Human Proton-coupled Folate Transporter Research Articles

SCREENING OF PROTON PUMP INHIBITOR ANTAGONIST FOR HUMAN FOLATE TRANSPORTER

Folate transport is crucial for proper proliferation of cells. Amongst humans, the folate is procured from external sources since it is not synthesized in the human body. Hence specific transporters are involved in uptake of folate. For the malignant cancerous cells, these folate are crucially required on very frequent rate to fulfil the rapid proliferation of the cancerous cells. In the present study, we have comparatively studied three antagonist drug molecules which could be a potential candidate to inhibit the folate uptake by the human Proton Coupled folate transporter (hPCFT). These candidate antagonist include Pemetrexed, 2-[(4-Aminobenzoyl)amino] pentanedioate-pABA-Glu and Methotrexate. Out of these three antagonist drug, the Pemetrexed molecule was observed to specifically bind to the key active site loop (G155XXG158) of the hPCFT transporter. The Binding energy of the Pemetrexed was also comparatively lowest (-7.6 kcal/mol). Hence from the present study we conclude that the Pemetrexed antagonist drug is more e fficient to inhibit folate transport as compared to the 2-[(4-Aminobenzoyl)amino]pentanedioate-pABA-Glu and Methotrexate antagonist drug molecules.

Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter

Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.

Regulation of differential proton-coupled folate transporter gene expression in human tumors: transactivation by KLF15 with NRF-1 and the role of Sp1

Tumors can be therapeutically targeted with novel antifolates (e.g. AGF94) that are selectively transported by the human proton-coupled folate transporter (hPCFT). Studies were performed to determine the transcription regulation of hPCFT in tumors and identify possible mechanisms that contribute to the highly disparate levels of hPCFT in HepG2 versus HT1080 tumor cells. Transfection of hPCFT-null HT1080 cells with hPCFT restored transport and sensitivity to AGF94 Progressive deletions of the hPCFT promoter construct (-2005 to +96) and reporter gene assays in HepG2 and HT1080 cells confirmed differences in hPCFT transactivation and localized a minimal promoter to between positions -50 and +96. The minimal promoter included KLF15, GC-Box and NRF-1 cis-binding elements whose functional importance was confirmed by promoter deletions and mutations of core consensus sequences and reporter gene assays. In HepG2 cells, NRF-1, KLF15 and Sp1 transcripts were increased over HT1080 cells by ∼5.1-, ∼44-, and ∼2.4-fold, respectively. In Drosophila SL2 cells, transfection with KLF15 and NRF-1 synergistically activated the hPCFT promoter; Sp1 was modestly activating or inhibitory. Chromatin immunoprecipitation and electrophoretic mobility shift assay (EMSA) and supershifts confirmed differential binding of KLF15, Sp1, and NRF-1 to the hPCFT promoter in HepG2 and HT1080 cells that paralleled hPCFT levels. Treatment of HT1080 nuclear extracts (NE) with protein kinase A increased Sp1 binding to its consensus sequence by EMSA, suggesting a role for Sp1 phosphorylation in regulating hPCFT transcription. A better understanding of determinants of hPCFT transcriptional control may identify new therapeutic strategies for cancer by modulating hPCFT levels in combination with hPCFT-targeted antifolates.

Development and validation of chemical features-based proton-coupled folate transporter/activity and reduced folate carrier/activity models (pharmacophores)

All clinically used antifolates lack transport selectivity for tumors over normal cells resulting in dose-limiting toxicities. There is growing interest in developing novel tumor-targeted cytotoxic antifolates with selective transport into tumors over normal cells via the proton-coupled folate transporter (PCFT) over the ubiquitously expressed reduced folate carrier (RFC). A lack of X-ray crystal structures or predictive models for PCFT or RFC has hindered structure-aided drug design for PCFT-selective therapeutics. Four-point validated models (pharmacophores) were generated for PCFT/Activity (HBA, NI, RA, RA) and RFC/Activity (HBD, NI, HBA, HBA) based on inhibition (IC50) of proliferation of isogenic Chinese hamster ovary (CHO) cells engineered to express only human PCFT or only RFC. Our results revealed substantial differences in structural features required for transport of novel molecules by these transporters which can be utilized for developing transporter-selective antifolates.

