Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

超出所提供的空间。 脂肪酸是细胞膜的重要组成部分，也是类二十烷酸的能量来源和前体。脂肪酸也可以作为第二信使和信号转导的调节剂。通过这些机制，FA在控制细胞事件如生长、分化、增殖和凋亡中发挥重要的生理作用。据记载，氧化脂肪酸（OFA），包括类二十烷酸，在人类病理条件下以葡萄糖醛酸苷的形式生物合成和排泄。游离脂肪酸（FFA）的葡萄糖醛酸化的生理功能还不太发达。然而，通过在人尿液中鉴定FFA的羧基连接的葡萄糖醛酸苷，证实了FFA的葡萄糖醛酸苷的体内生物合成。 作为本提案的一个主要目标，我们将表征人组织中内质网（ER）和核膜中的FA葡萄糖醛酸化。该项目的中心假设是，FA及其氧化衍生物是人UGT的生理学重要底物，葡萄糖醛酸化对某些FA水平升高起保护作用。这项研究计划将集中于确定UGT 2B亚家族的UDP-葡萄糖醛酸转移酶（UGT）的催化和分子特性，该酶可使人体中的OFA和FFA葡萄糖醛酸化。具体目标1和2将表征参与FA葡萄糖醛酸化的UGT的催化特性。我们将鉴定作为人UGT亚型底物的FA。将研究底物特异性和底物-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及其对组织培养中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。将研究有效葡萄糖醛酸化所需的UGT结构域。将鉴定位于底物结合位点或细胞靶向ER和核膜所需的氨基酸基序。将进行具有光活性的FA的光亲和标记研究，以鉴定参与FA结合的氨基酸。将使用绿色荧光蛋白融合和免疫荧光研究来研究UGT对ER和核膜的靶向作用。定点诱变研究将证实结构域的重要性。 从我们对FA葡萄糖醛酸化的研究中获得的信息不仅将为理解FA解毒对各种FA水平升高的反应提供理论依据，而且还将提供可用作重要治疗策略的信息，例如开发针对心血管疾病，炎症反应和癌症的药物。性能现场=

EXCEED THE SPACE PROVIDED. 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 biosynthesizeFA 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. PERFORMANCE SITE ========================================Section End===========================================