Functions of intracellular lipid-binding proteins

描述（由申请人提供）：维生素A代谢物视黄酸（RA）通过激活称为视黄酸受体（RAR）的配体诱导型转录因子来调节多种生物学功能。我们以前的研究令人惊讶地证明，RA也作为另一个核受体，即PPAR 4的内源性配体，因此，激素显示双重转录活性。除了核受体之外，疏水化合物如RA在细胞中与细胞内脂质结合蛋白（iLBP）家族的成员缔合，其中一些起到将特定化合物从胞质溶胶直接递送至细胞核中的特定核受体的作用，从而增强其配体的转录活性。我们最近的研究结果表明，RA在其两种受体之间的分配受两种iLBP调节，细胞视黄酸结合蛋白II（CRABP-II）将RA递送至RAR，脂肪酸结合蛋白5（FABP 5）将该配体穿梭至PPAR 4。在这里，我们建议进一步描绘的分子机制，RA的双重转录活性的基础上，调查新发现的类维生素A和PPAR 4信号之间的串扰在调节脂质代谢和能量稳态的潜在作用，并探讨的可能性，通过激活其两个受体，RA可能是防止肥胖和胰岛素抵抗的发展。拟议的实验将针对两个具体目标。1. RA的双重转录活性在脂肪功能中的作用。最近的报道暗示RAR和PPAR 4在调节脂肪细胞生物学和脂质稳态的不同方面。拟议的研究将利用培养的脂肪细胞描绘的两个RA受体在脂肪细胞分化和脂肪生物学的各个方面，如胰岛素反应和脂质代谢的作用。此外，将采用对肥胖和糖尿病易感的小鼠模型来研究RA及其相关核受体和结合蛋白在体内肥胖和胰岛素抵抗的发展中的参与。2. FABP 5激活PPAR 4的结构基础。在结合RA后，FABP 5被动员到细胞核，在那里它将配体递送到PPAR 4。有趣的是，虽然FABP 5结合许多亲脂性配体，但其核转位仅由特定化合物触发。为了深入了解FABP 5激活中配体特异性的结构特征，我们建议解决和比较与活化配体和非活化配体复合的蛋白质的晶体结构。此外，为了阐明FABP 5与PPAR 4有效结合的能力的结构基础，我们计划解决FABP 5与受体的复合物的结构。最后，我们将确定PPAR 4的结构及其目前已知的唯一生理配体RA。公共卫生相关性：说明：维生素A代谢物视黄酸通过激活两种核受体来调节多种生物学功能：视黄酸受体（RAR）和过氧化物酶体增殖物激活受体4（PPAR 4）。我们最近发现，这种激素在其两种受体之间的分配受细胞内脂质结合蛋白（iLBP）家族的两个成员调节，细胞视黄酸结合蛋白II（CRABP-II）将RA递送至RAR，脂肪酸结合蛋白5（FABP 5）将该配体转运至PPAR 4。本研究旨在探讨视黄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，视黄酸可能具有保护活动，对肥胖和胰岛素抵抗的发展。此外，我们建议描绘的结构特征，FABP 5和PPAR 4的能力，合作介导的视黄酸的转录活性的基础。

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.