Identification of the amino acid residue responsible for the flavonoid sensitivity of human proton-coupled folate transporter

Identification of the amino acid residue responsible for the flavonoid sensitivity of human proton-coupled folate transporter

Specific inhibitory effects of myricetin on human proton-coupled folate transporter: Comparison with its effects on rat proton-coupled folate transporter and human riboflavin transporter 3

Myricetin is a flavonoid that inhibits human proton-coupled folate transporter (hPCFT) in a transient manner, in which inhibition is manifested in its presence, and also in a sustained manner, in which inhibition induced in its presence persists after its removal. In an effort to elucidate the mechanisms involved in those, we examined if myricetin might or might not act similarly on some other transporters. Transporters examined for that, in comparison with hPCFT, were its rat ortholog (rPCFT) and human riboflavin transporter 3 (hRFVT3). Experiments were conducted, using human embryonic kidney 293 cells transiently expressing the transporter to be examined, to assess the effects of myricetin (100 μM) on the uptake of folate by the PCFTs and riboflavin by hRFVT3. For hPCFT, myricetin was confirmed to induce a transient inhibition and also a sustained inhibition. However, myricetin induced neither transient nor sustained type of rPCFT inhibition. hRFVT3 was inhibited by myricetin in a transient manner, but not in a sustained manner. These results suggest the involvement of a hPCFT-specific mechanism in the sustained inhibition. The transient inhibition may be induced by a mechanism specific to hPCFT and also hRFVT3.

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1).

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6-7 linker.

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate-proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236-265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6-7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6-7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1-6 and TMD7-12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252-265 from TMD7-12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6-7 loop (positions 236-250 or 251-265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT-ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6-7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

Can Yeast Salvage Folate? Bioinformatics Suggests Yes!

Folate is an important nutrient for cell growth and division in all organisms. Many microorganisms including prokaryotes and eukaryotes can synthesize their own folate while humans cannot. To assimilate folate into the cell a folate transporter is required for uptake through the cell membrane. Three types of folate transporters are known to exist in humans to facilitate the uptake of folate: Reduced Folate Carrier (RFC), Proton-Coupled Folate Transporter (PCFT) and Folate Receptors (FR). Although yeast cells can synthesize their own folate, laboratory evidence suggests that it can also utilize folate from exogenous source. However, little is known about the presence of folate transporters and other proteins involved in utilization of folate in yeasts. Hence using various bioinformatics analysis tools we attempted to determine the types of folate transporter present using genome sequences of well-studied yeasts from Entrez Database, Yeast Genome Database and Candia Genome Databases. Bioinformatics suggests the folate transporter present the yeast is similar to human PCFT with significant identity. All yeast possessed genes to encode the putative proteins which are similar to human PCFT. Saccharomyces cerevisiae’s putative protein had the highest identity match (identity=30.2%, E-value= <0.05) while Candida glabrata had the lowest (identity=22.6%, E-value: <0.05). Phylogenetic analysis showed yeast PCFT being distant from human PCFT but the conserved domain and hydropathy analysis indicated that it performs similar function to the human PCFT. Although bioinformatics suggests the presence of PCFT in yeast, genetic studies in the future could further help to understand the PCFT. If laboratory results are consistent with our finding the yeast could be used as a model organism to study many diseases in humans related to PCFT.

Expression, Purification and Functional Characterization of Human Proton-Coupled Folate Transporter (SLC46A1)

