Structural Insight into PPARγ Activation Through Covalent Modification with Endogenous Fatty Acids
Structural Insight into PPARγ Activation Through Covalent Modification with Endogenous Fatty Acids
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DOI：
10.1016/j.jmb.2008.10.039
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发表时间：
2009-01-09
影响因子：
5.6
通讯作者：
Morikawa, Kosuke
中科院分区：
文献类型：
--
作者：
Waku, Tsuyoshi;Shiraki, Takuma;Morikawa, Kosuke
过氧化物酶体增殖物激活受体（PPAR）γ是一种调节脂质稳态的核受体，几种脂肪酸代谢产物已被鉴定为PPAR γ配体。在这里，我们提出了四个晶体结构的过氧化物酶体增殖物激活受体γ配体结合域（LBD）共价结合内源性脂肪酸通过一个独特的半胱氨酸，据报道，这是至关重要的受体激活。与15-脱氧-δ（12，14）-前列腺素J（2）（15 d-PGJ（2））复合的LBD的结构分析显示，15 d-PGJ（2）的共价结合诱导螺旋H2 '后的环区域的构象变化，以及LBD中产生的共价键周围的侧链网络的重排。环和螺旋H3上这些重新定位的残基的点突变几乎完全消除了15 d-PGJ 2对PPAR γ的激活，表明所观察到的结构改变可能对内源性脂肪酸对PPAR γ的激活至关重要。为了解决内源性PPAR γ配体的部分激动作用的问题，我们利用了一系列氧化的二十碳四烯酸（oxoETEs）作为与PPAR γ共价结合的配体。尽管结构和化学性质相似，但这些脂肪酸表现出不同程度的转录活性。使用两种oxoETE/PPAR γ LBD复合物的晶体学研究表明，每个oxoETE的转录强度与环构象的差异有关，而不是每个配体和螺旋H12之间的相互作用。这些结果表明，环构象可能是负责的PPAR γ活性的调制。基于这些结果，我们通过计算机筛选和基于细胞的测定鉴定了与PPAR γ共价结合的新型激动剂。我们对LBD与硝基-233复合物的晶体学研究表明，这种新鉴定的激动剂和半胱氨酸之间确实形成了预期的共价键。这项研究提出了通过与内源性脂肪酸共价修饰的PPAR γ的激活和调节机制的结构基础。(C)2008爱思唯尔有限公司保留所有权利。
Peroxisome proliferator-activated receptor (PPAR) gamma is a nuclear receptor that regulates lipid homeostasis, and several fatty acid metabolites have been identified as PPAR gamma ligands. Here, we present four crystal structures of the PPAR gamma ligand binding domain (LBD) covalently bound to endogenous fatty acids via a unique cysteine, which is reportedly critical for receptor activation. The structure analyses of the LBD complexed with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) revealed that the covalent binding of 15d-PGJ2 induced conformational changes in the loop region following helix H2', and rearrangements of the side-chain network around the created covalent bond in the LBD. Point mutations of these repositioned residues on the loop and helix H3 almost completely abolished PPAR gamma activation by 15d-PGJ2, indicating that the observed structural alteration may be crucial for PPAR gamma activation by the endogenous fatty acid. To address the issue of partial agonism of endogenous PPAR gamma ligands, we took advantage of a series of oxidized eicosatetraenoic acids (oxoETEs) as covalently bound ligands to PPAR gamma. Despite similar structural and chemical properties, these fatty acids exhibited distinct degrees of transcriptional activity. Crystallographic studies, using two of the oxoETE/PPAR gamma LBD complexes, revealed that transcriptional strength of each oxoETE is associated with the difference in the loop conformation, rather than the interaction between each ligand and helix H12. These results suggest that the loop conformation may be responsible for the modulation of PPAR gamma activity. Based on these results, we identified novel agonists covalently bound to PPAR gamma by in silico screening and a cell-based assay. Our crystallographic study of LBD complexed with nitro-233 demonstrated that the expected covalent bond is indeed formed between this newly identified agonist and the cysteine. This study presents the structural basis for the activation and modulation mechanism of PPAR gamma through covalent modification with endogenous fatty acids. (C) 2008 Elsevier Ltd. All rights reserved.