Identification and characterization of WASP and FKBP-like protein

Abstract

Ulcerative colitis (UC) is a chronic inflammation of the colon which together with the related Crohn s disease (CD) represent the major forms of Inflammatory Bowel Disease (IBD). IBD is believed to arise in a dysfunctional intestinal barrier which allows the normal gut flora to trigger an unwanted chronic immune response in genetically susceptible individuals. The cause of these illnesses is unknown, and treatment today is solely to alleviate the symptoms.

In order to identify biomarkers that could help us to further our understanding of the pathophysiology of UC, we applied the subtractive suppression hybridization (SSH) method to identify UC-differentially regulated genes in colonic mucosal biopsy specimens (Paper I). The 331 differentially expressed genes were grouped into functional categories and when analyzed revealed a gene expression pattern consistent with UC as an inflammatory disorder with altered epithelial homeostasis. Interestingly, 21 genes were found to be involved in membrane trafficking. Moreover, 37 of the 331 genes were unknown genes with no predicted function. Based on the sequence homology to the WASP-protein (connected to the immunodeficiency disorder Wiskott-Aldrich syndrome) one unknown gene was selected for further investigation. We named the gene WAFL. While the initial characterization of WAFL revealed a broad tissue expression, qPCR analysis indicated that WAFL was upregulated in inflamed tissue of UC compared to inflamed tissue from CD patients (i.e. consistent with the SSH data). To learn about the function of this protein we initiated a detailed investigation. One approach was to obtain the structure of the protein.

In Paper II, the expression of regions of WAFL in E.coli, and in insect cells via a baculoviral system, is described. These peptides were purified and were found to be folded correctly. They have subsequently been used for functional tests of WAFL.

The major finding in my thesis, Paper III, links WAFL to regulation of early endosomes. WAFL interacts with actin and WASP-interacting protein (WIP), which implies a role in actin-based transport of the endosomes. Moreover, cells depleted of WAFL by RNAi exhibited a disturbed transport of endocytic cargo towards lysosomes. Protein mapping experiments showed that the endosome localization is mediated by WAFL s central coiled-coil domain. This domain of WAFL binds to the integral membrane components monophosphorylated phosphatidylinositols, in particular PtdIns(3)P which is found on early endosomes. Interestingly, WAFL appears also on the phagosome of invasive bacteria such as Salmonella, Yersinia, and Shigella indicating that WAFL may be involved in the phagocytosis process. The observation that induced expression is observed in macrophages support this possibility.

In conclusion, we have identified and characterized a novel gene, WAFL, from UC bioptic material. This interesting gene is upregulated in inflamed tissue of UC patients and has potential functions in the correct processing of phagosomes and early endosomes.