Characterization of the Biochemical Activities Associated with Human FATP2

与人类 FATP2 相关的生化活性的表征

• 批准号: 7810370
• 负责人: Elaina Marie Melton
• 金额: $ 3.67万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7810370
• 关键词: Acyl Coenzyme A; Address; Adipose tissue; Binding; Biochemical; Biological Process; Caco-2 Cells; Cardiovascular Diseases; Cell membrane; Cells; Ceramides; Coenzyme A Ligases; Complement component C1s; Complex; Coupled; Data; Diabetes Mellitus; Dyslipidemias; Energy Intake; Exhibits; Exons; Expenditure; Family; Fatty Acids; Goals; Hepatic; Hepatocyte; Homeostasis; Human; Image; Individual; Inhibitory Concentration 50; Integral Membrane Protein; Intestines; Kidney; Kinetics; Label; Lead; Libraries; Lipids; Liver; Mass Spectrum Analysis; Metabolic; Metabolic Diseases; Obesity; Oxidative Stress; Pathway interactions; Pattern; Physiological; Property; Protein Family; Protein Isoforms; Protein Region; Proteins; RNA Splicing; Research; Research Proposals; Resolution; Role; Substrate Specificity; Temperature; Testing; Tetracyclines; Therapeutic; Tissues; Triglycerides; Variant; Very Long Chain Fatty Acid; Work; Yeasts; adenylate; cell type; fatty acid metabolism; fatty acid oxidation; fatty acid transport; fatty acid-transport protein; high throughput screening; inhibitor/antagonist; insight; knock-down; lipid metabolism; long chain fatty acid; loss of function; member; novel; plasma membrane fatty acid-binding protein; research study; small hairpin RNA; small molecule; stem; therapeutic target; trafficking; translocase; uptake

DESCRIPTION (provided by applicant): Fatty acid transport proteins (FATPs) are a family of integral membrane proteins, several of which function in the transport and activation of exogenous fatty acids across the plasma membrane. FATP2 is expressed in the intestine and liver, yet its physiological role in normal fatty acid homeostasis in these cells types has not been defined. We have identified two splice variants of human FATP2, which exhibit distinguishing biochemical activities. The first form (FATP2a), also described as a very long chain acyl CoA synthetase has a Mr of 70,000 while the second form (FATP2b) obtained from the Image Consortium library has a Mr of 65,000. FATP2b lacks exon3, which encodes a region of the protein that contains part of the ATP binding domain and required for adenylate formation. Both forms of FATP2 are proficient in fatty acid transport yet FATP2b is unable to activate very long chain fatty acids. These studies thus define two naturally occurring variants of a member of the FATP family that distinguish their roles in fatty acid transport and activation. Using 293 T-REx cells, which allows regulated expression of FATP2a and FATP2b, experiments will be completed to [1] define their individual roles in fatty acid transport and activation and [2] address whether they direct fatty acids into different metabolic pools as assessed using stable isotopically-labeled fatty acids in combination with high resolution mass spectrometry. To access the physiological roles of FATP2 in the intestine and liver, shRNA will be used to knockdown FATP2 expression in Caco-2 (intestinal like) and HepG2 (hepatic like) cells, and patterns of fatty acid transport, activation and intracellular trafficking assessed. The individual roles of these two variants will be further assessed using five small molecule inhibitors selected from high throughput screens for small compounds that disrupt fatty acid transport. Collectively, these studies are expected to provide novel insights into the differential roles of FATP2a and FATP2b in fatty acid homeostasis. Dislipidemia has been shown to be a contributing factor to metabolic diseases such a diabetes, obesity, and cardiovascular disease. Characterizing the proteins involved in lipid metabolism can help elucidate the biological processes which govern these pathological states. The focus of this research proposal includes characterizing the function of FATP2 which is expected to give insight into the biological processes underpining lipid metabolism.

描述（由申请人提供）：脂肪酸转运蛋白（脂肪）是整体膜蛋白的家族，其中一些在跨质膜的外源脂肪酸的运输和激活中起作用。 FATP2在肠道和肝脏中表达，但尚未定义其在这些细胞类型的正常脂肪酸稳态中的生理作用。我们已经确定了人类FATP2的两个剪接变体，它们表现出区别生化活性。第一种形式（FATP2A），也被描述为非常长的链酰基COA合成酶的MR为70,000，而从图像联盟库中获得的第二种形式（FATP2B）的MR为65,000。 FATP2B缺乏exon3，它编码包含ATP结合结构域的一部分蛋白质的区域，并且需要腺苷酸形成。两种形式的FATP2都精通脂肪酸的运输，但FatP2B无法激活很长的链脂肪酸。因此，这些研究定义了FATP家族成员的两个自然发生的变体，它们区分了它们在脂肪酸传输和激活中的作用。使用293个T-Rex细胞，允许调节FATP2A和FATP2B的表达，实验将完成[1]以定义其在脂肪酸传输和激活中的各个角色，并且[2]解决它们是否将脂肪酸引导到不同的代谢池中，该脂肪酸是否使用稳定的同型脂肪酸与高分辨率相结合，以评估稳定的脂肪酸。为了获得FATP2在肠道和肝脏中的生理作用，SHRNA将用于敲低FATP2在CACO-2（肠类似）和HEPG2（肝喜欢）细胞中的表达，以及脂肪酸转运，激活和细胞内运输的模式评估。这两种变体的个别作用将使用从高吞吐量筛选中选择的五个小分子抑制剂进一步评估，以破坏脂肪酸转运的小化合物。总的来说，这些研究预计将提供有关FATP2A和FATP2B在脂肪酸稳态中的不同作用的新见解。障碍血症已被证明是代谢疾病，例如糖尿病，肥胖和心血管疾病的促成因素。表征参与脂质代谢的蛋白质可以帮助阐明控制这些病理状态的生物学过程。该研究建议的重点包括表征FATP2的功能，FATP2的功能有望深入了解脂质代谢的基础生物学过程。