BIOCHEMICAL CHARACTERIZATION OF PPAR

描述（改编自研究者摘要）：最近， 过氧化物酶体增殖物，一类重要的肿瘤 启动子，激活类固醇激素受体的新成员 过氧化物酶体增殖物激活受体（过氧化物酶体增殖物激活受体）。 已经发现了几种亚型的过氧化物酶体增殖物激活受体（α，β，γ），虽然 主要的肝脏亚型（PPAR α）是目前的重点， 调查 本提案将审查事件的顺序 由过氧化物酶体增殖物引发， 表情 首先，将开发适当的分子工具， PPARalpha功能研究。 最近，针对PPARalpha的特异性抗体 和表位标记的受体（PPARalpha-Flag）， 将有助于在其他可能的形式中描述阿尔法亚型。 光亲和配体探针和稳定的细胞系， PPARalpha-Flag将被追踪。 第二，亚细胞定位 将检查PPARalpha。 PPARalpha是否主要存在于 失活状态是受体功能的重要考虑因素。 将使用共聚焦显微镜检查PPARalpha在 失活和过氧化物酶体增殖物激活状态。 三是 将确定PPARa-蛋白质复合物的组成。 的 PPARalpha与热休克蛋白和其他蛋白的特异性相互作用 通常与类固醇激素受体相关的因素将被检查 使用几种互补的方法。 受体复合物的比较 在过氧化物酶体增殖剂治疗之前和之后， 对信号转导通路的全面认识。 第四，确定 如果磷酸化是PPARalpha功能的重要调节因子。 化学和酶消化，然后是磷酸氨基和磷酸肽 分析将用于检查哪些残基被磷酸化 过氧化物酶体增殖物对PPARalpha和PPARalpha的影响不同 治疗 每个磷酸化残基的重要性将被检查 通过在PPARalpha中引入特定的突变。 最后， 将检查PPARalpha穿过核膜的转运。 如果 共聚焦显微镜证实PPARalpha存在于胞质溶胶中， 控制其进入细胞核的过程将被检查。 将构建标记的PPARalpha，并在体外核 进行了吸收研究，并鉴定了作为 监护人将被追捕。 综合起来，拟议的研究将 大大增加了我们对PPARalpha调节机制的了解， 因此我们对一类重要致癌物质有了认识。

DESCRIPTION (Adapted from the Investigator's Abstract): Recently, it has been shown that peroxisome proliferators, an important class of tumor promoters, activate novel members of the steroid hormone receptor superfamily, the peroxisome proliferator-activated receptors (PPAR). Several subtypes of PPAR have been discovered (alpha, beta, gamma), although the predominant liver subtype (PPAR alpha) is the focus of the present investigation. The present proposal will examine the sequence of events initiated by peroxisome proliferators and culminated in altered gene expression. First, the appropriate molecular tools will be developed for the study of PPARalpha function. Recently, specific antibodies to PPARalpha and an epitope-labeled receptor (PPARalpha-Flag) have been developed which will help characterize the alpha subtype among the other possible forms. Photoaffinity ligand probes and stable cells lines which express PPARalpha-Flag will be pursued. Second, the subcellular localization of PPARalpha will be examined. Whether PPARalpha is primarily cytosolic in the inactivated state is an important consideration in receptor function. Confocal microscopy will be used to examine PPARalpha distribution in the inactivated and peroxisome proliferator-activated states. Third, the composition of the PPARalpha-protein complex will be determined. The specific interactions of PPARalpha with heat shock proteins and other factors often associated with steroid hormone receptors will be examined using several complementary approaches. Comparison of the receptor complex before and after peroxisome proliferator treatment will be important in the overall understanding of the signal transduction pathway. Fourth, determine if phosphorylation is an important regulator of PPARalpha function. Chemical and enzymatic digestion followed by phosphoamino and phosphopeptide analysis will be used to examine which residues are phosphorylated on PPARalpha and which are differentially affected by peroxisome proliferator treatment. The importance of each phosphorylated residue will be examined by introducing specific mutations in PPARalpha. Last, the process of transport of PPARalpha across the nuclear membrane will be examined. If confocal microscopy verifies that PPARalpha is present in the cytosol, the process that governs its transport into the nucleus will be examined. Fluorescently-labeled PPARalpha will be constructed and in vitro nuclear uptake studies implemented and identification of proteins acting as chaperones will be pursued. Taken together the proposed studies will greatly increase our knowledge of PPARalpha's mechanism of regulation and hence our understanding of an important class of carcinogen.