Folate cofactors play important roles in hundreds of metabolic reactions in a cell such as synthesis of protein and DNA precursors. The human proton-coupled folate transporter (PCFT) is the only means for absorption of dietary folates. PCFT also transports anti-cancer agents such as Pemetrexed and Methotrexate. Inadequate supply of folates and impaired PCFT function is associated with many disorders such as cancer, hereditary folate malabsorption, hypercysteinemia, heart diseases, obesity, anemia, neural tube defects and Alzheimer's disease. It is therefore important to study structural and functional characteristics of PCFT, to understand folate-homeostasis mechanisms and to develop better folate-based therapies. The primary requirement in structural and functional characterization of mammalian membrane proteins such as PCFT is the availability of good expression systems and methods to obtain large quantities of purified protein. To address this primary concern, we are establishing an efficient system to over-express PCFT and developing methods to obtain purified PCFT in good yields. We have expressed human PCFT in insect, yeast, and bacterial cells. We have successfully purified PCFT to homogeneity. Importantly, we also demonstrate that the heterologously-expressed PCFT is functional. Our findings are significant because 12-transmembrane helical, mammalian membrane proteins such as PCFT are notoriously difficult to express in versatile prokaryotic and single-celled eukaryotic systems. Furthermore, due to their predominant hydrophobic character and requirement of detergent and/or lipid environment for their stability, such membrane proteins are difficult to purify. Each of the three PCFT expression systems that we have developed offers unique advantages over another depending on the intended use of PCFT. Our findings will be significant for the advancement of knowledge concerning both the structure and function of PCFT through basic science research as well as for industrial applications including design and screening of PCFT-targeted agents.

Abstract 5403: Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT)

Abstract Pemetrexed (PMX) is a 5-substituted pyrrolo[2,3-d]pyrimidine antifolate used for first-line and maintenance therapy of NSCLC. Whereas correlations were reported between clinical responses in NSCLC and levels of folate receptor (FR) α, FRα is not a pharmacologically relevant PMX transporter. Rather, PMX is primarily transported by the reduced folate carrier (RFC) and PCFT. RFC is ubiquitously expressed, whereas PCFT shows more limited expression in normal tissues, although both are broadly expressed in tumor cells. Unlike RFC, PCFT is active at acidic pH, with significant levels at pH 6.8. Using patient cDNAs from normal lung (n = 8) and NSCLC samples (n = 37), we measured transcripts for RFC and PCFT, and for intracellular targets for PMX, including thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFTase), by real-time RT-PCR (qPCR). PCFT was detected at similar levels in normal lung and NSCLC specimens, while RFC was decreased in tumors compared to normal lung. Expression of TS and GARFTase were increased in NSCLC samples, compared to normal tissue. In NSCLC cell lines A549, CRL5872, H460, H1650 and H2030, PCFT and RFC, but not FRα, were detected by qPCR. We synthesized novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers related to PMX [AGF94 (2′,5′) and AGF154 (2′,4′)] as potential antitumor agents. Based on patterns of inhibition of proliferation of engineered Chinese hamster ovary cells expressing human RFC or PCFT, AGF94 and AGF154, unlike PMX, are not transported by RFC but are excellent substrates for PCFT. AGF94 and AGF154 showed potencies comparable to PMX toward all the NSCLC cell lines. We found that [3H]PMX and [3H]AGF154 were transported by PCFT into the NSCLC cell lines at pH 5.5 and pH 6.8 and uptake could be blocked by non-radioactive AGF94. AGF154 accumulation over sustained exposure at pH 5.5 was greater than that of PMX, whereas accumulations of AGF154 and PMX were nearly identical at pH 6.8. In H460 cells treated with PMX, thymidine (10 μM) partially protected cells from growth inhibition, whereas neither adenosine (10 μM) nor 5-aminoimidazole-4-carboxamide (AICA) (320 μM) were protective. In cells treated with AGF94 or AGF154, adenosine and AICA, but not thymidine, were completely protective, consistent with inhibition of de novo purine biosynthesis at GARFTase. Potent GARFTase inhibition by AGF94 and AGF154 was confirmed by an in situ assay which measures incorporation of [14C]glycine into formyl GAR (FGAR), the GARFTase product. Our results suggest that novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates may offer significant promise for treating NSCLC, reflecting their selective membrane transport by PCFT over RFC and potent inhibition of de novo purine biosynthesis. Citation Format: Mike R. Wilson, Zhanjun Hou, Lei Wang, Si Wang, Manohar Ratnam, Aleem Gangjee, Larry Matherly. Targeted therapy of non-small cell lung cancer (NSCLC) by novel 6-pyrrolo[2,3-d]pyrimidine thienoyl antifolates with selective transport by the proton-coupled folate transporter (PCFT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5403. doi:10.1158/1538-7445.AM2015-5403

