MOLECULAR REGULATION OF FXR ACTIVITY

FXR 活性的分子调控

• 批准号: 7895875
• 负责人: Jongsook Kim Kemper
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7895875
• 关键词: Acetylation; Acetylesterase; Bile Acids; Biological; Cholesterol; Chromatin; Cultured Cells; DNA Binding; Deacetylase; Deacetylation; Development; Diabetes Mellitus; Disease; Disease model; Down-Regulation; EP300 gene; Fatty Acids; Fatty Liver; Fc Receptor; Fibroblast Growth Factor; Gene Targeting; Genes; Glucose; Goals; Growth; Health; Histone Deacetylase Inhibitor; Histone H3; Homeostasis; In Vitro; Individual; Intestines; Leptin; Lipids; Lipoproteins; Liver; Liver Regeneration; Lysine; Mass Spectrum Analysis; Mediating; Metabolic; Metabolic Diseases; Metabolism; Molecular; Mus; Mutation; Nuclear; Obesity; Orphan; Pathology; Physiological; Physiology; Play; Post-Translational Protein Processing; Process; Proteins; Publishing; Receptor Down-Regulation; Recruitment Activity; Regulation; Role; SRE-1 binding protein; Site; Small Intestines; Stress; TP53 gene; Testing; Therapeutic Agents; Transactivation; Transcription Regulatory Protein; Ubiquitin; blood glucose regulation; cofactor; design; functional outcomes; glucose metabolism; histone modification; hypercholesterolemia; improved; in vivo; insight; lipoprotein triglyceride; mouse model; mutant; novel therapeutics; promoter; protein degradation; receptor; receptor function; small heterodimer partner protein; tumorigenesis

The overall goal of this project is to understand molecular mechanisms by which the nuclear bile acid receptor, farnesoid X receptor (FXR), regulates metabolic homeostasis in normal and diseased states. The specific goal of this application is to elucidate the role of post-translational acetylation and deacetylation of FXR mediated by p300 and SIRT1 in FXR function in normal physiology and in pathological conditions. FXR plays a central role in cholesterol/bile acid, fatty acid, lipoprotein, and glucose metabolism by regulating expression of numerous its target genes. Although such important biological roles of FXR are now known, how FXR activity is regulated remains relatively unknown. Transcriptional cofactors, such as p300 acetylase and SIRT1 deacetylase, modulate receptor activity, not only by histone modification of their target gene chromatin, but also by post-translational modification of the receptor itself. In preliminary studies, we found that FXR is acetylated and deacetylated by p300 and SIRT1, respectively. P300 and SIRT1 antagonize each other’s activity in the regulation of FXR transactivation. Down-regulation of p300 altered expression of FXR target genes, such that beneficial changes in lipid and glucose metabolic profiles would be expected. FXR acetylation was dynamically regulated under normal physiological states but surprisingly, FXR acetylation levels were substantially elevated in ob/ob mouse liver. Specific lysine residues in FXR were identified as acetylation sites by mass spectrometry and mutation analyses. Interestingly, mutation of these individual sites had different effects on FXR transactivation activity. These intriguing results led us to hypothesize first, that acetylation profoundly modulates FXR activity and is dynamically regulated by p300 and SIRT1 under normal physiological conditions but is highly elevated under metabolic disease and stress conditions and second, that FXR acetylation at different sites may have distinct functional outcomes in normal and disease states by regulating different FXR target genes. To test our hypothesis, we will: 1) determine whether p300 and SIRT1 are critical in vivo cofactors of FXR by down-regulation of these cofactors in cultured cells and in vivo ; 2) identify acetylated sites in FXR and determine the effects of mutations of these sites on FXR function in vitro, in cultured cells, and in vivo; and 3) determine whether FXR acetylation is dysregulated in pathophysiological conditions and the role of acetylation at specific sites in the disease pathology. Our studies will provide substantial insight into the molecular mechanism of FXR action in vivo and information that may be important for the development of novel therapeutic agents for metabolic disorders, such as fatty liver (liver steatosis), hypercholesterolemia, and diabetes. The bile acid receptor FXR has important biological roles in cholesterol and bile acid homeostasis, triglyceride and lipoprotein metabolism, and glucose regulation in the liver, but how the activity of FXR is regulated remains largely unknown. Our studies to define how FXR acetylation controls FXR activity in health and disease states will provide important information about the mechanisms regulating levels of cholesterol, triglycerides, lipoproteins, and glucose which are abnormal in diseases such as hypercholesterolemia, obesity, and diabetes. The studies may also facilitate the design of therapeutic agents for treating these metabolic disorders.

该项目的总体目标是了解核胆汁酸受体法尼西德X受体(FXR)在正常和疾病状态下调节代谢稳态的分子机制。本应用的具体目的是阐明在正常生理和病理条件下，p300和SIRT1介导的FXR翻译后乙酰化和去乙酰化在FXR功能中的作用。FXR播放 通过调节许多其靶基因的表达，在胆固醇/胆汁酸、脂肪酸、脂蛋白和葡萄糖代谢中发挥核心作用。尽管FXR如此重要的生物学作用已为人所知，但FXR活性是如何被调控的仍然相对未知。转录辅助因子，如p300乙酰基酶和SIRT1去乙酰基酶，不仅通过组蛋白修饰其靶基因染色质，而且还调节受体的活性。 也可以通过受体本身的翻译后修饰来实现。在初步研究中，我们发现FXR分别被p300和SIRT1乙酰化和去乙酰化。P300和SIRT1在调节FXR反式激活中相互拮抗。P300的下调改变了FXR靶基因的表达，从而预期脂肪和葡萄糖代谢谱将发生有益的变化。FXR乙酰化在正常生理状态下是动态调节的，但令人惊讶的是，在ob/ob小鼠的肝脏中，FXR乙酰化水平显著升高。通过质谱分析和突变分析，确定FXR中特定的赖氨酸残基为乙酰化位点。有趣的是，这些单个位点的突变对FXR反式激活活性有不同的影响。这些耐人寻味的结果让我们首先假设， 乙酰化深刻地调节FXR的活性，在正常生理条件下受p300和SIRT1的动态调节，但在代谢性疾病和应激条件下高度升高；第二，不同位置的FXR乙酰化可能通过调节不同的FXR靶基因在正常和疾病状态下具有不同的功能结果。为了验证我们的假设，我们将：1)通过下调培养细胞和体内的这些辅因子来确定p300和SIRT1是否是FXR的关键辅因子；2)确定FXR中的乙酰化位点，并确定这些位点的突变在体外、培养细胞和体内对FXR功能的影响；以及3)确定FXR乙酰化在病理生理条件下是否失调，以及特定位置的乙酰化在疾病病理中的作用。我们的研究将提供 深入了解FXR在体内作用的分子机制，以及可能对开发新的代谢紊乱治疗药物(如脂肪肝(肝脏脂肪变性)、高胆固醇血症和糖尿病)至关重要的信息。 胆汁酸受体FXR在胆固醇和胆汁酸稳态、甘油三酯和脂蛋白代谢以及肝脏中的葡萄糖调节等方面具有重要的生物学作用，但FXR的活性是如何调节的在很大程度上仍不清楚。我们的研究旨在确定FXR乙酰化如何在健康和疾病状态下控制FXR活性，这将为调节胆固醇、甘油三酯、脂蛋白和葡萄糖水平的机制提供重要信息，这些水平在高胆固醇血症、肥胖和糖尿病等疾病中是异常的。这些研究还可能有助于设计治疗这些代谢紊乱的治疗剂。