Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

超出所提供的空间。脂肪酸（FAs）是细胞膜的重要结构成分、能量来源和类二十烷的前体。FAs还可以作为信号转导的第二信使和调节因子。通过这些机制，FAs在控制细胞生长、分化、增殖和凋亡等事件中发挥着重要的生理作用。有文献记载，氧化脂肪酸（OFAs），包括二十烷酸，在人类病理状态下以葡萄糖醛酸盐的形式被生物合成和排泄。游离脂肪酸（FFAs）葡萄糖醛酸化的生理功能还不太发达。然而，通过在人体尿液中鉴定出FFAs的羧基连接的葡萄糖醛酸盐，证实了FFAs的葡萄糖醛酸盐在体内的生物合成。作为本提案的主要目标，我们将表征人体组织内质网（ER）和核膜中的FA葡萄糖醛酸化。该项目的中心假设是，FAs及其氧化衍生物是人体ugt生理上重要的底物，葡萄糖醛酸化对某些FAs水平升高起保护作用。本研究计划将侧重于确定UGT2B亚家族中葡萄糖醛酸化OFAs和FFAs的udp -葡萄糖醛酸基转移酶（UGTs）的催化和分子特性。具体目标1和2将表征ugt参与FA葡萄糖醛酸化的催化性质。我们将鉴定作为人类UGT同种异构体底物的FAs。底物特异性和底物-抑制剂相互作用将被研究。我们将生物合成efa glucuronides并研究其潜在毒性及其对组织培养中UGTs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。定位于底物结合位点或细胞靶向内质网和核膜所需的氨基酸基序将被识别。利用光活性FAs进行光亲和标记研究，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UGTs靶向内质网和核膜。定点诱变研究将证实结构域的重要性。从我们对FA糖醛酸化的研究中获得的信息不仅将为理解各种FA水平升高时FA解毒提供理论依据，而且还可以作为重要的治疗策略，例如针对心血管疾病，炎症反应和癌症的药物开发。网站性能 ======================================== 节结束 ===========================================

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===========================================