Abstract 4483: Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter

Abstract Major folate uptake mechanisms include the reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), and folate receptors α and β. Antifolates such as methotrexate or pemetrexed are excellent substrates for human RFC (hRFC) and variously for human PCFT (hPCFT) and human folate receptor α (hFRα). Recent attention has focused on developing tumor-targeted folate-based therapies, reflecting tumor-selective uptake by hFRα and/or hPCFT. Novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl regioisomers AGF94 (2′,5′) and AGF154 (2′,4′) were synthesized and characterized as potent inhibitors of cell proliferation of engineered Chinese hamster ovary (CHO) cells expressing hPCFT or hFRα, but not for CHO cells expressing hRFC. In 41 ovarian cancer specimens from patients, hFRα, hPCFT and hRFC were all expressed. FRα transcript levels increased with stage of disease, although hPCFT levels were constant. IGROV1 is a clinically relevant ovarian cancer cell line that expresses both hFRα and hPCFT. IGROV1 cells were highly sensitive to both AGF94 and AGF154, as reflected in inhibition of cell proliferation in vitro. Growth inhibition by AGF94 or AGF154 in IGROV1 cells was partially reversed by excess folic acid, likely reflecting drug uptake by hFRα, as well as by hPCFT. To evaluate the impact of loss of hFRα on AGF94 and AGF154 in vitro inhibition of IGROV1 cell proliferation, FRα was knocked down (&amp;gt;95%) by lentivirus shRNAs. Clonal isolates were generated (n = 17) and profiled by RT-PCR for expression of the folate transporters and key enzymes likely to impact antifolate sensitivities (e.g., folylpolyglutamyl synthetase, β-glycinamide ribonucleotide formyltransferase), relative to a non-targeted control (NTC) cells. Gene expression profiles were similar for IGROV1 hFRα knockdown (KD) clones and NTC cells. Two representative hFRα KD clones were assayed for inhibition of cell proliferation by AGF94 and AGF154, with IC50s for IGROV1 KD clones of 26±1.8 and 26±9 nM for AGF94, and 65±1.2 and 81±2.4 nM for AGF154. For comparison, IC50s in the IGROV1 NTC cells expressing both hFRα and hPCFT were 0.6±0.1 nM for AGF94 and 2.2±0.8 nM for AGF154. In contrast, the hFRα-specific antifolate, AGF102 showed IC50s of &amp;gt; 1000 nM and ∼2.5 nM for KD and NTC cells. The preservation of substantial inhibitory activity for AGF94 and AGF154, accompanying the nearly complete loss of FRα expression in the KD clones is likely due to the sustained uptake of these novel antifolates by hPCFT. For AGF154, this was confirmed by direct uptake assays with [3H]-AGF154. Our results suggest that 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with substantial substrate activities for hFRα and hPCFT may offer significant advantages in treating tumors that express both these transport systems. Citation Format: Zhanjun Hou, Steve Orr, Christina George, Adrianne Wallace, Larry H. Matherly, Lei Wang, Si Yang, Aleem Gandjee. Impact of loss of folate receptor alpha on antitumor effects of novel 6-substituted pyrrolo[2,3-d]pyrimidine antifolates with substrate activities for both folate receptors and the proton-coupled folate transporter. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4483. doi:10.1158/1538-7445.AM2015-4483

Abstract 4544: Synthesis and biological evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates

