Expression, purification and characterization of the human proton-coupled folate transporter
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The human proton-coupled folated transporter (PCFT, SLC46A1) is the primary means for the absorption of dietary folates. Folates (Vitamin B9) and folate cofactors are required for more than a hundred metabolic reactions in cells including synthesis of DNA, protein and the one-carbon donor S-adenosyl methionine. PCFT and folates are associated with numerous disorders including the rare autosomal disorder hereditary folate malabsorption, nerual tube defects, cancer, stroke and heart diseases, anemia, and Alzheimer's disease. Therefore, PCFT is referred to as a molecular player for life and dieseases. Folate-based agents have been in development for the treatment of diseases for more than seven decades, yet only a few agents are effective drugs. For folate-based agents that are a part of the chemotherapy regimen, undesired effects and drug resistance development constitute the major limitations. A significant obstacle in the development of new as well as more effective and improved drugs stems from the fact that a detailed structure as well as the molecular mechanism of folate translocation of PCFT are presently lacking. Despite the fact that PCFT works optimally at acidic pH, it is expressed in certain tissue environments with pH 7.4. This leaves a major gap in the understanding of folate homeostasis pathways. This dissertation addresses key questions about PCFT: (A) structural characterization of PCFT through mutagenesis based topological analysis; (B) identification of the functionally important Motif A that could be a novel target for PCFT-targeted drug design; and (C) identification of the folate-binding pocket and development of a click chemistry-based photoaffinity labeling approach to characterize the folate-binding pocket. Additionally, three heterologous expression systems for PCFT, and protocols to purify PCFT - importantly, in its functional form - have been developed. The expression systems established within the present dissertation represent crucial progress in the field of PCFT by providing access to many new biophysical-biochemical techniques such as high-throughput functional screening, and high-resolution structural studies where milligram quantities of purified PCFT are needed. Together these advancements are a significant step towards understanding the molecular players for life and diseases through folate pathways.