Molecular Regulation of Folate and Antifolate Transport

描述（由申请人提供）：叶酸是生命所必需的，叶酸缺乏会导致一系列健康问题，包括心血管疾病、胎儿异常、神经系统疾病和癌症。以甲氨蝶呤和最近的雷替曲塞和培美曲塞为代表的抗叶酸药，继续在现代药典中占据独特而重要的位置，用于治疗癌症和其他病理状况。该续期申请旨在更好地了解人类还原性叶酸载体（hRFC）和人类质子偶联叶酸转运体（hPCFT）的生物学和治疗潜力，这是人类细胞、组织和肿瘤中叶酸和抗叶酸的两种主要膜转运系统，它们广泛表达，但在功能和解剖学上是不同的。我们对hRFC的结构功能研究为其膜拓扑结构、n -糖基化、重要结构域和氨基酸以及三维包装关联提供了实质性的见解。最近，我们开始表征hRFC的四级结构，明确地鉴定了hRFC单体分子组成的hRFC同源低聚物，并获得了令人信服的证据，证明失活的hRFC突变体对野生型hRFC具有“负优势”，包括对细胞内运输和/或hRFC单体之间的功能“偶联”的下游影响。在2006年确定了低pH叶酸转运蛋白hPCFT之后，我们开始了一系列的研究，旨在直接评估这种转运蛋白相对于hRFC对癌症可能的治疗重要性。生成的关键试剂包括稳定转染的细胞系（包括tet调节模型），功能性“无半胱氨酸”hPCFT构建物，hPCFT蛋白抗体，以及可能最令人兴奋的，第一代不通过hRFC运输的特异性和强效hPCFT细胞毒性底物。我们提出的研究目的1将描述hRFC同聚物的结构、功能和调控特征。广泛的生化、细胞和分子方法将用于确定hRFC低聚物的最小功能单元和结构特征，并探索hRFC低聚物在运输机制、抗叶酸抗性和生化调节方面的意义。目的2将确定治疗潜力，以及hPCFT的结构和功能特征。重点是利用细胞毒性抗叶酸药物治疗靶向肿瘤的可能性，这些药物对hPCFT（和叶酸受体）具有选择性，但不通过hRFC运输。拟议的研究包括hPCFT和hRFC肿瘤表达和活性谱的测定，hRFC和hPCFT的功能化学计量学，以及这些运输系统和叶酸受体伴随肿瘤表达的治疗影响。我们提出的研究得到了我们初步和已发表的结果的有力支持，并因其新颖性和可能为这些生理和药理学上重要的膜转运蛋白的机制和调控提供关键的新见解而与众不同。公共卫生相关性：叶酸对生命至关重要，叶酸缺乏会导致一系列健康问题，包括心血管疾病、胎儿畸形、神经系统疾病和癌症。以甲氨蝶呤和最近的雷替曲塞和培美曲塞为代表的抗叶酸药，继续在现代药典中占据独特而重要的位置，用于治疗癌症和其他病理状况。该申请旨在更好地了解人类还原性叶酸载体（hRFC）和人类质子偶联叶酸转运体（hPCFT）的生物学和治疗潜力，这是人类细胞、组织和肿瘤中叶酸和抗叶酸的两种主要膜转运系统，它们广泛表达，但在功能和解剖学上不同。我们提出的研究为设计包括实体肿瘤在内的癌症的新疗法以及在膳食叶酸补充和抗叶酸治疗癌症和其他疾病的背景下治疗改变转运蛋白水平和功能的方法提供了重要的前奏曲。

DESCRIPTION (provided by applicant): Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. This renewal application for a grant currently in its 19th year proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our structure-function studies of hRFC have provided substantial insights into its membrane topology, N-glycosylation, important domains and amino acids, and three-dimensional packing associations. Most recently, we began to characterize hRFC quaternary structure and unambiguously identified hRFC homo-oligomers composed of monomeric hRFC molecules, and obtained compelling evidence for a "negative dominance" of wild type hRFC by inactive hRFC mutants, involving downstream effects on intracellular trafficking and/or functional "coupling" between hRFC monomers. Following identification of the low pH folate transporter, hPCFT, in 2006, we began a series of studies designed to directly assess the possible therapeutic importance of this transporter vis ` vis hRFC for cancer. Key reagents were generated including stably transfected cell lines (including Tet-regulated models), a functional "cysteine-less" hPCFT construct, antibodies to the hPCFT protein, and, perhaps most exciting, the first generation of specific and potent hPCFT cytotoxic substrates that are not transported by hRFC. Aim 1 of our proposed study will characterize the structural, functional, and regulatory features of hRFC homo- oligomers. A wide range of biochemical, cellular, and molecular approaches will be used to identify the minimum functional unit and characterize the structure of oligomeric hRFC, and to explore the implications of hRFC oligomers to transport mechanism, antifolate resistance, and biochemical modulation. Aim 2 will establish the therapeutic potential, along with the structural and functional features of hPCFT. The focus is overtly on possibilities for therapeutic targeting tumors with cytotoxic antifolate drugs that are selective for hPCFT (and folate receptors) but are not transported by hRFC. Proposed studies include assays of hPCFT and hRFC tumor expression and activity profiles, functional stoichiometries for hRFC and hPCFT, and the therapeutic impact of concomitant tumor expression of these transport systems, as well as folate receptors. Our proposed studies are strongly supported by our preliminary and published results, and are distinctive for their novelty and likelihood of providing critical new insights into mechanism and regulation of these physiologically and pharmacologically important membrane transporters. PUBLIC HEALTH RELEVANCE: Folates are essential for life and folate deficiency contributes to a range of health problems including cardiovascular disease, fetal abnormalities, neurologic disorders, and cancer. Antifolates, represented by methotrexate and, more recently, raltitrexed and pemetrexed, continue to occupy a unique and important niche among the modern day pharmacopoeia for cancer along with other pathologic conditions. The application proposes to better understand the biology and therapeutic potential of the human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT), two of the major membrane transport systems for folates and antifolates in human cells, tissues and tumors which are widely expressed, yet are functionally and anatomically distinct. Our proposed studies provide an important prelude for the design of new therapeutics for cancer including solid tumors and approaches for therapeutically altering transporter levels and function in the context of dietary folate supplementation and antifolate therapy of cancer and other diseases.