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、胎儿畸形、神经系统疾病和癌症。以甲氨蝶呤为代表的叶酸拮抗剂，以及最近的雷替曲塞和培美曲塞，继续在现代药典中占据独特而重要的位置，用于癌症沿着其他病理状况。本授权的更新申请目前已进入第19个年头，旨在更好地了解人还原叶酸载体（hRFC）和人质子偶联叶酸转运蛋白（hPCFT）的生物学和治疗潜力，这两种主要的膜转运系统是叶酸和抗叶酸剂在人类细胞、组织和肿瘤中广泛表达，但在功能和解剖学上不同。我们对hRFC的结构-功能研究为hRFC的膜拓扑结构、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四级结构，并明确确定hRFC单体hRFC分子组成的同源寡聚体，并获得了令人信服的证据，野生型hRFC的“负优势”的失活hRFC突变体，涉及下游影响细胞内运输和/或功能性“耦合”之间的hRFC单体。在2006年鉴定出低pH叶酸转运蛋白hPCFT后，我们开始了一系列旨在直接评估这种转运蛋白维斯维斯hRFC对癌症的可能治疗重要性的研究。产生了关键试剂，包括稳定转染的细胞系（包括Tet-regulated模型），功能性的“无半胱氨酸”hPCFT构建体，hPCFT蛋白的抗体，以及可能最令人兴奋的，第一代特异性和有效的hPCFT细胞毒性底物，其不被hRFC转运。本研究的目的一是探讨hRFC同源寡聚体的结构、功能和调控特性.广泛的生物化学，细胞和分子的方法将被用来确定最小的功能单元和寡聚体hRFC的结构特征，并探讨hRFC寡聚体的运输机制，抗叶酸剂抗性和生化调节的影响。目的2将确立hPCFT的治疗潜力，沿着hPCFT的结构和功能特征。焦点明显地集中在用细胞毒性抗叶酸药物治疗靶向肿瘤的可能性上，所述细胞毒性抗叶酸药物对hPCFT（和叶酸受体）具有选择性，但不被hRFC转运。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，以及这些转运系统以及叶酸受体的伴随肿瘤表达的治疗影响。我们提出的研究得到了我们的初步和已发表结果的有力支持，并且因其新奇和可能性而与众不同，这些新颖性和可能性为这些生理和非生理重要的膜转运蛋白的机制和调控提供了关键的新见解。公共卫生相关性：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、胎儿畸形、神经系统疾病和癌症。以甲氨蝶呤为代表的叶酸拮抗剂，以及最近的雷替曲塞和培美曲塞，继续在现代药典中占据独特而重要的位置，用于癌症沿着其他病理状况。本申请提出了更好地理解人还原叶酸载体（hRFC）和人质子偶联叶酸转运蛋白（hPCFT）的生物学和治疗潜力，这两种是人细胞、组织和肿瘤中广泛表达的叶酸和抗叶酸剂的两种主要膜转运系统，但在功能和解剖学上不同。我们提出的研究为癌症（包括实体瘤）的新疗法的设计以及在癌症和其他疾病的膳食叶酸补充剂和抗叶酸治疗的背景下治疗性改变转运蛋白水平和功能的方法提供了重要的前奏。

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.