44 research outputs found
Metabolism of 5-Fluorouracil in Cultured Cells. Protection from 5-Fluorouracil Cytotoxicity by Purines
Equilibrative Nucleoside Transporter Family Members from Leishmania donovani Are Electrogenic Proton Symporters
Leishmania donovani express two members of the equilibrative nucleoside transporter family; LdNT1 encoded by two closely related and linked genes, LdNT1.1 and LdNT1.2, that transport adenosine and pyrimidine nucleosides and LdNT2 that transports inosine and guanosine exclusively. LdNT1.1, LdNT1.2, and LdNT2 have been expressed in Xenopus laevis oocytes and found to be electrogenic in the presence of nucleoside ligands for which they mediate transport. Further analysis revealed that ligand uptake and transport currents through LdNT1-type transporters are proton-dependent. In addition to the flux of protons that is coupled to the transport reaction, LdNT1 transporters mediate a variable constitutive proton conductance that is blocked by substrates and dipyridamole. Surprisingly, LdNT1.1 and LdNT1.2 exhibit different electrogenic properties, despite their close sequence homology. This electrophysiological study provides the first demonstration that members of the equilibrative nucleoside transporter family can be electrogenic and establishes that these three permeases, unlike their mammalian counterparts, are probably concentrative rather than facilitative transporters
A quantitative proteomic screen to identify potential drug resistance mechanism in α-difluoromethylornithine (DFMO) resistant Leishmania donovani
Visceral leishmaniasis (VL) caused by Leishmania donovani is a systemic protozoan disease that is fatal if left untreated. The promastigote form of L. donovani is sensitive to growth inhibition by dl-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania (DFMO-16), which was capable of proliferating in 16 mM DFMO. To elucidate the molecular basis for this resistance, we undertook a comparative proteomic analysis of DFMO-resistant/sensitive parasites using isobaric tagging for relative and absolute quantification (iTRAQ/LC–MS/MS). Out of the 101 proteins identified in at least 2 of the 3 independent experiments, 82 proteins are 1.5- to 44.0-fold more abundant in DFMO-resistant strain (DFMO-16) while 19 are 2- to 5.0-fold less abundant as compared to the wild-type (DI700) parasites. Proteins with 2-fold or greater abundance in the DFMO-resistant strain include free radical detoxification, polyamine and trypanothione metabolic proteins, proteins involved in metabolism, intracellular survival and proteolysis, elongation factors, signaling molecules and mitochondrial transporters, and many with no annotated function. Differentially modulated proteins contribute to our understanding of molecular mechanism of DFMO-resistance and have the potential to act as biomarkers. Biological significance: This study will facilitate a deeper understanding of the phenomenon of acquired drug resistance and possible biomarkers in Leishmania against antiparasitic drug DFMO. Also it will provide information about the metabolic pathways modulated in resistant parasites as an adaptation mechanism to counter drugs. Studies like this are important to safeguard the efficacy of a limited repertoire of anti-parasitic drugs, and to lead the development of new drugs and drug combinations
The <i>Trypanosoma cruzi</i> Diamine Transporter Is Essential for Robust Infection of Mammalian Cells
<div><p><i>Trypanosoma cruzi</i> is incapable of synthesizing putrescine or cadaverine <i>de novo</i>, and, therefore, salvage of polyamines from the host milieu is an obligatory nutritional function for the parasite. A high-affinity diamine transporter (TcPOT1) from <i>T</i>. <i>cruzi</i> has been identified previously that recognizes both putrescine and cadaverine as ligands. In order to assess the functional role of TcPOT1 in intact parasites, <i>a</i> Δ<i>tcpot1</i> null mutant was constructed by targeted gene replacement and characterized. The Δ<i>tcpot1</i> mutant lacked high-affinity putrescine-cadaverine transport capability but retained the capacity to transport diamines via a non-saturable, low-affinity mechanism. Transport of spermidine and arginine was not impacted by the Δ<i>tcpot1</i> lesion. The Δ<i>tcpot1</i> cell line exhibited a significant but not total defect in its ability to subsist in Vero cells, although initial infection rates were not affected by the lesion. These findings reveal that TcPOT1 is the sole high-affinity diamine permease in <i>T</i>. <i>cruzi</i>, that genetic obliteration of TcPOT1 impairs the ability of the parasite to maintain a robust infection in mammalian cells, and that a secondary low-affinity uptake mechanism for this key parasite nutrient is operative but insufficient for optimal infection.</p></div
Identification and reconstitution of the nucleoside transporter of CEM human leukemia cells
The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75 000 ± 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55 000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47 000 ± 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 ± 11 μM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine \u3e thymidine \u3e cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles. © 1990
Parasite loads in mammalian cells infected with either wild type, Δ<i>tcpot1</i>, or Δ<i>tcpot1</i>[p<i>TcPOT1</i>] “add-back” parasites.
<p>Vero cells were infected with (A) wild type, (B) Δ<i>tcpot1</i>, or (C) Δ<i>tcpot1</i>[p<i>TcPOT1</i>] metacyclic trypomastigotes at a ratio of five parasites per cell and DAPI-stained amastigotes enumerated as indicated in the Experimental procedures section. Each color represents data from three independent experiments for the wild type and Δ<i>tcpot1</i> cell lines and is indicated as n1 (black), n2 (orange), or n3 (blue), and data from two independent experiments for the “add-back.” Within each experiment amastigotes enumeration was performed in duplicate. (D) Demonstration that putrescine transport capability has been conferred by ectopic expression of <i>TcPOT1</i> in the Δ<i>tcpot1</i>[p<i>TcPOT1</i>] “add-back” line. Putrescine transport was measured as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152715#pone.0152715.g002" target="_blank">Fig 2</a> in wild type (■), Δ<i>tcpot1</i> (grey dot), and Δ<i>tcpot1</i>[p<i>TcPOT1</i>::<i>GFP</i>] () cells, and the data presented are those of three replicates. Direct fluorescence microscopy (x63 magnification) (E) displays expression of <i>TcPOT1</i>.<i>1</i>::<i>GFP</i> in Vero cells infected with <i>T</i>. <i>cruzi</i> amastigotes. Shown are a phase contrast image (1), a merge of GFP and DAPI images (2), GFP fluorescence (3), and DAPI-stained parasites (4).</p
Comparison of the abilities of wild type and Δ<i>tcpot1 T</i>. <i>cruzi</i> to take up putrescine as a function of concentration and time.
<p>(A) The rates by which wild type (●) and Δ<i>tcpot1</i> (grey dot) epimastigotes incorporate extracellular [<sup>3</sup>H]putrescine over a range of putrescine concentrations from 5–200 μM were determined as described in Experimental procedures. Uptake measurements were determined after a 10 s exposure to the radioactive ligand. The graph is of a typical experiment that was performed in triplicate. (B) The rates of [<sup>3</sup>H]putrescine uptake into wild type (■) and Δ<i>tcpot1</i> (grey dot) epimastigotes were measured at 200 μM putrescine over a 120 minute time course. The data depicted are from one of three independent experiments, all with comparable results.</p