Characterization of the Biochemical Activities Associated with Human FATP2

描述(由申请人提供)：脂肪酸运输蛋白(FATP)是一个完整的膜蛋白家族，其中几个在外源脂肪酸通过质膜的运输和激活中发挥作用。FATP2在肠道和肝脏中表达，但它在这些细胞类型的正常脂肪酸稳态中的生理作用尚未确定。我们已经确定了人类FATP2的两个剪接变体，它们表现出不同的生化活性。第一种形式(FATP2a)也被描述为超长链酰基辅酶A合成酶，其MR为70,000，而从Image Consortium文库获得的第二种形式(FATP2b)的MR为65,000。FATP2b缺乏外显子3，外显子3编码蛋白质的一个区域，该区域包含部分ATP结合结构域，是腺苷形成所必需的。这两种形式的FATP2都精通脂肪酸的运输，但FATP2b无法激活超长链脂肪酸。因此，这些研究定义了FATP家族成员的两个自然发生的变体，它们在脂肪酸运输和激活中的作用有所不同。使用293 T-Rex细胞，它允许FATP2a和FATP2b的调控表达，实验将完成[1]确定它们在脂肪酸运输和激活中的单独作用，并[2]解决它们是否将脂肪酸引导到不同的代谢池中，如使用稳定的同位素标记脂肪酸结合高分辨率质谱仪进行评估。为了了解FATP2在肠道和肝脏中的生理作用，shRNA将被用来敲除Caco-2(肠样)和HepG2(肝样)细胞中FATP2的表达，并评估脂肪酸运输、激活和细胞内运输的模式。将使用从高通量筛选中选择的五种小分子抑制剂来进一步评估这两种变体的单独作用，筛选出干扰脂肪酸运输的小化合物。总而言之，这些研究有望为FATP2a和FATP2b在脂肪酸动态平衡中的不同作用提供新的见解。血脂紊乱已被证明是糖尿病、肥胖症和心血管疾病等代谢性疾病的一个促成因素。鉴定参与脂类代谢的蛋白质有助于阐明支配这些病理状态的生物过程。这项研究计划的重点包括表征FATP2的功能，这有望深入了解支撑脂质代谢的生物过程。

DESCRIPTION (provided by applicant): Fatty acid transport proteins (FATPs) are a family of integral membrane proteins, several of which function in the transport and activation of exogenous fatty acids across the plasma membrane. FATP2 is expressed in the intestine and liver, yet its physiological role in normal fatty acid homeostasis in these cells types has not been defined. We have identified two splice variants of human FATP2, which exhibit distinguishing biochemical activities. The first form (FATP2a), also described as a very long chain acyl CoA synthetase has a Mr of 70,000 while the second form (FATP2b) obtained from the Image Consortium library has a Mr of 65,000. FATP2b lacks exon3, which encodes a region of the protein that contains part of the ATP binding domain and required for adenylate formation. Both forms of FATP2 are proficient in fatty acid transport yet FATP2b is unable to activate very long chain fatty acids. These studies thus define two naturally occurring variants of a member of the FATP family that distinguish their roles in fatty acid transport and activation. Using 293 T-REx cells, which allows regulated expression of FATP2a and FATP2b, experiments will be completed to [1] define their individual roles in fatty acid transport and activation and [2] address whether they direct fatty acids into different metabolic pools as assessed using stable isotopically-labeled fatty acids in combination with high resolution mass spectrometry. To access the physiological roles of FATP2 in the intestine and liver, shRNA will be used to knockdown FATP2 expression in Caco-2 (intestinal like) and HepG2 (hepatic like) cells, and patterns of fatty acid transport, activation and intracellular trafficking assessed. The individual roles of these two variants will be further assessed using five small molecule inhibitors selected from high throughput screens for small compounds that disrupt fatty acid transport. Collectively, these studies are expected to provide novel insights into the differential roles of FATP2a and FATP2b in fatty acid homeostasis. Dislipidemia has been shown to be a contributing factor to metabolic diseases such a diabetes, obesity, and cardiovascular disease. Characterizing the proteins involved in lipid metabolism can help elucidate the biological processes which govern these pathological states. The focus of this research proposal includes characterizing the function of FATP2 which is expected to give insight into the biological processes underpining lipid metabolism.