1. HLA-DQA1 and Celiac Disease Presented by Cassie Bac Image: http://www.scientificamerican.com/article.cfm?id=celiac-disease- insightshttp://www.scientificamerican.com/article.cfm?id=celiac-disease- insights

2. Background on Celiac Disease (CD) Autoimmune disease that creates a toxic immune response to gluten-- damaging the lining of the small intestine Affects 1 in 133 healthy individuals in the United States Can appear at anytime throughout life! No known cure Image: http://www.reallygreatfoods.com/http://www.reallygreatfoods.com/ No wheat, rye, or barley allowed!!

3. What are the classic symptoms of CD? Malnutrition Abdominal cramping Intestinal gas and stomach bloating Chronic diarrhea Weight loss or weight gain Dermatitis Herpetiformis Images: http://healthhype.com; http://www.ehow.com/about_5141982_pediatric-celiac-disease-symptoms.htmlhttp://healthhype.combout_5141982_pediatric-celiac-disease-symptoms.html

4. HLA-DQA1 protein structure cell-cell recognition cell surface receptors immune system cellular interaction

5. How is HLA-DQA1 associated with CD? Peptide Cell Surface Images: http://en.wikipedia.org/wiki/Gluten_immunochemistryhttp://en.wikipedia.org/wiki/Gluten_immunochemistry T-Cell T-cell Receptor DQA1 Antigen Presenting Cell Peptide

6. What is still unknown? What mechanism causes the toxic immune system response in Celiac Disease? Image: http://jaymecarleton.com/secondnature/green-news/2011/04/toxic-toys-r-us/

7. What do we know about gliadin? Gliadin= a protein derived from gluten and the toxic factor involved in Celiac Disease! Question: How does gliadin act??

8. Experimental Approach 1. Chemical Genetics How does gliadin affect HLA- DQA1 expression levels? 2. Phenotype How does gliadin affect the phenotype of the small intestine? 3. Interactions How is gliadin acting at the molecular level?

9. Experimental Approach 1. Chemical Genetics How does gliadin affect HLA- DQA1 expression levels? 2. Phenotype How does gliadin affect the phenotype of the small intestine? 3. Interactions How is gliadin acting at the molecular level?

10. Experimental Approach How does gliadin affect HLA- DQA1 expression levels? 2. Phenotype How does gliadin affect the phenotype of the small intestine? 3. Interactions How is gliadin acting at the molecular level? 1. Chemical Genetics

11. (1) How does gliadin affect HLA-DQA1 expression levels? Method: Inject mouse intestinal cells with gliadin (+ other varieties) and measure HLA-DQA1 expression levels with microarray

12. Why use a mouse model? Mouse protein domains H2-Aa Human protein domains HLA-DQA1 71.8% conserved protein structure Images: From SMART. HLA-DQA1 sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.pl; SMART. H2-Aa sequence search results. Retrieved from http://smart.embl-heidelberg.de/smart/show_motifs.plhttp://smart.embl-heidelberg.de/smart/show_motifs.pl

13. Microarray data http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000& gmax=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1 WT patient expression levels Celiac patient expression levels

14. (1) Hypothesis The addition of gliadin to mouse intestinal cells should show decreased HLA-DQA1 expression.

15. (1) Expected Data Mouse Intestinal Cells HLA-DQA1 expression + +/ - WTWT + gliadin Celiac patient Celiac patient + HLA-DQA1 Celiac patient + Gliadin -- - = High HLA-DQA1 expression (+) = Low HLA-DQA1 expression ( - )

16. (2) How does gliadin affect small intestine phenotype? Method: Observe phenotype of mouse intestinal cells following gliadin treatment

17. What do we know about intestinal cells? Images: http://en.wikipedia.org/wiki/Small_intestine; http://en.wikipedia.org/wiki/Microvillushttp://en.wikipedia.org/wiki/Small_intestine Microvilli line intestinal cell membrane; involved in absorption. Small intestine

18. CD and the small intestine... Image: Children’s Hospital Boston. 2009. http://www.youngmenshealthsite.org/gluten_free_diet.ht ml http://www.youngmenshealthsite.org/gluten_free_diet.ht ml

19. (2) Hypothesis The addition of gliadin to mouse intestinal cells should cause physical damage to the microvilli. In addition, the cells that showed low HLA-DQA1 expression should show increased intestinal damage.

