Glucuronidation of Fatty Acids by Human ER & Nuclear UGT

人内质网对脂肪酸的葡萄糖醛酸化

• 批准号: 6787636
• 负责人: Anna Radominska-Pandya
• 金额: $ 27.51万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-15 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6787636
• 关键词: Glucuronidation; Fatty; Acids; Human; ER

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.

描述（由申请人提供）：脂肪酸（FAS）是重要的结构 细胞膜的成分，eicosanoids的能源和前体。 fas 还可以充当信号转导的第二使者和调节器。经过 这些机制，FA在控制中起着重要的生理作用 细胞事件，例如生长，分化，增殖和凋亡。 已经有记录在包括类花生酸的氧化脂肪酸（OFAS）， 在病理学中以生物合成和排泄的葡萄糖醛酸形式排出 人类的条件。游离葡萄糖醛酸化的生理功能 脂肪酸（FFA）的发达较少。但是，体内生物合成 通过鉴定，FFA的葡萄糖醛酸苷已得到证实 FFA在人尿中的FFA的羧基连接葡萄糖醛酸。 作为该提案的主要目的，我们将表征FA葡萄糖醛酸化 在人体组织中的内质网（ER）和核膜中。这 该项目的中心假设是FAS及其氧化衍生物 人类UGT和葡萄糖醛酸化的生理上重要的底物是 针对某些FA的水平升高起保护作用。这项研究 提案将集中于催化和分子的测定 UGT2B亚家族的UDP-葡萄糖糖基转移酶（UGT）的特性 人类中的葡萄糖醛酸酯和FFA。具体目标1和2将是特征 参与FA葡萄糖醛酸化的UGT的催化特性。我们将 识别是人类UGT同工型底物的FA。底物特异性 将研究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毒性及其 对组织培养物中UGT表达的影响。在特定目标3和4中， 结构功能关系研究将进行。结构 将研究有效的葡萄糖醛酸化所需的UGT域。氨基 酸基序位于底物结合位点或细胞所需的酸基序 将确定针对ER和核膜的靶向。光性 将使用光活性FAS进行标记研究以识别氨基 涉及FA结合的酸。将UGT靶向ER和核膜 将使用绿色荧光蛋白融合和 免疫荧光研究。位置定向的诱变研究将确认 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排毒的理由 各种FA的水平升高，但也可以用作信息 重要的治疗策略，例如靶向药物的开发 心血管疾病，炎症反应和癌症。