105 research outputs found
Post-transcriptional regulation of the human reduced folate carrier as a novel adaptive mechanism in response to folate excess or deficiency
A unique role of GATA1S in down syndrome acute megakaryocytic leukemia biology and therapy
Background: Acute megakaryocytic leukemia (AMkL) in Down syndrome (DS) children is uniformly associated with somatic GATA1 mutations, which result in the synthesis of a shorter protein (GATA1s) with altered transactivation activity compared to the wild-type GATA1. It is not fully established whether leukemogenesis and therapeutic responses in DS AMkL patients are due to loss of the wild-type GATA1 or due to a unique function of GATA1s. Methodology: Stable clones of CMK cells with decreased GATA1s or Bcl-2 levels were generated by using GATA1- or BCL-2-specific lentivirus shRNAs. In vitro ara-C, daunorubicin, and VP-16 cytotoxicities of the shRNA stable clones were determined by using the Cell Titer-blue reagent. Apoptosis and cell cycle distribution were determined by flow cytometry analysis. Changes in gene transcript levels were determined by gene expression microarray and/or real-time RT-PCR. Changes in protein levels were measured by Western blotting. In vivo binding of GATA1s to IL1A promoter was determined by chromatin immunoprecipitation assays. Results: Lentivirus shRNA knockdown of the GATA1 gene in the DS AMkL cell line, CMK (harbors a mutated GATA1 gene and only expresses GATA1s), resulting in lower GATA1s protein levels, promoted cell differentiation towards the megakaryocytic lineage and repressed cell proliferation. Increased basal apoptosis and sensitivities to ara-C, daunorubicin, and VP-16 accompanied by down-regulated Bcl-2 were also detected in the CMK GATA1 shRNA knockdown clones. Essentially the same results were obtained when Bcl-2 was knocked down with lentivirus shRNA in CMK cells. Besides Bcl-2, down-regulation of GATA1s also resulted in altered expression of genes (e.g., IL1A, PF4, and TUBB1) related to cell death, proliferation, and differentiation. Conclusion: Our results suggest that GATA1s may facilitate leukemogenesis and potentially impact therapeutic responses in DS AMkL by promoting proliferation and survival, and by repressing megakaryocytic lineage differentiation, potentially by regulating expression of Bcl-2 protein and other relevant genes. © 2011 Xavier et al
Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.
The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1-8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9-12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOua(R)2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4'maleimidylstilbene-2,2'-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 microM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N -ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein
The evolving biology of the proton-coupled folate transporter: New insights into regulation, structure, and mechanism
The human proton-coupled folate transporter (PCFT; SLC46A1) or hPCFT was identified in 2006 as the principal folate transporter involved in the intestinal absorption of dietary folates. A rare autosomal recessive hereditary folate malabsorption syndrome is attributable to human SLC46A1 variants. The recognition that hPCFT was highly expressed in many tumors stimulated substantial interest in its potential for cytotoxic drug targeting, taking advantage of its high-level transport activity under acidic pH conditions that characterize many tumors and its modest expression in most normal tissues. To better understand the basis for variations in hPCFT levels between tissues including human tumors, studies have examined the transcriptional regulation of hPCFT including the roles of CpG hypermethylation and critical transcription factors and cis elements. Additional focus involved identifying key structural and functional determinants of hPCFT transport that, combined with homology models based on structural homologies to the bacterial transporters GlpT and LacY, have enabled new structural and mechanistic insights. Recently, cryo-electron microscopy structures of chicken PCFT in a substrate-free state and in complex with the antifolate pemetrexed were reported, providing further structural insights into determinants of (anti)folate recognition and the mechanism of pH-regulated (anti)folate transport by PCFT. Like many major facilitator proteins, hPCFT exists as a homo-oligomer, and evidence suggests that homo-oligomerization of hPCFT monomeric proteins may be important for its intracellular trafficking and/or transport function. Better understanding of the structure, function and regulation of hPCFT should facilitate the rational development of new therapeutic strategies for conditions associated with folate deficiency, as well as cancer
Functional interactions between arginine-133 and aspartate-88 in the human reduced folate carrier: evidence for a charge-pair association
Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons
The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter
Biology and therapeutic applications of the proton-coupled folate transporter
Introduction: The proton-coupled folate transporter (PCFT; SLC46A1) was discovered in 2006 as the principal mechanism by which folates are absorbed in the intestine and the causal basis for hereditary folate malabsorption (HFM). In 2011, it was found that PCFT is highly expressed in many tumors. This stimulated interest in using PCFT for cytotoxic drug targeting, taking advantage of the substantial levels of PCFT transport and acidic pH conditions commonly associated with tumors. Areas covered: We summarize the literature from 2006 to 2022 that explores the role of PCFT in the intestinal absorption of dietary folates and its role in HFM and as a transporter of folates and antifolates such as pemetrexed (Alimta) in relation to cancer. We provide the rationale for the discovery of a new generation of targeted pyrrolo[2,3-d]pyrimidine antifolates with selective PCFT transport and inhibitory activity toward de novo purine biosynthesis in solid tumors. We summarize the benefits of this approach to cancer therapy and exciting new developments in the structural biology of PCFT and its potential to foster refinement of active structures of PCFT-targeted anti-cancer drugs. Expert opinion: We summarize the promising future and potential challenges of implementing PCFT-targeted therapeutics for HFM and a variety of cancers
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