scholarly journals Cysteine-accessibility analysis of transmembrane domains 11–13 of human concentrative nucleoside transporter 3

2006 ◽  
Vol 394 (2) ◽  
pp. 389-398 ◽  
Author(s):  
Jing Zhang ◽  
Tracey Tackaberry ◽  
Mabel W. L. Ritzel ◽  
Taylor Raborn ◽  
Gerry Barron ◽  
...  
Keyword(s):  
Expression System ◽  
Transmembrane Domains ◽  
Nucleoside Transport ◽  
Cysteine Residues ◽  
Nucleoside Transporter ◽  
Yeast Expression System ◽  
Cysteine Substitution ◽  
Concentrative Nucleoside Transporter ◽  
Translocation Pathway ◽  
Nucleoside Drugs

hCNT3 (human concentrative nucleoside transporter 3) is a nucleoside–sodium symporter that transports a broad range of naturally occurring purine and pyrimidine nucleosides as well as anticancer nucleoside drugs. To understand its uridine binding and translocation mechanisms, a cysteine-less version of hCNT3 was constructed and used for cysteine-accessibility and permeant-protection assays. Cysteine-less hCNT3, with 14 endogenous cysteine residues changed to serine, displayed wild-type properties in a yeast expression system, indicating that endogenous cysteine residues are not essential for hCNT3-mediated nucleoside transport. A series of cysteine-substitution mutants spanning predicted TMs (transmembrane domains) 11–13 was constructed and tested for accessibility to thiol-specific reagents. Mutants M496C, G498C, F563C, A594C, G598C and A606C had no detectable transport activity, indicating that a cysteine substitution at each of these positions was not tolerated. Two functional mutants in putative TM 11 (L480C and S487C) and four in putative TM 12 (N565C, T557C, G567C and I571C) were partially inhibited by MTS (methanethiosulphonate) reagent and high concentrations of uridine protected against inhibition, indicating that TMs 11 and 12 may form part of the nucleoside translocation pathway. The lack of accessibility of MTS reagents to TM 13 mutants suggests that TM 13 is not exposed to the nucleoside translocation pathway. Furthermore, G567C, N565C and I571C mutants were only sensitive to MTSEA (MTS-ethylammonium), a membranepermeant thiol reagent, indicating that these residues may be accessible from the cytoplasmic side of the membrane, providing evidence in support of the predicted orientation of TM 12 in the current putative topology model of hCNT3.

Role of cysteine 416 in N-ethylmaleimide sensitivity of human equilibrative nucleoside transporter 1 (hENT1)

2018 ◽  
Vol 475 (20) ◽  
pp. 3293-3309 ◽  
Author(s):  
Sylvia Y.M. Yao ◽  
Amy M.L. Ng ◽  
Carol E. Cass ◽  
James D. Young
Keyword(s):  
Expression System ◽  
Cysteine Residue ◽  
Cysteine Residues ◽  
Nucleoside Transporter ◽  
Wild Type ◽  
Binding Characteristics ◽  
Equilibrative Nucleoside Transporter ◽  
C Terminus ◽  
Nucleoside Drugs

Human equilibrative nucleoside transporter 1 (hENT1), the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for cellular uptake of physiologic nucleosides and many antineoplastic and antiviral nucleoside drugs. hENT1, which is potently inhibited by nitrobenzylthioinosine (NBMPR), possesses 11 transmembrane helical domains with an intracellular N-terminus and an extracellular C-terminus. As a protein with 10 endogenous cysteine residues, it is sensitive to inhibition by the membrane permeable sulfhydryl-reactive reagent N-ethylmaleimide (NEM) but is unaffected by the membrane impermeable sulfhydryl-reactive reagent p-chloromercuriphenyl sulfonate. To identify the residue(s) involved in NEM inhibition, we created a cysteine-less version of hENT1 (hENT1C-), with all 10 endogenous cysteine residues mutated to serine, and showed that it displays wild-type uridine transport and NBMPR-binding characteristics when produced in the Xenopus oocyte heterologous expression system, indicating that endogenous cysteine residues are not essential for hENT1 function. We then tested NEM sensitivity of recombinant wild-type hENT1, hENT1 mutants C1S to C10S (single cysteine residues replaced by serine), hENT1C- (all cysteine residues replaced by serine), and hENT1C- mutants S1C to S10C (single serine residues converted back to cysteine). Mutants C9S (C416S/hENT1) and S9C (S416C/hENT1C-) were insensitive and sensitive, respectively, to inhibition by NEM, identifying Cys416 as the endofacial cysteine residue in hENT1 responsible for NEM inhibition. Kinetic experiments suggested that NEM modification of Cys416, which is located at the inner extremity of TM10, results in the inhibition of hENT1 uridine transport and NBMPR binding by constraining the protein in its inward-facing conformation.

