Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

描述（由申请人提供）：脂肪酸（FA）是重要的结构 细胞膜的组分、能量来源和类二十烷酸的前体。FAS 也可以作为第二信使和信号转导的调节剂。通过 在这些机制中，FAs在控制 细胞事件如生长、分化、增殖和凋亡。 已经证明氧化脂肪酸（OFA），包括类二十烷酸， 是生物合成和排泄的形式葡糖苷酸在病理 人类的条件。葡萄糖醛酸化的生理功能 脂肪酸（FFA）的开发较少。然而，在体内生物合成的 FFA的葡萄糖醛酸苷已通过鉴定 人尿中游离脂肪酸的羧基连接葡糖苷酸。 作为本提案的主要目标，我们将表征FA葡萄糖醛酸化 在人组织的内质网（ER）和核膜中。的 该项目的中心假设是，脂肪酸及其氧化衍生物 是人UGT和葡萄糖醛酸化的重要生理底物 对某些脂肪酸水平升高起保护作用。本研究 建议将集中在催化和分子的测定 UGT 2B亚家族的UDP-葡萄糖醛酸基转移酶（UGT）的性质， 葡萄糖醛酸酯OFA和FFA。具体目标1和2将描述 参与FA葡萄糖醛酸化的UGT的催化性质。我们将 鉴定作为人UGT同种型底物的FA。底物特异性 并研究底物-抑制剂相互作用。我们将 生物合成FA葡萄糖醛酸苷，并研究其潜在的毒性和 对组织培养物中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 将研究有效葡萄糖醛酸化所需的UGT结构域。氨基 酸基序位于底物结合位点或细胞生长所需 将鉴定靶向ER和核膜。光亲和 将进行光活性FA的标记研究以鉴定氨基 参与FA结合的酸。UGT靶向ER和核膜 将使用绿色荧光蛋白融合物进行研究， 免疫荧光研究。定点诱变研究将证实 结构域的重要性。 从我们的FA葡萄糖醛酸化研究中获得的信息将提供 不仅是理解FA解毒反应的理由 各种脂肪酸的水平升高，而且还可以作为 重要的治疗策略，如开发靶向药物 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.