ABSTRACT
Ulcerative colitis (UC) is one of the major forms of inflammatory bowel disease with unknown cause. A molecular marker, WAFL, has recently been found to be up-regulated in the inflamed colonic mucosa of UC patients. Towards understanding biological function of WAFL, we analyzed proteins interacting with WAFL in HEK-293 cells by immunoprecipitation and mass spectrometry. Among four proteins found to specifically interact with WAFL, both KIAA0196 and KIAA1033 bind to alpha-appendage of the adaptor protein complex 2 (AP2), which acts as an interaction hub for accessory proteins in endocytosis mediated by clathrin-coated vesicle (CCV). The specific interaction between WAFL and KIAA0196 was also confirmed in human colorectal carcinoma HCT-116 cells by co-immunoprecipitation with specific antibodies. Meta-analyses of the databases of expressed genes suggest that the three genes are co-expressed in many tissues and cell types, and that their molecular function may be classified in the category of 'membrane traffic protein'. Therefore, these results suggest that WAFL may play an important role in endocytosis and subsequent membrane trafficking by interacting with AP2 through KIAA0196 and KIAA1033.
Subject(s)
Colitis, Ulcerative/pathology , Endocytosis , Tacrolimus Binding Proteins/metabolism , Transcription Factor AP-2/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Cell Line , Cell Line, Tumor , Chromatography, Liquid/methods , Colorectal Neoplasms/pathology , Humans , Macrophages/metabolism , Mass Spectrometry/methods , Models, Biological , Proteins/metabolism , Proteomics/methods , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
We have previously identified a new gene with sequence homology to the WASP-family of actin regulators denoted WAFL (WASP and FKBP-like). Here we report a possible biological function for WAFL, by demonstrating an association to early endosomes via its central coiled-coil domain. Further we show by functional and structural studies that WAFL is associated with both microtubules and the actin filament system, the two means of transport of early endosomes. In addition, WAFL interacts with WASP-interacting protein (WIP) and actin, thus linking WAFL to actin dynamics. The use of RNAi depletion of WAFL shows that WAFL-deficient cells display delayed transport of endosomal cargo. Our findings are compatible with a model whereby WAFL is involved in the transport of early endosomes at the level of transition between microfilament-based and microtubule-based movement.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Endocytosis/physiology , Microtubules/metabolism , Tacrolimus Binding Proteins/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Animals , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Endosomes/metabolism , HeLa Cells , Humans , Mice , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tacrolimus Binding Proteins/chemistry , Tacrolimus Binding Proteins/genetics , Thiazolidines/metabolism , Wiskott-Aldrich Syndrome Protein Family/chemistry , Wiskott-Aldrich Syndrome Protein Family/genetics , Wiskott-Aldrich Syndrome Protein, Neuronal/genetics , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolismABSTRACT
BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) is a complex inflammatory disease of the gastrointestinal tract with unknown cause that lacks molecular markers for diagnosis. Crohn's disease (CD) and ulcerative colitis (UC) are the two major forms of IBD. The aim of this study was to investigate gene expression patterns in UC and characterize newly identified marker genes potentially linked to disease pathogenesis of UC. MATERIALS AND METHODS: Biopsies were taken from eight UC patients, from inflamed and non-inflamed parts of the colon. Gene expression was investigated by subtractive suppression hybridization (SSH), and further study of a selected gene was performed by Northern blot, immunohistochemistry, immunocytochemistry, and in vitro monocyte differentiation. RESULTS: Three hundred thirty-one differentially expressed genes were found and classified into functional groups. In this paper, we report one gene with unknown function to be differentially expressed in UC but not Crohn's disease by real-time reverse transcriptase polymerase chain reaction. Due to its predicted protein architecture, we call this gene Wiskott-Aldrich syndrome protein and FKBP-like (WAFL). Initial pilot experiments suggest WAFL to participate in innate immune functions. CONCLUSION: The SSH result supports the current view of UC to be a chronic inflammatory disorder with aberrant expression of epithelial barrier proteins, cell fate-related factors, and disturbed metabolism. The new gene, WAFL, reported in this study, appears to be conditionally regulated in myeloid cells. This indicates that WAFL may be connected to innate immune-host responses. As such, it represents an interesting, hitherto unknown player in IBD where there is a need for further elucidation on the molecular and cellular level.