NOD2 and Crohn’s Disease Jonathan Peterson. What is Crohn’s Disease(CD)? Intestinal inflammation Immune dysfunction Symptoms – Malnutrition – Dehydration.

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Presentation transcript:

NOD2 and Crohn’s Disease Jonathan Peterson

What is Crohn’s Disease(CD)? Intestinal inflammation Immune dysfunction Symptoms – Malnutrition – Dehydration – Diarrhea – Intestinal blockage

Who is at risk? European Descent Industrialized areas crohns_n_ html#s318538&title=Dwight_D_Eisenhower

What causes CD? Environmental factors – microbiome – “let them eat dirt” Genetics – MZ Twin studies 56% genetic – Complex Disease 71 variants=30% of heritability – 3 strongest in NOD2

But what is NOD2? Nucleotide-binding oligomerization domain-containing protein 2 Intestinal macrophages and epithelial cells Immune response

NOD2 Domains 2 CARD domains 1 NACHT(NOD) domain Leucine Rich repeats

MDP induced oligomerization LRR Domain NACHT Domain Inactive Active

Bacteria Alarm System working-heres-how-to-fix-it/

NOD2 and CD 3 NOD2 variants – SNP8(702 R  W) – SNP12 (908 G  R) – SNP13 (1007 fs) SNP8 SNP13 SNP12

What goes wrong? Faulty MDP recognition leads to altered immune response MDPNOD2 RIP2- kinase NFkB Immune response

Why Inflammation? Contradiction? Main theories – Faulty alarm – NOD2 negative NFkB feedback Still lots of Debate

Why add to the controversy? Address the problem at NOD2 instead Current treatments act downstream of NFkB IDEA: Look for targets upstream – Directly affect NOD2

String Analysis

NLRP1(aka NALP1) NLRP1 Interacts with MDP AND NOD2!!!

NLRC4(aka CLAN) NOD2-CLAN and NLRP1-Clan hetero-oligomers!!!

Common Domains NLRP1 NOD2 CLAN

Main Hypothesis NOD2 oligomerizes with several proteins in MDP dependent fashion, resulting in diverse immune response Altered Immune Response

Experiment 1.Identify potential NOD2 oligomerization partners using BLAST analysis 2.Construct model system 3.Use TAP/MS in presence and absence of MDP to test for interactions. 4.Microarray for expression of interacting proteins

1. Potential NOD2 partners Blast Analysis T-Coffee Alignment NACHT Domain LRR Domain

Proteins identified via BLAST NOD2 NLRC5 NLRC3 CIITA NLRP2 NLRX1 NLRP4 NLRP3

Experiment 1.Identify potential NOD2 oligomerization partners using BLAST analysis 2.Construct model system 3.Use TAP/MS in presence and absence of MDP to test for interactions. 4.Analyze CD Microarray for expression of interacting proteins

Model organism?

NCM460 cell line Model IT’S MADE OF PEOPLE! Epithelial colon cells Transfect with TAP-NOD2 ½ in MDP ½ in Saline TAP

Check expression Lyse transfected cells and run PCR Confirm transfection and normal expression mRNA 2NOD2 3NLRC5 4NLRC3 5CIITA 6NLRP2 7NLRP3 8NLRP4 TAP-NOD2

Experiment 1.Identify potential NOD2 oligomerization partners using BLAST analysis 2.Construct model system 3.Use TAP/MS in presence and absence of MDP to test for interactions. 4.Analyze CD Microarray for expression of interacting proteins

TAP/MS Results Type 1: Type 2: Type 3: RIP2

CD Biopsy Microarray: Expression downstream of Type 1 Type2 Type3 “X” Region of interest

Future Directions Identify affected “X” genes and their pathways More Microarray investigation More TAP GWAS Microbiome research – Fecal transplant

Questions?

References "CCFA.org: About Crohn's Disease." CCFA.org: Home. Web.. Chiodini, RJ. "Crohn's Disease and the Mycobacterioses: A Review and Comparison of Two Disease Entitie." Ameican Society for Microbiology 2 (1989): Print. "Crohn's Disease - Causes, Symptoms & Treatment." EMedicineHealth.com. Web. "Crohn's Disease Symptoms." Tree. Web. disease-symptoms.aspxhttp:// disease-symptoms.aspx Staff, Mayo Clinic. "Crohn's Disease: Causes - MayoClinic.com." Mayo Clinic. Web.. Vignal, Cecile, Elisabeth Singer, Laurent Peyrin-Biroulet, Pierre Desreumaux, and Mathias Chamaillard. "How NOD2 Mutations Predispose to Crohn's Disease?" Microbes and Infection 9.5 (2007): Print. Yazdanyar, Shiva, Maren Weischer, and Borge G. Nordestgaard. "Genotyping for NOD2 Genetic Variants and Crohn Disease: A Metaanalysis." Clinical Chemistry (2009): Print.