BIOCHEMICAL CHARACTERIZATION OF PPAR

描述(改编自《调查者摘要》)：最近，它 已有研究表明，过氧化体增殖物是一类重要的肿瘤 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(PPAR)超家族。 已经发现了几种PPAR亚型(阿尔法、贝塔、伽马)，尽管 主要的肝脏亚型(PPARα)是目前关注的焦点 调查。本提案将审查事件的先后顺序 由过氧化物体增殖物引发并最终导致基因改变 表情。首先，将开发适当的分子工具来 PPARAlpha函数的研究。最近，针对PPARpha的特异性抗体 并开发了一种表位标记的受体(PPARα-Flag)，该受体 将有助于在其他可能的形式中确定阿尔法子类型的特征。 光亲和配体探针及稳定表达的细胞系 PPAR Alpha-Flag将被追查。第二，细胞的亚细胞定位 将对PPARpha进行检查。PPARpha是否主要是胞质的 失活状态是受体功能的重要考虑因素。 共聚焦显微镜将被用来研究PPARα在 失活状态和过氧化物体增殖物激活状态。第三， PPARα-蛋白质复合体的组成将被确定。这个 PPARpha与热休克蛋白等的特异性相互作用 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。受体复合体的比较 过氧化物酶体增殖物治疗前后将在 对信号转导途径有全面的了解。第四，确定 如果磷酸化是PPARpha功能的重要调节因子。 化学和酶消化，然后是磷氨基和磷肽 分析将被用来检查哪些残基被磷酸化 PPARα和受过氧化物酶体增殖物不同影响的基因 治疗。将检查每个磷酸化残基的重要性。 通过在PPARpha中引入特定的突变。最后，这一过程 将研究PPARpha通过核膜的运输。如果 共聚焦显微镜证实PPARpha存在于胞浆中， 我们将研究控制其进入原子核的过程。 将构建荧光标记的PPARpha，并在体外进行核 实施摄取研究和鉴定起作用的蛋白质 监护人将被追查。综合起来，拟议的研究将 大大增加了我们对PPARAlpha的调节机制和 因此，我们理解了一类重要的致癌物。

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.