Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

超出提供的空间。脂肪酸(FAs)是细胞膜的重要结构成分，是二十烷类化合物的能源和前体。Fas还可以作为信号转导的第二信使和调节器。通过这些机制，FAs在控制细胞生长、分化、增殖和凋亡等方面发挥着重要的生理作用。已有文献证明，氧化脂肪酸(Ofas)，包括二十烷类化合物，是在人类的病理条件下以葡萄糖醛酸苷的形式生物合成和排泄的。游离脂肪酸的葡萄糖醛酸化作用(FFAs)的生理功能还不是很发达。然而，通过对人体尿液中游离脂肪酸的羧基连接的葡萄糖醛酸类化合物的鉴定，已经证实了游离脂肪酸在体内的葡萄糖醛酸类化合物的生物合成。作为这项提案的一个主要目标，我们将表征人体组织中内质网(ER)和核膜中的FA葡萄糖醛酸化反应。该项目的中心假设是FAs及其氧化的衍生物是人类UGT的重要生理底物，而葡萄糖醛酸化作用对某些FAs水平的升高具有保护作用。这项研究计划将集中在测定UGT2B亚家族的UGT2B亚家族的UDP-葡萄糖醛酸基转移酶(UGT)的催化和分子性质。具体的目标1和2将表征参与FA葡萄糖醛酸化的UGT的催化性能。我们将鉴定作为人类UGT亚型底物的FA。将研究底物专一性和底物-抑制物相互作用。我们将生物合成FA葡萄糖醛酸类化合物，并研究它们的潜在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。我们将研究有效的葡萄糖醛酸化作用所需的UGT的结构域。定位于底物结合部位或细胞靶向内质网和核膜所需的氨基酸基序将被确定。将进行光活性FA的光亲和标记研究，以确定参与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靶向内质网和核膜。定点突变研究将证实结构域的重要性。从我们对FA糖醛酸化作用的研究中获得的信息不仅将为理解FA在各种FA水平升高时的解毒作用提供理论基础，而且还将提供可用于重要治疗策略的信息，如针对心血管疾病、炎症反应和癌症的药物的开发。表演网站========================================Section End===========================================

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