Tomoo Keigo, Zhang Yi, Mohallem Rodrigo, Aryal Uma K, Klauda Jeffery B, Henderson Gregory C
Department of Nutrition Science, Purdue University, West Lafayette, Indiana, USA.
Department of Comparative Pathobiology, Purdue University, West Lafayette, Indiana, USA.
FASEB J. 2025 Jun 30;39(12):e70732. doi: 10.1096/fj.202501328R.
Free fatty acid (FFA) transport through the bloodstream had been believed to mainly depend on binding to albumin. However, we recently discovered that albumin knockout (Alb) mice still have meaningful levels of FFAs in the bloodstream, indicating the presence of other FFA carrier proteins in plasma. To identify these proteins, we collected plasma from wildtype (WT) and Alb mice. Plasma proteins that can bind to FFAs were isolated by a fatty acid pulldown assay and identified by proteomic analysis. Additionally, plasma proteins were separated by fast protein liquid chromatography (FPLC), and the FPLC fractions were used to measure FFAs, cholesterol, triacylglycerol, and plasma proteins of interest. Furthermore, in silico docking simulations were used to identify potential FFA binding sites on the more highly expressed proteins isolated by the fatty acid pulldown assay to ascertain the likelihood that these proteins may have binding sites for FFA. Proteomic analysis on proteins isolated by the pulldown assay detected 202 proteins, collectively termed the FFA transportome. Examples of highly expressed proteins that showed FFA binding in the assay include albumin, transferrin, and apolipoprotein A-I. Notably, FFA levels in the FPLC fractions positively correlated with albumin, transferrin, HDL cholesterol, and Apo AI levels. The docking results further suggested that indeed many of the discovered proteins may have FFA binding sites. In conclusion, we elucidated the FFA transportome in mice, and this collection of FFA carrier proteins may facilitate FFA transport through the bloodstream along with albumin.
游离脂肪酸（FFA）通过血液循环的运输一直被认为主要依赖于与白蛋白的结合。然而，我们最近发现，白蛋白基因敲除（Alb）小鼠的血液中仍有相当水平的游离脂肪酸，这表明血浆中存在其他游离脂肪酸载体蛋白。为了鉴定这些蛋白，我们收集了野生型（WT）小鼠和Alb小鼠的血浆。通过脂肪酸下拉实验分离出能与游离脂肪酸结合的血浆蛋白，并通过蛋白质组学分析进行鉴定。此外，通过快速蛋白质液相色谱（FPLC）分离血浆蛋白，并用FPLC级分来测量游离脂肪酸、胆固醇、三酰甘油以及感兴趣的血浆蛋白。此外，利用计算机对接模拟来确定通过脂肪酸下拉实验分离出的高表达蛋白上潜在的游离脂肪酸结合位点，以确定这些蛋白可能具有游离脂肪酸结合位点的可能性。对通过下拉实验分离出的蛋白进行蛋白质组学分析，检测到202种蛋白，统称为游离脂肪酸转运组。在实验中显示出游离脂肪酸结合能力的高表达蛋白实例包括白蛋白、转铁蛋白和载脂蛋白A-I。值得注意的是，FPLC级分中的游离脂肪酸水平与白蛋白、转铁蛋白、高密度脂蛋白胆固醇和载脂蛋白AI水平呈正相关。对接结果进一步表明，确实许多已发现的蛋白可能具有游离脂肪酸结合位点。总之，我们阐明了小鼠的游离脂肪酸转运组，并且这组游离脂肪酸载体蛋白可能与白蛋白一起促进游离脂肪酸在血液循环中的运输。
