FOLATE BINDERS--CONSTITUTIVE/FOLATE DEPENDENT EXPRESSION

叶酸结合剂——组成型/叶酸依赖性表达

• 批准号: 2227218
• 负责人: SHELDON P ROTHENBERG
• 金额: $ 14.69万
• 依托单位: SUNY DOWNSTATE MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2227218
• 关键词: FOLATE; BINDERS; CONSTITUTIVE; DEPENDENT; EXPRESSION

Folate is an essential nutrient that functions as a carrier of single carbon units for the biosynthesis of nucleic acids, the methylation of homocysteine for the biosynthesis of methionine, for the metabolism of histidine, and for the serine-glycine interconversion. This important role of folate in intracellular metabolism is reflected in the clinical disorder of folate deficiency and megaloblastic anemia that occurs with nutritional deficiency malabsorption (acquired and congenital), augmented consumption (pregnancy, hemolytic anemia) and disorders of metabolism (drugs and toxins). It also has been the basis for the development and therapeutic application of antifolate drugs to inhibit the growth of neoplastic cells. An essential component of folate homeostasis is the process by which folate is transported into cells and this occurs by two mechanisms: 1) an invariant pathway which utilizes a transmembrane anion channel that is selective for reduced anionic folates and the antifol, methotrexate, and excludes pteroyglutamic acid, an anionic but non-reduced folate; and 2) a variant mechanism which utilizes a folate binding protein (FBP) (or receptor) that is anchored to the external surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) adduct. In contrast to the invariant transmembrane channel system, the GPI-anchored FBP is constitutively expressed on very few cells but may be induced by slow conditioning in a low folate environment. However, cells that constitutively express the GPI-FBP can grow under low folate conditions (approximately 1 nM) without such conditioning. The purification and structural and functional properties of a number of human FBP(s) including the GPI-FBP, the cloning of the complementary DNA (cDNA) encoding one GPI- FBP, the isolation of an unprocessed FBP pseudogene and the isolation and characterization of a gene encoding a FBP expressed in human placenta have been reported from this laboratory. This gene (assigned FBP/PL-1 gene) has an unusual cis promoter element comprised of a G-rich SP1 binding sequence linked to tandemly duplicated GGAAG pentameric sequences that are the motifs for the ets oncogene encoding GA binding nuclear transcription factor and which have been identified as regulatory elements in the genes encoding subunits of cytochrome c oxidase. We now propose to continue our studies with the following two objectives: 1) To isolate and characterize additional genes that constitute the multigene family that encode the membrane-associated and other cellular FBP(s); 2) To characterize cis promoter elements and enhancer sequences that regulate transcription, identify and purify trans-active nuclear factors that interact with these regulatory genomic elements and characterize cytoplasmic factors that regulate expression at the level of translation. These studies will enhance our understanding of the cellular transport and intracellular metabolism of folate cofactors and could provide a basis for up-modulation of these mechanisms to maintain normal folate homeostasis when environmental factors may induce folate deficiency or impaired metabolism.

叶酸是一种必需的营养素，作为一种载体， 用于核酸生物合成的碳单元， 同型半胱氨酸用于甲硫氨酸的生物合成，用于 组氨酸和丝氨酸-甘氨酸相互转化。这一重要作用 叶酸在细胞内代谢中的作用反映在临床上， 叶酸缺乏症和巨幼细胞性贫血， 营养缺乏吸收不良（后天和先天性），加重 消耗（怀孕，溶血性贫血）和代谢紊乱 （毒品和毒素）。它也是发展和 抗叶酸剂药物抑制肿瘤生长的治疗应用 肿瘤细胞 叶酸稳态的一个重要组成部分是 叶酸被转运到细胞中，这通过两种机制发生：1） 利用跨膜阴离子通道的不变途径， 对还原阴离子叶酸和抗真菌药、甲氨蝶呤具有选择性， 不包括蝶酰谷氨酸，一种阴离子但非还原的叶酸;和2）a 利用叶酸结合蛋白（FBP）的变体机制（或 受体），其锚定在质膜的外表面 通过糖基磷脂酰肌醇（GPI）加合物。相对于 恒定跨膜通道系统，GPI锚定的FBP是 在极少数细胞上组成型表达，但可由缓慢的 在低叶酸环境中调理。然而， 组成型表达GPI-FBP可以在低叶酸条件下生长 （约1 nM）。纯化和 许多人FBP的结构和功能特性，包括 GPI-FBP，克隆编码一个GPI-FBP的互补DNA（cDNA）， 未加工的FBP假基因的分离以及未加工的FBP假基因的分离和 在人胎盘中表达的编码FBP的基因的表征具有 从这个实验室报告。该基因（指定的FBP/PL-1基因）具有 一个不寻常的顺式启动子元件，由富含G的SP1结合序列组成 连接到串联重复的GGAAG五聚体序列， 编码GA结合核转录的ets癌基因的基序 因子，并已被确定为基因中的调控元件 编码细胞色素c氧化酶的亚基。 我们现建议继续进行研究，以达致以下两个目标： 1)为了分离和表征构成这些基因的其他基因， 多基因家族，编码膜相关和其他细胞 2）表征顺式启动子元件和增强子序列 其调节转录、鉴定和纯化反式活性核 与这些调控基因组元件相互作用的因子， 表征细胞质因子，其在以下水平调节表达： 翻译.这些研究将增强我们对细胞的理解， 叶酸辅因子的转运和细胞内代谢，并可 为这些机制的上调提供了基础， 当环境因素可能导致叶酸缺乏时，叶酸稳态 或者新陈代谢受损