Abstract Reduced folates are essential cofactors for the biosynthesis of purines and pyrimidines. Since humans do not synthesize folate, it is necessary to obtain these cofactors from dietary sources. In mammals, three specialized systems exist that mediate membrane transport of folates and antifolates across biological membranes. These include the reduced folate carrier (RFC), the primary route for the uptake of folates and antifolates in mammalian cells, folate receptors (FRs) α and β, and the proton-coupled folate transporter (PCFT). Whereas RFC is ubiquitously expressed, FRs and PCFT show a narrower pattern of tissue expression. Toxicity of clinically used antifolates is attributed to their lack of selectivity for tumor cells over normal cells, likely due in part to their RFC transport. Antifolates with tumor-selective FR and/or PCFT drug uptake would circumvent major toxicities of currently used antifolates. We previously described the synthesis and biological activity of AGF23, a 6-substituted pyrrolo[2,3-d]pyrimidine. Against FRα- and PCFT-expressing KB human tumor cells, AGF23 was potently inhibitory (IC50 = 1.9 nM). Two novel isosteres of AGF23, AGF215 and AGF234 with oxygen and nitrogen in place of carbon at C11, respectively, were designed and synthesized. This simple isosteric replacement of carbon with heteroatoms in the bridge affords compounds with different chain lengths, angles, conformations and extra hydrogen bond donors and/or acceptors compared to the parent carbon chain analog. AGF215 and AGF234 were compared to AGF23 as inhibitors of proliferation of engineered Chinese hamster ovary (CHO) cells expressing human PCFT (R2/PCFT4), FRα (RT16), or RFC (PC43-10). AGF215 and AGF234, like AGF23, were inactive toward PC43-10 cells. AGF23 and AGF234 showed low level activity toward R2/PCFT4 cells (IC50∼200 nM), whereas AGF215 was inactive. Toward FRα-expressing RT16 cells, all the analogs were potently active with IC50 values of 1-6 nM. Toward KB tumor cells, AGF215 and AGF234 were highly potent with IC50 values of 3 and 1.3 nM, respectively. AGF215 and AGF234 are potential analogs for further preclinical studies and analog design. Citation Format: Aleem Gangjee, Lalit Golani, Adrianne Wallaceb, Larry H. Matherly. Synthesis and biological evaluation of novel 6-substituted pyrrolo[2,3-d]pyrimidines as targeted antifolates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4544. doi:10.1158/1538-7445.AM2015-4544

Structural determinants of human proton-coupled folate transporter oligomerization: role of GXXXG motifs and identification of oligomeric interfaces at transmembrane domains 3 and 6.

The human proton-coupled folate transporter (hPCFT) is expressed in solid tumours and is active at pHs characterizing the tumour microenvironment. Recent attention focused on exploiting hPCFT for targeting solid tumours with novel cytotoxic anti-folates. hPCFT has 12 transmembrane domains (TMDs) and forms homo-oligomers with functional significance. The hPCFT primary sequence includes GXXXG motifs in TMD2 (G(93)XXXG(97)) and TMD4 (G(155)XXXG(159)). To investigate roles of these motifs in hPCFT function, stability and surface expression, we mutated glycine to leucine to generate single or multiple substitution mutants. Only the G93L and G159L mutants preserved substantial [(3)H]methotrexate (Mtx) transport when expressed in hPCFT-null (R1-11) HeLa cells. Transport activity of the glycine-to-leucine mutants correlated with surface hPCFT by surface biotinylation and confocal microscopy with ECFP*-tagged hPCFTs, suggesting a role for GXXXG in hPCFT stability and intracellular trafficking. When co-expressed in R1-11 cells, haemagglutinin-tagged glycine-to-leucine mutants and His10-tagged wild-type (WT) hPCFT co-associated on nickel affinity columns, suggesting that the GXXXG motifs are not directly involved in hPCFT oligomerization. This was substantiated by in situ FRET experiments with co-expressed ECFP*- and YFP-tagged hPCFT. Molecular modelling of dimeric hPCFT structures showed juxtaposed TMDs 2, 3, 4 and 6 as potential structural interfaces between monomers. hPCFT cysteine insertion mutants in TMD3 (Q136C and L137C) and TMD6 (W213C, L214C, L224C, A227C, F228C, F230C and G231C) were expressed in R1-11 cells and cross-linked with 1,6-hexanediyl bismethanethiosulfonate, confirming TMD juxtapositions. Altogether, our results imply that TMDs 3 and 6 provide critical interfaces for formation of hPCFT oligomers, which might be facilitated by the GXXXG motifs in TMD2 and TMD4.

