Scientific Abstract

Proposal No.  IBD-0119R2
Principal Investigator:  Xuhang Li, Ph.D.
Applicant Organization:  Johns Hopkins University (Baltimore, Maryland, U.S.A.)
Project Title:  Initial proteomic studies of inflammatory bowel diseases
Period of Award:  January 1, 2005 – December 31, 2007

Inflammatory bowel diseases (IBD) are chronic and frequently disabling intestinal inflammatory disorders that affect more than a million Americans.  Initial proteomic studies of IBD patients with ulcerative colitis (UC) and Crohn’s disease (CD) are proposed. The hypothesis to be tested is that by identifying proteins that are differentially expressed or modified in IBD vs. normal intestine, we will increase our current understanding of the pathogenesis of IBD and of the associated diarrhea.  Using 1-D and 2-D gel electrophoresis, mass spectrometry and Western blot screening, our preliminary studies have demonstrated that: 1) a large number of proteins are differentially expressed in IBD intestinal mucosa vs. normal mucosa and some of these protein have been identified; and 2) three membrane transport proteins, Na⁺/H⁺ exchanger 3 (NHE3), ClC Cl^- channel 5 (ClC5) and Na⁺/K⁺-ATPase, are significantly downregulated in IBD mucosa, suggesting a potential explanation for IBD-associated diarrhea.

We propose in this application to extend our preliminary studies with two specific aims:

AIM 1.  Identify proteins that are changed in expression and post-translational modification in the intestinal mucosa of patients with active UC or CD compared to i) uninvolved intestinal mucosa from the same patients, ii) normal intestinal mucosa in control subjects, and iii) infectious/inflammatory colitis (C. difficile colitis).  To increase the chances of identifying changes in relatively low abundant proteins, we will first separate total cellular proteins into several subfractions according to their detergent-solubility and the sizes of protein complexes.  Protein expression and modification will then be analyzed by either 1-D/2-D gel/DIGE/MS technology (DIGE, Differential Image Gel Electrophoresis; MS, Mass Spectrometry), or electrospray LC-MS/MS (liquid chromatography MS/MS), from either total mucosa, or villus and crypt cells or lamina proprial cells isolated by laser capture microdissection (LCM).

AIM 2.  Identify changes in the expression of intestinal membrane transporters for Na absorption and Cl secretion, including NHE3, in the intestinal mucosa of patients with active UC or CD compared to i) uninvolved intestinal mucosa from the same patients, ii) normal intestinal mucosa in control subjects, and iii) infectious/inflammatory colitis (C. difficile colitis). Transporters of interest will be identified using large-scale targeted screening by Western blot analysis.  The targeted screening will also include several intestinal epithelial brush border-associated PDZ-containing proteins that have been recently shown to regulate trafficking and activity of membrane transporters. 

The ultimate goal is to identify potential diagnostic biomarkers and therapeutic targets to cure IBD or alleviate the symptoms of IBD.