Kalidas Ramamoorthy, Subrata Sabui, George Kim, James M Flekenstein, Alaullah Sheikh, Hamid M Said
{"title":"IQGAP-2：人类结肠硫胺素焦磷酸转运体（hcTPPT）的新型相互作用伙伴。","authors":"Kalidas Ramamoorthy, Subrata Sabui, George Kim, James M Flekenstein, Alaullah Sheikh, Hamid M Said","doi":"10.1152/ajpcell.00484.2024","DOIUrl":null,"url":null,"abstract":"<p><p>The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B<sub>1</sub> (i.e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine, and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (<i>P</i> < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (<i>P</i> < 0.01 and <i>P</i> < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.<b>NEW & NOTEWORTHY</b> This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology.</p>","PeriodicalId":7585,"journal":{"name":"American journal of physiology. 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Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (<i>P</i> < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (<i>P</i> < 0.01 and <i>P</i> < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.<b>NEW & NOTEWORTHY</b> This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology.</p>\",\"PeriodicalId\":7585,\"journal\":{\"name\":\"American journal of physiology. 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IQGAP-2: a novel interacting partner with the human colonic thiamin pyrophosphate transporter.
The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B1 (i.e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine, and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (P < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (P < 0.01 and P < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.NEW & NOTEWORTHY This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology.
期刊介绍：
The American Journal of Physiology-Cell Physiology is dedicated to innovative approaches to the study of cell and molecular physiology. Contributions that use cellular and molecular approaches to shed light on mechanisms of physiological control at higher levels of organization also appear regularly. Manuscripts dealing with the structure and function of cell membranes, contractile systems, cellular organelles, and membrane channels, transporters, and pumps are encouraged. Studies dealing with integrated regulation of cellular function, including mechanisms of signal transduction, development, gene expression, cell-to-cell interactions, and the cell physiology of pathophysiological states, are also eagerly sought. Interdisciplinary studies that apply the approaches of biochemistry, biophysics, molecular biology, morphology, and immunology to the determination of new principles in cell physiology are especially welcome.
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