The Human Proton-Coupled Folate Transporter: Determination of Conformation and Identification of the Folate-Binding Pocket

Folate cofactors play crucial roles in hundreds of reactions in cells including DNA and protein synthesis. The human proton-coupled folate transporter (PCFT) is the only means of absorption of dietary folates in humans. PCFT expression and function is associated with many disorders including hereditary folate malabsorption, neural tube defects, Down syndrome, cancer, heart diseases, Alzheimer's and Parkinson's disease. Upregulation of PCFT expression in tumor cells is of significant consideration for development of PCFT-targeted chemotherapeutic agents. However, not much is known about the structure and function of PCFT, which contributes to the low clinical success rate of folate-based agents. To address this gap in the knowledge we performed extensive Cys-mutagenesis studies. We analyzed 40 residues towards the extracellular face of PCFT, 35 positions towards the cytoplasmic face of PCFT and 28 positions along the proposed folate-binding pocket of PCFT. Based on the accessibility studies of the extracellular face of PCFT we determined loop-helix boundaries of this face and identified the glycerol-3-phosphate transporter (PDB#1PW4) and tripeptide-proton symporter (PDB#4APS) as the best templates for modeling PCFT. Based on the accessibility studies of the cytoplasmic face of PCFT, we identified loop-helix boundaries of this face. Here, we also show that our accessibility studies support the hypothesis that PCFT is present predominantly in an inward-open conformation in the absence of substrate (pH 7.5 and no folic acid). We also show that the folate-binding pocket of PCFT is formed by residues present in PCFT transmembrane helices I, IV, V, X and XI. Our results are of high significance in understanding the details of folate-homeostasis mechanisms and in design of PCFT-targeted therapeutic and diagnostic agents.

Sustained inhibition of proton-coupled folate transporter by myricetin

Myricetin is a flavonoid that has recently been suggested to interfere with the intestinal folate transport system. To examine that possibility, focusing on its sustained inhibitory effect on proton-coupled folate transporter (PCFT), the uptake of folate was examined in Caco-2 cells, in which PCFT is known to be in operation, in the absence of myricetin in the medium during uptake period after preincubation of the cells with the flavonoid (100 μM) for 1 h. This pretreatment induced an extensive and sustained reduction in the carrier-mediated component of folate uptake, which was attributable to a reduction in the maximum transport rate (Vmax). Although the affinity of the transporter for folate was increased at the same time as indicated by a reduction in the Michaelis constant (Km), the change in Km was overwhelmed in extent by that in Vmax. Consistent with the finding, folate transport by human PCFT stably expressed in Madin-Darby canine kidney II cells was reduced in a similar manner with simultaneous reductions in Vmax and Km by myricetin pretreatment. Attention may need to be given for a possibility that such a sustained inhibition of PCFT could potentially be a cause of the malabsorption of folate and also antifolate drugs.

Abstract 782: Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important

Abstract The proton coupled folate transporter (PCFT) is a proton-folate symporter with a twelve transmembrane domain (TMD) structure. Human PCFT (hPCFT) is highly expressed in many solid tumors and has an acidic pH optimum, suggesting its high activity in the acidic tumor microenvironment. Our recent studies have focused on exploiting PCFT for targeting solid tumors with novel cytotoxic antifolates. Predicted topology models suggest that the loop domain between TMDs 2 and 3 resides in the cytosol. However, mutations in TMD2-3 loop residues Asp109 or Arg113 result in loss of PCFT activity, suggesting an important role in transport activity. Additionally, TMD2 may itself form a part of the substrate binding domain, suggesting functional importance for TMD2 residues. Using site-directed mutagenesis, we replaced each of the seven cysteine (cys) residues of hPCFT with serine, creating functional “cys-less” PCFT for use in substituted cys accessibility methods (SCAM). We then used site-directed mutagenesis to create 34 single cys mutants spanning TMD2 and the TMD 2-3 loop region (positions 85-118) in a cys-less PCFT background. These mutants were transfected into R1-11 HeLa cells, which express no endogenous folate transporters. We measured [3H]methotrexate uptake at pH 5.5 as a readout of PCFT activity and confirmed PCFT protein expression by western blotting. All 34 mutants were detected on westerns and 29 mutants were active, while W107C, D109C, G112C, R113C, and R114C were inactive. Using functionally active mutants, we performed pull-down assays with 2-aminoethyl methanethiosulfonate-biotin (MTSEA-biotin) and steptavidin beads, in order to determine the aqueous accessibility of each mutant. We found that mutants G93C, F94W, G105C, A106C, S110C, and L116C reacted with MTSEA-biotin, suggesting that these residues are aqueous-accessible. For G93C, F94C, and L116C, MTSEA-biotin labeling was inhibited by pemetrexed pretreatment, and MTSEA-biotin treatment inhibited transport activity of G93C and F94C. Our results suggest that the TMD 2-3 loop domain is aqueous accessible and must form a novel functionally important “reentrant loop” structure. Additionally, residues located in TMD2 (Gly93 and Phe94) appear to play a key role in substrate binding to PCFT. Better understanding of functional and structural determinants of PCFT membrane transport will be critical to the development of novel PCFT-selective antifolate drugs for targeting solid tumors. Citation Format: Michael R. Wilson, Zhanjun Hou, Larry H. Matherly. Transmembrane 2-3 loop domain of the proton coupled folate transporter (PCFT) forms a novel “reentrant loop” structure and is functionally important. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2014-782

