Identification of IQ Motif Containing GTPase Activating Protein 2, IQGAP-2 as an interacting partner with the Human Colonic Thiamin Pyrophosphate Transporter (hcTPPT): Effects on physiology and Cell Biology of the Transporter

Background:Humans/mammals obtain vitamin B1 from two sources: the diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of thiamin pyrophosphate (TPP). We have previously shown that colonocytes process an effcient and specific carrier-mediated process for uptake of the generated TPP that involves the human colonic thiamine pyrophosphate transporter (hcTPPT, product of the SLC44A4 gene). We have also shown that expression of the cTPPT along the GI tract is restricted to the large intestine and have characterized different aspects of its regulation at both the transcriptional and post-transcriptional levels.Aim:To determine whether the hcTPPT has interacting protein partner(s) in human colonocytes that affect its functionality and/or cell biology.Methods: Yeast Two-Hybrid system (Y2H) was used to screen a random-primed normal human colonic cDNA library using the full-length SLC44A4 as a bait. Effect of knocking-down (with gene-specific siRNA) and over-expressing the putative interactor was examined using human-derived colonic epithelial NCM460 cells and human primary differentiated colonoid monolayers (obtained form organ donors). We also used confocal imaging, RT-qPCR and western blotting in our investigations. Results:Our Y2H screening have identified three putative interactors with the hcTPPT, namely: IQGAP-2, SNX-6, and DMXL-1. While all these putative interactors are expressed in human colonocytes, expression of the IQGAP-2 was the highest, and thus, we focused our investigation on this putative interactor. First, we verified the co-localization of IQGAP-2 with the hcTPPT in human colonic NCM460 cells using confocal imaging. We then examined the effect of expressing IQGAP-2 in NCM460 cells and in differentiated colonoids on carrier-mediated TPP uptake and observed significant induction with both models. Knock-down IQGAP-2 (with gene-specific siRNA), on the other hand, led to a significant decrease in TPP uptake by colonocytes. We also found that expressing the IQGAP-2 in colonocytes lead to a marked enhancement in hTPPT protein stability (increased protein half-life). Interestingly, we also found that expression of IQGAP-2 was suppressed by factors/conditions (e.g. lipopolysaccharide, pro-inflammatory cytokine TNF-α and hypoxia, respectively) that inhibit colonic TPP uptake.Conclusion: The accessory protein IQGAP-2 is an interacting partner with the hcTPPT in human colonocytes and that the interaction has physiological/cell biological consequences. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.