IQGAP-2: a novel interacting partner with the human colonic thiamin pyrophosphate transporter.

IF 5 2区 生物学 Q2 CELL BIOLOGY
Kalidas Ramamoorthy, Subrata Sabui, George Kim, James M Flekenstein, Alaullah Sheikh, Hamid M Said
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引用次数: 0

Abstract

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.

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。
人类结肠硫胺素焦磷酸转运体(hcTPPT)在大肠中介导微生物群生成的磷酸化形式的维生素 B1(即硫胺素焦磷酸)的吸收。hcTPPT 沿着吸收道的表达仅限于大肠,而且该转运体只定位在极化上皮细胞/结肠细胞的顶端膜域。以前的研究已经描述了 hcTPPT 系统的不同生理/病理生理学特征,但目前还不知道该转运体是否有影响其生理/生物学的相互作用伙伴。我们利用 Y2H 筛选人类结肠 cDNA 文库来解决这个问题,并确定了 3 个推定的相互作用者,即 IQGAP-2、SNX-6 和 DMXL-1。以 IQGAP-2(其在人类结肠细胞中的表达量最高)为重点,我们发现(使用荧光显微镜成像和共沉淀免疫方法)该推定相互作用因子与 hcTPPT 共定位,并与该转运体直接相互作用。研究还发现,在 NCM460 细胞和人类原代分化结肠单层中过度表达 IQGAP-2 会显著(P 0.01)诱导 TPP 的吸收,而敲除 IQGAP-2(使用基因特异性 siRNAs)则会显著(P < 0.01 和 0.05)降低 TPP 的吸收。此外,我们还发现在 NCM460 细胞中过度表达 IQGAP-2 会显著增强 hcTPPT 蛋白的稳定性。最后,我们发现在对结肠 TPP 吸收有负面影响的条件/因素下,IQGAP-2 的表达受到明显抑制。这些结果确定了 IQGAP-2 是人结肠细胞中 hcTPPT 的相互作用伙伴,并表明这种相互作用具有生理和生物学后果。
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来源期刊
CiteScore
9.10
自引率
1.80%
发文量
252
审稿时长
1 months
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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