Abstract 783: Identification of structural determinants of human proton-coupled folate transporter oligomerization

Abstract The human proton-coupled folate transporter (hPCFT) is expressed in human solid tumors and is active at pHs characterizing the human microenvironment. hPCFT has 459 amino acids with 12 transmembrane domains and cytosolic N- and C-termini. Recent attention has focused on exploiting hPCFT for targeting solid tumors with novel cytotoxic antifolates (Kugel Desmoulin et al., Cancer Biol Ther 13: 1355-1373, 2012). hPCFT forms homo-oligomers with functional significance (Hou et al., J Biol Chem 287: 4982-4995, 2012). The hPCFT primary sequence includes GXXXG motifs in transmembrane domain (TMD) 2 (G93XXXG97) and 4 (G155XXXG159) that are analogous to dimerization motifs in other amphipathic proteins. To investigate the role of these GXXXG motifs in hPCFT oligomerization, we mutated Gly to Leu to obtain single (G93L, G97L, G155L, G159L) or multiple (G93L/G97L, G155L/G159L, G93L/G97L/G155L/G159L) mutants. Only with the G159L and G93L mutants, was [3H]methotrexate transport at pH 5.5 substantially preserved. Transport activity correlated closely with surface hPCFT levels by sulfo-NHS-SS-biotin labeling and Western blots. When hemagglutin (HA)-tagged G-to-L hPCFT mutants were co-expressed with FLAG-His10-tagged wild-type (WT) hPCFT in hPCFT-null HeLa cells, both mutant and WT proteins were retained on nickel affinity columns, suggesting that the hPCFT GXXXG motifs are not involved in protein oligomerization. This was substantiated by fluorescent resonance energy transfer (FRET) with N-terminal YFP-tagged and C-terminal CFP-tagged hPCFT constructs ectopically expressed in hPCFT-null HeLa cells. The GXXXG motifs in hPCFT are important for proper hPCFT folding and intracellular trafficking. A functional Cys-less (CL) hPCFT construct was generated in which the 7 Cys residues were replaced by Ser. Individual Cys insertion mutants were generated in which Cys was inserted from positions 115-137 in TMD3, and from positions 213-236 in TMD6. Constructs were transfected into hPCFT-null HeLa cells, followed by cross-linking with MTS-6-MTS (TMD3) or MTS-1-MTS (TMD6). Cross-links were detected for V118, A128, V132, Q136 and L137 in TMD 3, and for W213C, A227C, F228C, L234C, K235C, and E236C in TMD6, establishing proximities between vicinal TMDs in separate hPCFT monomers. These results strongly imply that TMD3 and TMD6 provide critical polypeptide interfaces for the formation of hPCFT oligomers. Identification of structural motifs or domains involved in hPCFT oligomerization may lead to novel approaches for therapeutically “rescuing” functionally impaired hPCFT mutants, or enhancing surface expression of hPCFT in tumors treated with hPCFT-selective antifolates. Key words: proton-coupled folate transporter, folate, oligomerization, cross-linking, transporter. Citation Format: Zhanjun Hou, M. Roy Wilson, Lucas Wilson, Sita Kugel Desmoulin, Jenny Huang, Larry H. Matherly. Identification of structural determinants of human proton-coupled folate transporter oligomerization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 783. doi:10.1158/1538-7445.AM2014-783

