Molecular Regulation of Folate and Antifolate Transport

描述（由申请人提供）：本项目的目标是表征还原叶酸载体（RFC）的结构、功能和调节，RFC是哺乳动物细胞中还原叶酸辅因子的主要膜转运系统。充足的叶酸供应对所有组织的生长和发育至关重要，越来越多的证据表明叶酸缺乏会导致染色体不稳定和恶性转化。RFC转运对甲氨蝶呤和以Tomudex和培美曲塞为代表的新一代抗叶酸剂的抗肿瘤活性也至关重要，RFC改变导致药物摄取减少并导致抗叶酸剂耐药性。该更新申请扩展了我们实验室最近的重大进展，包括：（a）人类RFC（hRFC）基因的表征和涉及6个交替剪接的5 '非编码外显子的转录和转录后控制的显着复杂性的证明（A1/2、A、B、C、D、E）和定位在翻译起始点上游35 kb以上的独特启动子;（B）鉴定功能上或结构上重要的带电氨基酸（D88，R133，R373，K411）或结构域[跨膜结构域（TMD）6/7接头;残基204-214];（c）开发功能性“无半胱氨酸”hRFC，并通过用硫醇反应剂的取代的半胱氨酸可及性方法（SCAM）将转运功能定位于TMD 1侧翼的外表面环结构域;和（e）纯化表位（His 10）标记的hRFC。 对于我们的继续研究，我们将在目标1中（i）建立TMD 9-12和TMD环边界的拓扑结构，以及（ii）通过SCAM、小鼠-人嵌合体RFC的表达、His 10标记的hRFC蛋白的放射亲和标记和定点诱变绘制（抗）叶酸底物的推定结合位点。(iii)hRFC寡聚体结构将通过HA-/His 10-标记的hRFC的共分级分离、分析凝胶过滤和蛋白脂质体重构来表征。在目标2中，我们将通过实时PCR和RNA酶保护建立（iv）人组织、肿瘤和细胞系中5 'UTR和启动子利用的模式。其他研究将描述：（五）主要的监管特点（例如，hRFC-A1/2、-D和-E启动子的顺式元件、转录因子）;（vi）转录后控制，包括主要5'非翻译区（UTR）和剪接形式对翻译效率和转录稳定性以及对hRFC水平和功能的影响;和（vii）从A1/2和A5' UTR中的框内AUC翻译的新hRFC同种型的意义。基于这些结果，我们将（viii）表征细胞叶酸和/或改变叶酸/核苷酸池的药理学操作对hRFC的转录和转录后调节。我们的研究应确定的主要调控特性的hRFC基因和异质性hRFC转录在人类肿瘤和组织，叶酸和抗叶酸底物结合和膜转位的hRFC蛋白的分子决定因素。

DESCRIPTION (provided by applicant): The goal of this project is to characterize the structure, function, and regulation of the reduced folate carrier (RFC), the major membrane transport system for reduced folate cofactors in mammalian cells. An adequate supply of folates is essential for growth and development of all tissues and there is increasing evidence that folate deficiency contributes to chromosomal instability and malignant transformation. RFC transport is also critical to antitumor activities of methotrexate and a new generation of antifolates typified by Tomudex and Pemetrexed, and RFC alterations result in decreased drug uptake and contribute to antifolate resistance. This renewal application expands on recent significant advances by our laboratory including: (a) characterization of the human RFC (hRFC) gene and demonstration of a remarkable complexity of transcriptional and posttranscriptional controls involving 6 alternately spliced 5'non-coding exons (A1/2,A,B,C,D,E) and unique promoters mapping over 35 kb upstream from the translational start; (b) identification of functionally or structurally important charged amino acids (D88,R133,R373,K411) or domains [transmembrane domain (TMD) 6/7 linker; residues 204-214]; (c) development of a functional "cysteine-less" hRFC and localization of transport function to an exofacial loop domain flanking TMD 1 by substituted cysteine accessibility methods (SCAM) with thiol reactive agents; (d) partial determination of hRFC membrane topology (TMDsl-8); and (e) purification of epitope (His10)-tagged hRFC. For our continued studies, we will in Aim 1 (i) establish the topology of TMDs 9-12 and TMD-loop boundaries, and (ii) map putative binding sites for (anti)folate substrates by SCAM, expression of mouse-human chimera RFCs, radioaffmity labeling of His10-tagged hRFC protein, and site-directed mutagenesis. (iii) hRFC oligomeric structures will be characterized by co-fractionation of HA-/His10-tagged hRFCs, analytical gel filtration, and proteoliposome reconstitution. In Aim 2, we will establish (iv) the patterns of 5'UTR and promoter utilization in human tissues, tumors, and cell lines by real-time PCR and RNAse protection. Other studies will characterize: (v) the major regulatory features (e.g., cis elements, transcription factors) of the hRFC-A1/2, -D, and -E promoters; (vi) posttranscriptional controls including effects of the major 5' untranslated regions (UTRs) and splice forms on translational efficiencies and transcript stabilities, and on hRFC levels and function; and (vii) the significance of novel hRFC isoforms translated from in-frame AUGs in the A1/2 and A 5'UTRs. Based on these results, we will (viii) characterize the transcriptional and posttranscriptional regulation of hRFC by cellular folates and/or by pharmacologic manipulations that alter folate/nucleotide pools. Our studies should identify on the major regulatory properties of the hRFC gene and heterogeneous hRFC transcripts in human tumors and tissues, and the molecular determinants of folate and antifolate substrate binding and membrane translocation by the hRFC protein.