Molecular Analysis of Fatty Acid Transport in Eukaryotes

真核生物中脂肪酸运输的分子分析

• 批准号: 6618519
• 负责人: Paul N. Black
• 金额: $ 29.3万
• 依托单位: ORDWAY RESEARCH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-06 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6618519
• 关键词: Saccharomyces cerevisiae; acid thiol ligase; enzyme substrate complex; fatty acid binding protein; fatty acid metabolism; fatty acid synthase; fatty acid transport; fungal proteins; long chain fatty acid; membrane transport proteins; protein isoforms; protein localization; protein structure function; site directed mutagenesis

DESCRIPTION (provided by applicant): Abnormalities in lipid metabolism are major contributing factors to various disease states including obesity, diabetes, and cardiomyopathies. Together, these diseases are the leading cause of death in the United States and most other developed countries. It is hypothesized that high circulating levels of lipids lead to excessive intraceliular accumulation of fatty acids and the resultant lipoatrophies of cells and tissues contributing to these disease states. Prevention and therapeutic intervention are therefore desirable and will require an understanding of the underlying molecular mechanisms leading to aberrant import and trafficking of fatty acids. Therefore, the focus of the present work is to determine the components and mechanisms governing fatty acid transport across a biological membrane. Current models suggest fatty acid import occurs by a mechanism involving simple diffusion and protein mediation. The present proposal employs Saccharomyces cerevisiae as a model system to define the transport mechanism. In S. cerevisiae long chain fatty acid import requires the putative transport protein Fat1p, a member of the highly conserved FATP family of proteins, and fatty acyl CoA synthetase (Faa1p or Faa4p). The FATP family shares amino acid identities with the very long-chain acyl CoA synthetase enzyme family. Current evidence supports the conclusion the protein has two activities: one in fatty acid transport and the other in activation of very long-chain fatty acids. Previous work from our laboratory demonstrated Fat1p and the murine homologue, mmFATP1, are functional orthologues. Fat1p is required when yeast are grown under anaerobic conditions to facilitate import of unsaturated fatty acids and is required for growth on media containing long chain fatty acids when fatty acid synthase is inhibited chemically. Growth and biochemical deficiencies resulting from mutations in the gene encoding Fat1p are alleviated by expression of mmFATP1 in yeast. Additional studies using directed mutagenesis of FAT1 have defined, through analyses of altered Fat1p proteins, functional domains and distinguished transport of long chain fatty acids from activation of very long chain fatty acids. The goals of the current experimental plan are to: [I] Define substrate specificity of different isoforms of mammalian FATP (mmFATP1, 2, 3, 4, and 5) expressed in S. cerevisiae for fatty acid transport and activation. [II] Further delimit subdomains and specific amino acid residues within Fat1p, which are essential for fatty acid transport, very long-chain fatty acyl CoA synthetase activity or both. [Ill] Define biochemical role of Fat1p in the trafficking of exogenous long-chain fatty acids using plasma membrane vesicles.

描述（由申请人提供）：脂质代谢异常是导致肥胖、糖尿病和心肌病等各种疾病状态的主要因素。这些疾病加在一起是美国和大多数其他发达国家的主要原因。据推测，高循环脂质水平导致脂肪酸在细胞内过度积累，并导致细胞和组织的脂肪萎缩，从而导致这些疾病状态。因此，预防和治疗干预是可取的，并且需要了解导致脂肪酸异常输入和运输的潜在分子机制。因此，目前工作的重点是确定控制脂肪酸跨生物膜转运的成分和机制。目前的模型表明脂肪酸输入是通过涉及简单扩散和蛋白质介导的机制发生的。本提案采用酿酒酵母作为模型系统来定义运输机制。在酿酒酵母中，长链脂肪酸的输入需要假定的转运蛋白 Fat1p（高度保守的 FATP 蛋白家族的成员）和脂酰辅酶 A 合成酶（Faa1p 或 Faa4p）。 FATP 家族与极长链酰基 CoA 合成酶家族具有相同的氨基酸特性。目前的证据支持该蛋白质具有两种活性的结论：一种是脂肪酸运输，另一种是极长链脂肪酸的激活。我们实验室之前的工作证明 Fat1p 和小鼠同源物 mmFATP1 是功能性直系同源物。当酵母在厌氧条件下生长以促进不饱和脂肪酸的输入时，Fat1p 是必需的，并且当脂肪酸合酶受到化学抑制时，Fat1p 是在含有长链脂肪酸的培养基上生长所必需的。酵母中 mmFATP1 的表达可以缓解由 Fat1p 编码基因突变引起的生长和生化缺陷。使用 FAT1 定向诱变的其他研究通过分析改变的 ​​Fat1p 蛋白、功能域和区分长链脂肪酸的运输和极长链脂肪酸的激活来定义。当前实验计划的目标是： [I] 定义酿酒酵母中表达的哺乳动物 FATP（mmFATP1、2、3、4 和 5）不同亚型对于脂肪酸转运和激活的底物特异性。 [II] 进一步界定 Fat1p 内的子结构域和特定氨基酸残基，这些残基对于脂肪酸运输、极长链脂肪酰基 CoA 合成酶活性或两者都是必需的。 [III] 定义 Fat1p 在使用质膜囊泡运输外源长链脂肪酸中的生化作用。