Substituted Cysteine Accessibility Reveals a Novel Transmembrane 2–3 Reentrant Loop and Functional Role for Transmembrane Domain 2 in the Human Proton-coupled Folate Transporter

The proton-coupled folate transporter (PCFT) is a folate-proton symporter highly expressed in solid tumors that can selectively target cytotoxic antifolates to tumors under acidic microenvironment conditions. Predicted topology models for PCFT suggest that the loop domain between transmembrane domains (TMDs) 2 and 3 resides in the cytosol. Mutations involving Asp-109 or Arg-113 in the TMD2-3 loop result in loss of activity. By structural homology to other solute carriers, TMD2 may form part of the PCFT substrate binding domain. In this study we mutated the seven cysteine (Cys) residues of human PCFT to serine, creating Cys-less PCFT. Thirty-three single-Cys mutants spanning TMD2 and the TMD2-3 loop in a Cys-less PCFT background were transfected into PCFT-null HeLa cells. All 33 mutants were detected by Western blotting, and 28 were active for [(3)H]methotrexate uptake at pH 5.5. For the active residues, we performed pulldown assays with membrane-impermeable 2-aminoethyl methanethiosulfonate-biotin and streptavidin beads to determine their aqueous-accessibilities. Multiple residues in TMD2 and the TMD2-3 loop domain reacted with 2-aminoethyl methanethiosulfonate-biotin, establishing aqueous accessibilities. Pemetrexed pretreatment inhibited biotinylation of TMD2 mutants G93C and F94C, and biotinylation of these residues inhibited methotrexate transport activity. Our results suggest that the TMD 2-3 loop domain is aqueous-accessible and forms a novel reentrant loop structure. Residues in TMD2 form an aqueous transmembrane pathway for folate substrates, and Gly-93 and Phe-94 may contribute to a substrate binding domain. Characterization of PCFT structure is essential to understanding the transport mechanism including the critical determinants of substrate binding.

Experimentally Defined Structural Model of the Human Proton-Coupled Folate Transporter

Folate cofactors, Vitamin B9, play crucial roles in more than a hundred one-carbon metabolism reactions in mammalian cells. Humans cannot synthesize folates de novo and absorption through the diet is the only source of this vitamin. The proton-coupled folate transporter (PCFT) mediates this uptake in the upper small intestine by a pH-dependent process. PCFT also transports folates into the central nervous system. Point mutations in the PCFT gene cause Hereditary Folate Malabsorption (HFM), with associated hematological and neurological defects due to impaired folate transport. Certain solid tumor cell lines express high levels of PCFT mRNA. Importantly, the overall expression of PCFT is limited to only certain tissues in humans. Therefore, PCFT is a molecular target for specific delivery of anti-folate chemotherapeutic agents to tumor cells. Unfortunately, the success rate of anti-folate agents to reach clinical use is very low due to their side-effects. A structural model of PCFT can aid the development of specific anti-folate agents for their PCFT-targeted delivery and thus minimize their side-effects. To optimize and verify an initial structural model of PCFT, we applied a wide array of experimental approaches: solvent accessibility profiling through substituted cysteine accessibility scanning, studying the helix packing, and assaying the functionality of PCFT mutants. We complemented the experimental data with theoretical approaches for structure prediction such as homology modeling/threading, ligand docking and an extensive review of other secondary transporters and folate transport proteins. With these combined approaches, we have developed a structural model of PCFT that accurately reflects our experimental observations. This model forms a basis to understand the impacts of HFM mutations, and to develop folate analogues for folate deficiency intervention. Additionally, it will aid the design of PCFT structure-based anti-folate agents for the treatment of cancer.