Functions of intracellular lipid-binding proteins

描述（由申请人提供）：维生素A代谢物维甲酸（RA）通过激活称为维甲酸受体（RAR）的配体诱导转录因子来调节多种生物功能。我们之前的研究令人惊讶地表明，RA还可以作为另一种核受体（即PPAR4）的内源性配体，因此该激素表现出双重转录活性。除了核受体，疏水性化合物，如RA，在细胞中与细胞内脂质结合蛋白（iLBP）家族成员结合，其中一些功能直接将特定化合物从细胞质溶胶传递到细胞核中的特定核受体，从而增强其配体的转录活性。我们最近的研究结果表明，RA在两个受体之间的分配是由两个ilbp调节的，一个是将RA传递给RAR的细胞维甲酸结合蛋白II (CRABP-II)，另一个是将RA转运到PPAR4的脂肪酸结合蛋白5 （FABP5）。在这里，我们建议进一步描述RA的双重转录活性的分子机制，研究新发现的类维生素a和PPAR4信号之间的串导在调节脂质代谢和能量稳态中的潜在作用，并探讨通过激活其两个受体，RA可能对肥胖和胰岛素抵抗的发展有保护作用的可能性。拟议的实验将解决两个具体目标。类风湿性关节炎双转录活性在脂肪功能中的作用。最近的报道表明，RAR和PPAR4都参与了脂肪细胞生物学和脂质稳态的不同方面的调节。拟议的研究将利用培养的脂肪细胞来描述两种RA受体在脂肪细胞分化和脂肪生物学的各个方面（如胰岛素反应性和脂质代谢）中的作用。此外，我们还将利用肥胖和糖尿病易感小鼠模型来研究RA及其相关核受体和结合蛋白在体内肥胖和胰岛素抵抗发展中的作用。2. FABP5激活PPAR4的结构基础。在与RA结合后，FABP5被动员到细胞核中，在那里它将配体递送给PPAR4。有趣的是，虽然FABP5结合了许多亲脂配体，但其核易位仅由特定化合物触发。为了深入了解FABP5激活中配体特异性的结构特征，我们提出解决并比较活化配体与非活化配体复合物的蛋白质晶体结构。此外，为了阐明FABP5与PPAR4有效结合能力的结构基础，我们计划解决FABP5与受体复合物的结构。最后，我们将确定PPAR4及其唯一已知的生理配体RA的结构。叙事：维生素A代谢物维甲酸通过激活两种核受体来调节多种生物功能：维甲酸受体（RAR）和过氧化物酶体增殖物激活受体4 （PPAR4）。我们最近发现，这种激素在两个受体之间的分配是由细胞内脂质结合蛋白（iLBP）家族的两个成员调节的，细胞维甲酸结合蛋白II （CRABP-II）将RA传递到RAR，脂肪酸结合蛋白5 （FABP5）将这种配体运送到PPAR4。本项目旨在探讨维甲酸及其受体和结合蛋白在脂肪组织生物学和调节能量稳态中的作用。一个特别的重点将放在调查的可能性，通过激活这两个受体，维甲酸可能具有保护作用，防止肥胖和胰岛素抵抗的发展。此外，我们建议描述FABP5和PPAR4协同介导维甲酸转录活性的结构特征。

DESCRIPTION (provided by applicant): The vitamin A metabolite retinoic acid (RA) regulates multiple biological functions by activating the ligand-inducible transcription factors termed retinoic acid receptors (RAR). Our previous studies surprisingly demonstrated that RA also serves as an endogenous ligand for another nuclear receptor, namely PPAR4, and thus that the hormone displays a dual transcriptional activity. In addition to nuclear receptors, hydrophobic compounds, such as RA, associate in cells with members of the family of intracellular lipid binding proteins (iLBP), some of which function to directly deliver specific compounds from the cytosol to particular nuclear receptors in the nucleus, thereby augmenting the transcriptional activities of their ligands. Our recent findings revealed that the partitioning of RA between its two receptors is regulated by two iLBPs, cellular retinoic acid binding protein II (CRABP-II) which delivers RA to RAR, and fatty acid binding protein 5 (FABP5), which shuttles this ligand to PPAR4. Here, we propose to further delineate the molecular mechanisms that underlie the dual transcriptional activity of RA, to investigate potential roles of the newly discovered cross-talk between retinoid and PPAR4 signalling in regulating lipid metabolism and energy homeostasis, and to explore the possibility that, by activating its two receptors, RA may be protective against the development of obesity and insulin resistance. Proposed experiments will address two Specific Aims. 1. Roles of the dual transcriptional activity of RA in adipose function. Recent reports implicated both RAR and PPAR4 in regulation of different aspects of adipocyte biology and lipid homeostasis. Proposed studies will utilize cultured adipocytes to delineate roles of the two RA receptors in adipocyte differentiation and in various aspects of adipose biology such as insulin responsiveness and lipid metabolism. In addition, a mouse model susceptible to obesity and diabetes will be employed to investigate the involvement of RA and its associated nuclear receptors and binding proteins in the development of obesity and insulin resistance in vivo. 2. Structural basis for the activation of PPAR4 by FABP5. Upon binding of RA, FABP5 is mobilized to the nucleus where it delivers the ligand to PPAR4. Interestingly, while FABP5 binds numerous lipophilic ligands, its nuclear translocation is triggered only by specific compounds. To gain insight into the structural features that underlie the ligand-specificity in activation of FABP5, we propose to solve and compare the crystal structures of the protein complexed with activating vs. non-activating ligands. In addition, to elucidate the structural basis for the ability of FABP5 to productively associate with PPAR4, we plan to solve the structure of the complex of FABP5 with the receptor. Finally, we will determine the structure of PPAR4 with its only currently known physiological ligand, RA. PUBLIC HEALTH RELEVANCE: NARRATIVE: The vitamin A metabolite retinoic acid regulates multiple biological functions by activating two nuclear receptors: the retinoic acid receptor (RAR), and the peroxisome proliferator-activated receptor 4 (PPAR4). We recently found that the partitioning of this hormone between its two receptors is regulated by two members of the family of intracellular lipid binding proteins (iLBP), cellular retinoic acid binding protein II (CRABP-II) which delivers RA to RAR, and fatty acid binding protein 5 (FABP5), which shuttles this ligand to PPAR4. This project aims to explore the roles of retinoic acid and its receptors and binding proteins in adipose tissue biology and in regulating energy homeostasis. A special focus would be placed on investigating the possibility that, by activating the two receptors, retinoic acid may possess protective activities against the development of obesity and insulin- resistance. In addition, we propose to delineate the structural features that underlie the ability of FABP5 and PPAR4 to cooperate in mediating the transcriptional activity of retinoic acid.