Molecular Regulation of Folate and Antifolate Transport

描述(申请人提供)：这个项目的目标是描述还原叶酸载体(RFC)的结构、功能和调控，RFC是哺乳动物细胞中还原叶酸辅助因子的主要膜运输系统。充足的叶酸供应对所有组织的生长和发育都是至关重要的，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的抗肿瘤活性也是至关重要的，RFC的改变导致药物摄取减少并导致抗叶酸耐药性。这一新的应用扩展了我们实验室最近的重大进展，包括：(A)人类RFC(HRFC)基因的特征以及涉及6个交替拼接的5‘非编码外显子(A1/2、A、B、C、D、E)和独特的启动子的转录和转录后调控的显著复杂性；(B)鉴定功能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)开发功能性的“无半胱氨酸”HRFC，并通过取代半胱氨酸可及性方法(SCAM)和硫醇反应试剂定位转运功能到TMD 1两侧的外环结构域；(D)HRFC膜拓扑结构的部分测定(TMDs1-8)；以及(E)标记表位(His10)的HRFC的纯化。 为了继续我们的研究，我们将在目标1(I)建立TMD 9-12和TMD-环边界的拓扑，以及(Ii)通过SCAM定位(抗)叶酸底物的可能结合位点，鼠-人嵌合体RFC的表达，His10标记的HRFC蛋白的放射亲和标记，以及定点突变。(Iii)HRFC寡聚体结构将通过共分离HA-/His10标记的hRFCs、分析性凝胶过滤和蛋白脂体重组来表征。在目标2中，我们将通过实时定量聚合酶链式反应和核糖核酸酶保护，建立(Iv)人类组织、肿瘤和细胞系中5‘非编码区和启动子的利用模式。其他研究将描述：(V)HRFC-A1/2、-D和-E启动子的主要调控特征(如顺式元件、转录因子)；(Vi)转录后调控，包括主要5‘非翻译区(UTRs)和剪接形式对翻译效率和转录稳定性的影响，以及对HRFC水平和功能的影响；(Vii)从A1/2和A5’UTRs框内Aug翻译的新的HRFC异构体的意义。基于这些结果，我们将()通过细胞叶酸和/或通过改变叶酸/核苷酸池的药物操作来表征HRFC的转录和转录后调控。我们的研究应该确定HRFC基因的主要调控特性和在人类肿瘤和组织中的异质性HRFC转录本，以及HRFC蛋白与叶酸和抗叶酸底物结合和膜转位的分子决定因素。

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.