell

• Nucleotide binding Oligomerisation Domains (NODs) are prevalent in Intestinal epithelial cells and macrophages

• Intracellular bacterial infection by E. coli, S. flexneri, S. typhimurium, L. monocytogenes and H. pylori activate the NODs

• NODs are cytosolic receptors that detect components of Peptidoglycan of Gram +ve and Gram -ve bacterial cell walls Nucleus

Introduction

NOD Signalling

• Activation of NOD1 by γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 by muramyl dipetide (MDP) induces a signalling cascade that activates the MAP kinase and NF-κB pathways.

• This leads to the activation of transcription factors that mediate gene expression of adaptive and innate immunity e.g. cytokines etc.

Diagram modified from Franchi et al., 2006

Introduction

NOD Signalling: A role for Optineurin?

• Originally found in the eye and implicated in glaucoma

• Optineurin is ubiquitously expressed in the body

• It is negative regulator of the TNFα pathway, binding to poly-ubiquitinylated RIP1 and acting as a competitive inhibitor for downstream pathway components

Diagram taken from Chalasani et al., 2008

Introduction

NOD Signalling: A role for Optineurin?

Diagram taken from Zhu et al., 2007

(B) Vectors expressing wild-type optineurin and/or RIP1 with reporter constructs were cotransfected into HEK293 cells, and reporter assays were performed.

• RIP1 is homologous to RIP2 and is an adaptor protein in TNFα signalling

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Introduction

Therapeutic Applications

• Genetic variation in NOD2 is associated with susceptibility to several inflammatory diseases.

• Crohn’s disease (CD), a chronic inflammatory disorder of the intestinal wall, is associated with three common mutations (R702W, G908R, and L1007insC) involving amino acid residues near or within the Leucine Rich Repeat Regions of NOD2

•There is no cure for Crohn’s disease

• Characterisation of this pathway and possible role of Optineurin could lead to therapeutic applications for NOD2 implicated diseases.

Introduction

Project Strategy

1.To set up reporter systems at the levels of:

- Transcriptional (Luciferase reporter assays)

- Post translational (Western Blots of cell lysates)

2.To set up delivery systems for gene of interest using appropriate cell lines:

- HEK293 for overexpression

- HT29/ RAW macrophages for endogenous protein expression

- HEK293T for lentivirus delivery system

Results

Proof of PrincipleNf-κB luciferase assay, HEK293 cells

• Overexpression of NOD2 enhances Nf-κB signalling

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Control NOD2

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Results

Western Blot of MDP/ PGN time course, RAW macrophages

MDP PGN LPS C 15 30 60 180

TRAF2

55.8kDa

C 15 30 60 180

50kDa

P-TAK1 76kDa 75kDa

P-P38

41kDa 36kDa

P-ERK

45kDa50kDa

P-RIP2

S180/181

59kDa 50kDa

P-cJun S63

41kDa 36kDa

Results

What role for Optineurin?Nf-κB luciferase assay, HEK293 cells

• Shows that Optineurin inhibits NOD2 signalling

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Control NOD2 NOD2 +Optn

Results

NOD1 response to MDP/PGNNf-κB luciferase assay, HEK293 cells

• Optineurin inhibited MDP and PGN stimulated NOD1 signalling

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CONTROL

NOD1

NOD1+

MDP

NOD1+

PGN

C+OPT

NOD1+

OPT

NOD1+

OPT+M

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NOD1+

OPT+PGN

Results

NOD2 response to MDP/PGNNf-κB luciferase assay, HEK293 cells

• Optineurin inhibited MDP and PGN stimulated NOD2 signalling

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CONTROL

NOD2

NOD2+

MDP

NOD2+

PGN

C+OPT

NOD2+

OPT

NOD2+

OPT+M

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NOD2+

OPT+PGN

Results

Defining the mechanism of actionNf-κB luciferase assay, HEK293 cells

• RIP1 is a positive control, results confirmed by literature• Optineurin inhibits TRAF2, RIP2 and RIP1 signalling

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Control RIP1 RIP2 TRAF2

Results

Defining the mechanism of actionNf-κB luciferase assay, HEK293 cells

• Both Optineurin WT and Optineurin D474N mutant inhibited RIP2 signalling• N=1 for the RIP2 + Optineurin D474N mutant

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control RIP2 RIP2+OPTN WT RIP2+OPTN DN

Summary

- Optineurin disrupts NOD1 and NOD2 signalling

- NOD1 and NOD2 signalling are stimulated by MDP and PGN, that are disrupted by Optineurin

- Various MAP kinases of the pathway and RIP2 are activated by MDP and PGN stimulation:

Question: is this downregulated in Optineurin cells?

- RIP2 signalling is inhibited by Optineurin and Optineurin D474N mutant (N=1)

Future Work

- Repeat RIP2 and Optineurin D474N experiments and expand to include Optineurin truncations (1-127, 127-577)

- Repeating RAW macrophage MDP and PGN stimulation timecourse but including Optineurin using lentivirus delivery system.

- Repeating MDP and PGN stimulations for 24hrs (Windheim et al. 2007)

- Pulldown experiments: Determining interactions between NOD1/2, RIP2 and Optineurin and in response to MDP/PGN stimulation

- Longer term : looking at NEMO interactions with Optineurin

: examining effects of bacterial infections of cells

Acknowledgements

Dr Andrew Macdonald

Adam Dale

Gabriel Nunez (University of Michigan, USA) (NOD1 and NOD2 plasmids)

Virology Group

Structural and Cellular Biology Group

Funding: