Association of TM4SF4 with the human thiamine transporter-2 in intestinal epithelial cells
| Autor: | Subramanian, Veedamali S, Nabokina, Svetlana M, Said, Hamid M |
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| Rok vydání: | 2014 |
| Předmět: |
Vitamin B-1
Microscopy Cultured Membrane Glycoproteins Gastroenterology & Hepatology Duodenum Blotting Cells 1.1 Normal biological development and functioning Clinical Sciences Yeast split-ubiquitin two-hybrid Membrane Transport Proteins Real-Time Polymerase Chain Reaction Intestine Protein-protein interaction Underpinning research Confocal Humans hTHTR-2 Thiamine Generic health relevance Intestinal Mucosa Western Biomarkers Gene Library |
| Zdroj: | Digestive diseases and sciences, vol 59, iss 3 |
| Popis: | BackgroundThe human thiamine transporter-2 (hTHTR-2) is involved in the intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology.AimsThe aim of this study was to identify protein partner(s) that interact with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction.MethodsThe yeast split-ubiquitin two-hybrid approach was used to screen a human intestinal cDNA library. GST-pull-down and cellular co-localization approaches were used to confirm the interaction between hTHTR-2 and the associated protein(s). The effect of such an interaction on hTHTR-2 function was examined by (3)H-thiamine uptake assays.ResultsOur screening results identified the human TransMembrane 4 SuperFamily 4 (TM4SF4) as a potential interactor with hTHTR-2. This interaction was confirmed by an in vitro GST-pull-down assay, and by live-cell confocal imaging of HuTu-80 cells co-expressing hTHTR-2-GFP and mCherry-TM4SF4 (the latter displayed a significant overlap of these two proteins in intracellular vesicles and at the cell membrane). Co-expression of hTHTR-2 with TM4SF4 in HuTu-80 cells led to a significant induction in thiamine uptake. In contrast, silencing TM4SF4 with gene-specific siRNA led to a significant decrease in thiamine uptake.ConclusionsThese results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter. |
| Databáze: | OpenAIRE |
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