Intestinal Biotin Absorption: Physiological, Cellular and Molecular Aspects

描述（由申请人提供）：本次续期申请的长期目标是继续我们对水溶性维生素生物素肠道吸收过程的生理学和细胞/分子生物学的研究。我们还旨在描述受慢性酒精消耗影响的肠道生物素吸收过程的细胞和分子参数。生物素是正常细胞功能所必需的，缺乏生物素会导致各种临床异常。人类和其他哺乳动物不能合成生物素，因此必须通过肠道吸收获得维生素。在目前的资助期内的研究已经描述了肠道生物素摄取过程的许多方面。这包括在体外和体内对人类SLC5A6基因（编码生物素转运体hSMVT的基因）的5'调控区进行表征，描述hSMVT的细胞内转运和膜靶向机制，以及确定生物素缺乏时肠道生物素摄取过程上调的分子机制。我们实验室先前使用基因特异性siRNA进行的体外研究表明，hSMVT的重要作用是培养的人肠上皮Caco-2细胞对生物素的摄取。SMVT是否在体内的天然肠道中发挥同样的作用尚不清楚。因此，在旨在产生条件SMVT缺陷（敲除）小鼠的新的初步研究中，我们已经产生了杂合子Slc5a6 loxP小鼠，目前正在使用它们产生纯合子Slc5a6 loxP小鼠，然后将用于产生全局和肠道特异性SMVT敲除小鼠。在其他初步研究中，利用酵母双杂交系统筛选人类肠道cDNA文库，并鉴定出与hSMVT相互作用的两个蛋白（PDZD11和EPAS1）。此外，获得的分子证据表明，特定的组氨酸残基参与hSMVT功能。最后，发现慢性酒精喂养小鼠导致肠道SMVT mRNA水平显著降低。我们的目的是：1)表征Slc5a6基因敲除小鼠肠道生物素吸收过程，即确定SMVT在体内肠道生物素吸收中的作用；2)鉴定与hSMVT相互作用并影响其生理/生物学的辅助蛋白；3)调查hSMVT系统的结构-功能/监管方面；4)研究慢性饮酒对肠道生物素转运生理/分子参数的影响。这些研究的结果将继续为肠道生物素摄取的生理/生物学和影响这一过程的因素提供新的和有价值的信息。这将最终帮助我们设计有效的策略来优化生物素体内稳态，特别是在生物素缺乏和次优水平的情况下。

DESCRIPTION (provided by applicant): The long-term objective of this renewal application is to continue our investigations into the physiology and cell/molecular biology of the intestinal absorption process of the water-soluble vitamin biotin. We also aim at delineating the cellular and molecular parameters of the intestinal biotin absorption process that are affected by chronic alcohol consumption. Biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Humans and other mammals cannot synthesize biotin, and thus, must obtain the vitamin via intestinal absorption. Studies during the current funding period have characterized many aspects of the intestinal biotin uptake process. This include characterization of the 5' -regulatory region of the human SLC5A6 gene (the gene that encodes the biotin transporter hSMVT) both in vitro and in vivo, delineation of the mechanisms involved in intracellular trafficking and membrane targeting of hSMVT, and determination of the molecular mechanism involved in the up-regulation of intestinal biotin uptake process in biotin deficiency. Previous in vitro studies from our laboratory using gene-specific siRNA have suggested an important role for hSMVT is biotin uptake by cultured human intestinal epithelial Caco-2 cells. Whether SMVT plays the same role in native intestine in vivo is not clear. Thus, in new preliminary studies aimed at generating conditional SMVT deficient (knockout) mice, we have already generated heterozygote Slc5a6 loxP mice and are currently using them to generating homozygous Slc5a6 loxP mice that will then use to generate global and intestinal-specific SMVT knockout mice. In other preliminary studies, a yeast two-hybrid system was used to screen a human intestinal cDNA library and two proteins (PDZD11 and EPAS1) that interact with hSMVT were identified. Furthermore, molecular evidence was obtained to suggest the involvement of specific histidine residues in hSMVT function. Finally, chronic alcohol feeding of mice was found to lead to a significant reduction in intestinal SMVT mRNA level. Our aims in this proposal are: 1) to characterize the intestinal biotin absorption process in Slc5a6 knockout mice, i.e., to determine the role of SMVT in intestinal biotin absorption in native intestine in vivo; 2) To identify accessory protein(s) that interacts with hSMVT and influences its physiology/ biology; 3) To investigate structure - function/regulatory aspects of the hSMVT system; and 4) to examine the effect of chronic alcohol consumption on physiological/molecular parameters of intestinal biotin transport. Results of these studies should continue to provide novel and valuable information regarding the physiology/biology of intestinal biotin uptake and the factors that affect the process. This should ultimately assist us in the designing of effective strategies to optimize biotin body homeostasis, especially in conditions associated with biotin deficiency and sub-optimal levels. PUBLIC HEALTH RELEVANCE: Humans and other mammals cannot synthesize the vitamin biotin (an essential micronutrient for normal health) and thus, must obtain it from exogenous sources via intestinal absorption. The aims of this proposal since its inception ten years ago continue to be the delineation of the mechanisms involved in intestinal biotin absorption, how the process is regulated, and what factors affect its function leading to deficiency and sub-optimal levels. Results of these investigations should help in the designing of effective strategies to optimize biotin body levels, especially in conditions associated with biotin deficiency and sub-optimal levels. 1