The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells. A biochemical basis of the selectivity of leucovorin rescue

This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Neuropsychopharmacology and Neurogenetic Aspects of Executive Functioning: Should Reward Gene Polymorphisms Constitute a Diagnostic Tool to Identify Individuals at Risk for Impaired Judgment?

Executive functions are processes that act in harmony to control behaviors necessary for maintaining focus and achieving outcomes. Executive dysfunction in neuropsychiatric disorders is attributed to structural or functional pathology of brain networks involving prefrontal cortex (PFC) and its connections with other brain regions. The PFC receives innervations from different neurons associated with a number of neurotransmitters, especially dopamine (DA). Here we review findings on the contribution of PFC DA to higher-order cognitive and emotional behaviors. We suggest that examination of multifactorial interactions of an individual's genetic history, along with environmental risk factors, can assist in the characterization of executive functioning for that individual. Based upon the results of genetic studies, we also propose genetic mapping as a probable diagnostic tool serving as a therapeutic adjunct for augmenting executive functioning capabilities. We conclude that preservation of the neurological underpinnings of executive functions requires the integrity of complex neural systems including the influence of specific genes and associated polymorphisms to provide adequate neurotransmission.     

Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION*

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the synthesis of nucleotide precursors, serine, and methionine in one-carbon transfer reactions (1). Because mammals cannot synthesize folates de novo, cellular uptake of these derivatives is essential for cell growth and tissue regeneration (2, 3). Folates are hydrophilic anionic molecules that do not cross biological membranes by diffusion alone, so it is not surprising that sophisticated membrane transport systems have evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier (RFC)² is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues (3, 4). RFC plays a generalized role in folate transport and provides specialized tissue functions such as transport across the basolateral membrane of renal proximal tubules (5), transplacental transport of folates (6), and folate transport across the blood-brain barrier (7), although the contribution of RFC to intestinal absorption of folates remains controversial (8, 9). Loss of RFC expression or function portends potentially profound physiologic and developmental consequences associated with folate deficiency (10). RFC is also a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate (Mtx), pemetrexed, and raltitrexed (4). Loss of RFC expression or synthesis of mutant RFC protein in tumor cells results in antifolate resistance caused by incomplete inhibition of cellular enzyme targets and low levels of antifolate substrate for polyglutamate synthesis (4, 11).Reflecting its particular physiologic and pharmacologic importance, interest in RFC structure and function has been high. Since 1994, when murine RFC was first cloned (12), application of state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins has led to a progressively detailed picture of the molecular structure of the carrier, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of α-helix transmembrane domains (TMDs) (4, 13). Although no crystal structure for RFC has yet been reported, a detailed homology model for human RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol 3-phosphate/inorganic phosphate antiporter GlpT was generated (13, 14) that permits testing of hypotheses related to hRFC structure and mechanism in a manner not previously possible.Considerations of hRFC structure and mechanism to date have all been based on the notion that a single 591-amino acid hRFC molecule is sufficient to mediate concentrative uptake of folate and antifolate substrates. However, a growing literature suggests that quaternary structure involving the formation of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly an important feature of the structure and function of many membrane transporters (15-18). For major facilitator superfamily proteins, both monomeric (e.g. LacY, GlpT, UhpT, and GLUT3) (19-22) and oligomeric (e.g. LacS, AE1, GLUT1, and TetA) (23-28) structures have been reported, establishing the lack of a clear structural consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a homo-oligomeric species composed of multiple hRFC monomers. Based on results with an assortment of biochemical methods with wt and a collection of mutant hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but also establish the probable importance of these higher order structures to intracellular trafficking and carrier function.