Association of TM4SF4 with the Human Thiamine Transporter-2 in Intestinal Epithelial Cells

Abstract

Background The 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. Aims The aim of this study was to identify protein partner(s) that interacts with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction. Methods The 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 3H-thiamine uptake assays. Results Our 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. Conclusions These results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter.

Distinct metabolic network states manifest in the gene expression profiles of pediatric inflammatory bowel disease patients and controls

“…Along the same vein, the TM4SF4 gene encodes a transmembrane protein that stimulates thiamine resorption in intestinal epithelial cells32. Thiamine, in turn, is an essential component of several co-enzyme complexes, including pyruvate dehydrogenase that catalyzes the formation of Acetyl CoA as a first step in fatty acid synthesis.…”

“…IL6 is a cytokine, known to promote intestinal inflammation, that has a clear role in fatty acid metabolism, for example, by stimulating apolipoprotein (a) expression and lipoprotein (a) synthesis in hepatocytes 31 . Along the same vein, the TM4SF4 gene encodes a transmembrane protein that stimulates thiamine resorption in intestinal epithelial cells 32 . Thiamine, in turn, is an essential component of several co-enzyme complexes, including pyruvate dehydrogenase that catalyzes the formation of Acetyl CoA as a first step in fatty acid synthesis.…”

Multiomics data analysis identifies TM4SF4 as a cell surface target in hepatocellular carcinoma

“…TM4SF4, originally identified in human intestine epithelium and liver tissues [94], is specifically expressed in the gastrointestinal tract and pancreas. In the intestines, TM4SF4 plays physiological roles in the absorption and uptake of thiamine by interacting with the human thiamine transporter-2 [95], and in the liver, TM4SF4 is required for regenerating liver cells through regulation of cellular proliferation [96]. In the pancreas, TM4SF4 is highly expressed in pancreatic α cells, and the protein is used as a cell surface marker to differentiate pancreatic α from β cells [97, 98].…”

Identification of transmembrane protein 237 as a novel interactor with the intestinal riboflavin transporter-3 (RFVT-3): role in functionality and cell biology

“…The human-derived intestinal epithelial HuTu-80 cells grown on glass-bottomed petri dishes (MatTek, Ashland, MA) were transiently cotransfected with GFP-hRFVT-3 and TMEM237-mCherry constructs (3 g each plasmid) at 70 -80% confluency using Lipofectamine 2000. Live cell confocal imaging was performed 48 h after transfection as described previously (38,39). Briefly, fluorophores were excited by using a 488-nm line from an argon ion laser, and a 543-nm line from a HeNe ion laser-emitted fluorescence was monitored with a 515 Ϯ 30 nm band-pass (GFP) or a 630 Ϯ 60-nm long-pass (mCherry) filter.…”