FOLATE BINDERS--CONSTITUTIVE/FOLATE DEPENDENT EXPRESSION

叶酸是一种基本的营养物质，其功能是作为单一营养物质的载体 核酸生物合成的碳单位，甲基化 同型半胱氨酸用于蛋氨酸的生物合成，用于 组氨酸和丝氨酸-甘氨酸的相互转化。这一重要角色 叶酸在细胞内代谢中的作用体现在临床上 叶酸缺乏症和巨幼细胞性贫血 营养缺乏性吸收不良(后天和先天性)，加重 消耗(妊娠、溶血性贫血)和代谢紊乱 (毒品和毒素)。它也是发展和发展的基础 抗叶酸药物在抑制卵巢癌生长中的应用 肿瘤细胞。 叶酸动态平衡的一个重要组成部分是通过以下过程 叶酸通过两种机制转运到细胞内：1) 利用跨膜阴离子通道的不变途径 选择性地选择还原的阴离子叶酸和安非他明、甲氨蝶呤和 不包括翼谷氨酸，一种阴离子但非还原的叶酸；和2)a 利用叶酸结合蛋白(FBP)(或 受体)固定在质膜外表面 通过糖基磷脂酰肌醇(GPI)加合物。与之相对的是 不变的跨膜通道系统，GPI锚定的FBP是 在极少数细胞上组成性表达，但可能被慢速诱导 在低叶酸环境中进行调节。然而，细胞 结构性表达GPI-FBP能在低叶酸条件下生长 (约1海里)，没有这样的条件。提纯和 S等人纤维蛋白原的结构和功能性质 GPI-FBP，编码一个GPI-FBP的互补DNA的克隆 FBP，一个未加工的FBP假基因的分离和分离 人胎盘中表达的FBP编码基因的鉴定 已经从这个实验室报告了。该基因(命名为FBP/PL-1基因)具有 一种由富含G的SP1结合序列组成的不寻常的顺式启动子元件 链接到重复的GGAAG五聚体序列，这些序列是 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节元件 编码细胞色素c氧化酶的亚基。 我们现在建议继续研究以下两个目标： 1)分离和鉴定构成基因的其他基因 编码膜相关和其他细胞的多基因家族 FBP(S)；2)鉴定顺式启动子元件和增强子序列 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和 描述在细胞水平上调节表达的细胞质因子 翻译。这些研究将加深我们对细胞 叶酸辅助因子的转运和细胞内代谢 为上调这些机制以维持正常提供基础 环境因素可能导致叶酸缺乏时的叶酸稳态 或新陈代谢受损。

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.