Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

描述(申请人提供)：脂肪酸(FA)是重要的结构物质 细胞膜的成分，二十烷类化合物的能源和前体。Fas 也可以作为信号转导的第二信使和调节器。通过 这些机制中，FAs起着重要的生理控制作用 细胞事件，如生长、分化、增殖和凋亡。 已有文献证明，氧化脂肪酸(OFA)，包括二十烷类化合物， 在病理上以葡萄糖醛酸苷的形式被生物合成和排泄 人类的状况。游离葡萄糖糖醛酸化的生理功能 脂肪酸(FFAs)还不够发达。然而，在体内的生物合成 FFAs中的葡萄糖醛酸内酯已被鉴定为 人尿中游离脂肪酸的羧基葡萄糖醛酸类化合物。 作为这项提案的一个主要目标，我们将描述FA葡萄糖醛酸化反应 在人体组织的内质网和核膜中。这个 该项目的中心假设是FA及其氧化衍生物 是人体UGT和葡萄糖醛酸化的重要生理底物 对某些脂肪酸水平的升高起到保护作用。这项研究 建议将重点放在催化剂和分子的测定上 UGT2B亚家族的UDP-葡萄糖醛酸基转移酶特性 人体内的葡萄糖醛酸脂、脂肪酸和游离脂肪酸。具体目标1和2将描述 UGT在FA葡萄糖醛酸化反应中的催化性能。我们会 鉴定作为人类UGT亚型底物的FA。底物专一性 并将研究底物与抑制剂的相互作用。我们会 生物合成脂肪酸葡萄糖醛酸类化合物及其潜在毒性研究 对组织培养中UGT表达的影响。在具体目标3和4中， 将进行结构-功能关系研究。结构性的 我们将研究有效的葡萄糖醛酸化作用所需的UGT结构域。氨基 定位于底物结合部位或细胞所需的酸性基序 将确定以内质网和核膜为靶点。光亲和力 将进行光活性脂肪酸的标记研究以识别氨基 与FA结合有关的酸。UGT靶向内质网和核膜的研究 将使用绿色荧光蛋白融合和 免疫荧光研究。定点突变研究将证实 结构域的重要性。 从我们对FA葡萄糖醛酸化反应的研究中获得的信息将提供 不仅是理解FA戒毒的一个基本原理 各种FA水平升高，但也有信息可用于 重要的治疗策略，如开发药物靶向 心血管疾病、炎症反应和癌症。

DESCRIPTION (Provided by applicant): Fatty acids (FAs) are important structural components of cell membranes, energy sources and precursors of eicosanoids. FAs can also act as second messengers and regulators of signal transduction. By these mechanisms, FAs play a significant physiological role in controlling cellular events such as growth, differentiation, proliferation and apoptosis. It has been documented that oxidized fatty acids (OFAs), including eicosanoids, are biosynthesized and excreted in the form of glucuronides in pathological conditions in humans. The physiological function of glucuronidation of free fatty acids (FFAs) is less developed. However, the in vivo biosynthesis of glucuronides of FFAs has been confirmed by the identification of carboxyl-linked glucuronides of FFAs in human urine. As a major objective of this proposal, we will characterize FA glucuronidation in endoplasmic reticulum (ER) and nuclear membranes in human tissues. The central hypothesis for this project is that FAs and their oxidixed derivatives are physiologically important substrates for human UGTs and glucuronidation plays a protective role against elevated levels of certain FAs. This research proposal will focus on the determination of the catalytic and molecular properties of UDP-glucuronosyltransferases (UGTs) of the UGT2B subfamily that glucuronidate OFAs and FFAs in humans. Specific Aims 1 and 2 will characterize the catalytic properties of UGTs involved in FA glucuronidation. We will identify FAs that are substrates for human UGT isoforms. Substrate specificity and substrate-inhibitor interactions will be investigated. We will biosynthesize FA glucuronides and study their potential toxicity and their effect on the expression of UGTs in tissue cultures. In Specific Aims 3 and 4, structure-function relationship studies will be performed. The structural domains of UGTs required for effective glucuronidation will be studied. Amino acid motifs localized in substrate binding sites or required for cellular targeting to the ER and nuclear membranes will be identified. Photoaffinity labeling studies with photoactive FAs will be carried out to identify amino acids involved in FA binding. Targeting of UGTs to ER and nuclear membranes will be investigated using green fluorescent protein fusions and immunofluorescence studies. Site-directed mutagenesis studies will confirm the importance of structural domains. The information obtained from our studies on FA glucuronidation will provide not only a rationale for understanding FA detoxification in response to elevated levels of various FAs but also information which can be used as important therapeutic strategies, such as the development of drugs targeting cardiovascular disease, inflammatory responses and cancer.