Molecular Basis of Propionate Toxicity

描述（由申请人提供）：丙酸酯是一种三碳短链脂肪酸，在土壤和包括人类在内的动物胃肠道中含量非常丰富。丙酸盐被广泛用作食品防腐剂，因为它能抑制致病性和非致病性原核生物和低等真核生物的生长，但它不影响人体细胞。丙酸的抑制作用归因于丙酸的活化形式，即丙酰辅酶A （Pr-CoA）的积累。根据从我们小组获得的已发表和未发表的数据，我们对这一观点提出了挑战。在这个应用中，我们打算研究丙酸毒性的分子基础。我们对数据的解释表明丙酸引起的生长停滞是由克雷布斯循环的柠檬酸合成酶（GltA）酶合成2-甲基柠檬酸的非生理性异构体的结果；我们把这种抑制剂称为2-MCGltA。我们的数据表明，2-MCGltA抑制糖异生酶。缺乏葡萄糖有多种负面影响。例如，细胞壁和核糖的合成被阻断，使细胞对渗透变化敏感，无法产生核酸和辅酶。一个具体的目标集中在鉴定和表征2-MCGltA的目标。我们试图了解2-MCGltA如何抑制靶标的活性，以及2-MCGltA的抑制作用是否具有立体特异性。第二个特定目标集中在鉴定和分析防止丙酸毒性所需的辅助功能，以及参与将丙酸转运到细胞中的蛋白质。公共卫生相关性：本申请中提出的项目将深入了解丙酸盐（一种食品防腐剂）如何在分子水平上对细胞生长产生抑制作用。这项工作与人类健康和营养有关，因为丙酸对致病和非致病微生物都有影响。

DESCRIPTION (provided by applicant): Propionate is a three-carbon short-chain fatty acid that is very abundant in soil and the gastrointestinal tracts of animals, including humans. Propionate is widely used as a food preservative because it inhibits the growth of pathogenic and non-pathogenic prokaryotes and lower eukaryotes, but it does not affect human cells. The inhibitory effects of propionate have been attributed to the build up of the activated form of propionate, i.e., propionyl-coenzyme A (Pr-CoA). We have challenged this idea on the basis of published and unpublished data obtained from our group. In this application we propose to investigate the molecular basis of propionate toxicity. Our interpretation of the data suggests that growth arrest caused by propionate is the result of the synthesis of a non-physiological isomer of 2-methylcitrate by the citrate synthase (GltA) enzyme of the Krebs cycle; we refer to this inhibitor as 2-MCGltA. Our data suggest that 2-MCGltA inhibits an enzyme of gluconeogenesis. The lack of glucose has multiple negative effects. For example, cell wall and ribose syntheses are blocked, rendering the cell sensitive to osmotic changes and unable to make nucleic acids and coenzymes. One specific aim focuses on the identification and characterization of the target of 2-MCGltA. We seek to learn how 2-MCGltA inhibits the activity of the target, and whether the inhibitory effect of 2-MCGltA is stereospecific. A second specific aim focuses on the identification and analysis of ancillary functions needed to prevent propionate toxicity, and proteins involved in transporting propionate into the cell. PUBLIC HEALTH RELEVANCE: The project proposed in this application will provide insights into how propionate (a food preservative) exerts its inhibitory effects on cell growth at the molecular level. This work is relevant to human health and nutrition since propionate affects pathogenic and non-pathogenic microbes alike.