Molecular Regulation of Folate and Antifolate Transport

描述（由申请人提供）：该项目的目标是表征还原性叶酸载体（RFC）的结构、功能和调控，RFC是哺乳动物细胞中还原性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转运对于甲氨蝶呤和以Tomudex和培美曲塞为代表的新一代抗叶酸药物的抗肿瘤活性也至关重要，RFC的改变导致药物摄取减少，并有助于抗叶酸药物耐药性。该更新申请扩展了我们实验室最近取得的重大进展，包括：(a)人类RFC （hRFC）基因的表征，并展示了涉及6个交替剪接的5'非编码外显子（A1/2, a，B，C，D，E）的转录和转录后控制的显着复杂性，以及从翻译开始上游超过35 kb的独特启动子；(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连接体]；残留204 - 214];(c)开发功能性“无半胱氨酸”hRFC，并通过巯基试剂替代半胱氨酸可及性方法（SCAM）将转运功能定位于TMD 1侧边的外表面环域；(d) hRFC膜拓扑结构的部分测定（TMDsl-8）；(e)纯化表位（His10）标记的hRFC。

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.