20. (2) Expected Data Mouse intestinal cells WTWT + gliadin Celiac patient Celiac patient + HLA-DQA1 Celiac patient + gliadin Images: http://health.uml.edu/thc/HealthIssues/Meghan_Donovan/Meghans_Homepage.htmlhttp://health.uml.edu/thc/HealthIssues/Meghan_Donovan/Meghans_Homepage.html HLA-DQA1 Expression Levels

21. (3) How is gliadin acting at the molecular level? Method: TAP-tag Label gliadin and determine how it is affecting HLA-DQA1

22. Gliadin may function in this way... Gliadin peptide? Cell Surface T-Cell T-cell Receptor DQA1 Antigen Presenting Cell Gliadin peptide?

23. HLA-DQA1 protein interactions Image: From STRING 8.3. HLA-DQA1 evidence map. Retrieved from: http://stringdb.org/newstring_cgi/show_network_section.pltaskId=8jlOaZSDt oSa&interactive=yes&advanced_menu=_unassigned&network_flavor=evide nc Question: Is gliadin regulating/binding HLA-DQA1?

24. What role do the predicted functional partners play? Question: Is gliadin regulating/binding any of these interacting proteins?

25. (3) Hypothesis Using Tandem Affinity Purification, gliadin should be found to regulate HLA-DQA1 protein at the molecular level. Image: http://www.nature.com/nrm/journal/v4/n1/fig_tab/nrm1007_F1.html

26. (3) Expected Data Mass spectrometry to analyze the protein- protein interactions Gliadin binds HLA-DQA1 Gliadin binds interacting proteins Image: http://www.fda.gov/Food/ScienceResearch/LaboratoryMethod s/DrugChemicalResiduesMethodology/ucm113209.htm http://www.fda.gov/Food/ScienceResearch/LaboratoryMethod s/DrugChemicalResiduesMethodology/ucm113209.htm

27. Future Directions MUDPIT- Multidimensional Protein Identification Technology Hypothesis: HLA-DQA1 protein observed in WT patient tissue samples, not observed in celiac samples. Experiment: Take intestinal tissue samples from WT and celiac patients and observe specific protein identifications from the two samples.

28. Future Directions Microarray primary HLA-DQA1 interacting proteins- look at expression levels in WT vs. Celiac Disease patients. Are their levels expressed in similar patterns as HLA-DQA1?

29. THE END Image: http://www.herbalgranny.com/2009/09/06/herbal-treatment-for-celiac-disease/http://www.herbalgranny.com/2009/09/06/herbal-treatment-for-celiac-disease/

30. References University of Chicago Celiac Disease Center. Celiac Disease Facts and Figures. Print. SMART- http://smart.embl-heidelberg.de/http://smart.embl-heidelberg.de/ GeneBee- http://www.genebee.msu.su/genebee.htmlhttp://www.genebee.msu.su/genebee.html http://knol.google.com/k/celiac ‐ disease# http://knol.google.com/k/celiac ‐ disease# Bizzaro, N., R. Tozzoli, D. Villalta, M. Fabris, and E. Tonutti. "Cutting ‐ Edge Issues in Celiac Disease and in Gluten Intolerance." Clinical Reviews in Allergy and Immunology 15.1 (1997): n. pag. Web. 7 Feb 2011http://www.springerlink.com.ezproxy.library.wisc.edu/content/j17wu631711u1027/fulltext.pdf.http://www.springerlink.com.ezproxy.library.wisc.edu/content/j17wu631711u1027/fulltext.pdf http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001280 PubChem- http://pubchem.ncbi.nlm.nih.gov/http://pubchem.ncbi.nlm.nih.gov/ STRING- http://string.embl.de/http://string.embl.de/ Homologene- http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=AlignmentScores&list_uids=123820 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=homologene&dopt=AlignmentScores&list_uids=123820 Microarrays- http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gma x=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1 http://www.ncbi.nlm.nih.gov/geo/gds/profileGraph.cgi?&dataset=zAAqAB7R9Q&dataset=yLSxM0vrwr$&gmin=108.726000&gma x=3651.000000&absc=&uid=24204411&gds=2042&idref=213831_at&annot=HLA-DQA1