scholarly journals Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells

1996 ◽  
Vol 317 (2) ◽  
pp. 457-465 ◽  
Author(s):  
Xiao FANG ◽  
Fiona E. PARKINSON ◽  
Delores A. MOWLES ◽  
James D. YOUNG ◽  
Carol E. CASS
Keyword(s):  
Mammalian Cells ◽  
Expression System ◽  
Functional Characterization ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Single Type ◽  
Nucleoside Transporter ◽  
Pyrimidine Nucleosides ◽  
Transporter Protein ◽  
Sodium Dependent

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the ‘equilibrative sensitive’ or ‘es’ transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximate Km values of 21 μM, 12.5 μM and 15 μM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.

Cysteine engineering of actin self-assembly interfaces

2009 ◽  
Vol 87 (4) ◽  
pp. 663-675 ◽  
Author(s):  
Kate Pengelly ◽  
Ana Loncar ◽  
Alex A. Perieteanu ◽  
John F. Dawson
Keyword(s):  
Self Assembly ◽  
Expression System ◽  
Cysteine Residues ◽  
Filamentous Actin ◽  
Mutant Proteins ◽  
Yeast Expression System ◽  
Atomic Interactions ◽  
Pointed End ◽  
Actin Protein ◽  
Chemical Groups

The Holmes model of filamentous actin (F-actin) and recent structural studies suggest specific atomic interactions between F-actin subunits. We tested these interactions through a cysteine-engineering approach with the goal of inhibiting filament formation by introducing chemical groups at sites important for polymerization. We substituted surface amino acids on the actin molecule with cysteine residues and tested the effect of producing these actin mutant proteins in a yeast expression system. The intrinsic folding and polymerization characteristics of the cysteine-engineered actin proteins were measured. The effect of chemical modification of the introduced cysteine residues on the polymerization of the actin mutant proteins was also examined. Modification of cysteine residues with large hydrophobic reagents resulted in polymerization inhibition. We examined the finding that the D288C actin protein does not polymerize under oxidizing conditions and forms protein aggregates when magnesium and EGTA are present. Chemical crosslinking experiments revealed the presence of a lower dimer when only D288C actin was present. When both D288C and A204C actin were present, crosslinking experiments support the proximity of Asp288 on the barbed end of one subunit to Ala204 on the pointed end of a neighboring subunit in the Holmes model of F-actin.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium

2001 ◽  
Vol 280 (5) ◽  
pp. C1076-C1082 ◽  
Author(s):  
George P. H. Leung ◽  
Jeffrey L. Ward ◽  
Patrick Y. D. Wong ◽  
Chung-Ming Tse
Keyword(s):  
Nucleoside Transport ◽  
Transport Systems ◽  
Thymidine Uptake ◽  
Nucleoside Transporter ◽  
Equilibrative Nucleoside Transporter ◽  
Functional Studies ◽  
Uridine Uptake ◽  
Concentrative Nucleoside Transporter ◽  
Nucleoside Uptake

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.

Expression of the human concentrative nucleoside transporter-3 (hCNT3) increases sensitivity towards cytotoxic nucleoside drugs

2003 ◽  
Vol 124 (4) ◽  
pp. A308
Author(s):  
Maria Desouza ◽  
George Ph Leung ◽  
Kenneth Kw To ◽  
Shuywang Toan ◽  
Chung-Ming Tse
Keyword(s):  
Nucleoside Transporter ◽  
Concentrative Nucleoside Transporter ◽  
Nucleoside Drugs

Molecular biology and regulation of nucleoside and nucleobase transporter proteins in eukaryotes and prokaryotes

2002 ◽  
Vol 80 (5) ◽  
pp. 623-638 ◽  
Author(s):  
Miguel A Cabrita ◽  
Stephen A Baldwin ◽  
James D Young ◽  
Carol E Cass
Keyword(s):  
Molecular Biology ◽  
Vital Role ◽  
Nucleoside Transporters ◽  
Nucleoside Transport ◽  
Nucleoside Transporter ◽  
Concentration Gradients ◽  
Transporter Proteins ◽  
Equilibrative Nucleoside Transporters ◽  
Nucleoside Drugs ◽  
Nucleobase Transport

The molecular cloning of cDNAs encoding nucleoside transporter proteins has greatly advanced understanding of how nucleoside permeants are translocated across cell membranes. The nucleoside transporter proteins identified thus far have been categorized into five distinct superfamilies. Two of these superfamilies, the equilibrative and concentrative nucleoside transporters, have human members and these will be examined in depth in this review. The human equilibrative nucleoside transporters translocate nucleosides and nucleobases bidirectionally down their concentration gradients and are important in the uptake of anticancer and antiviral nucleoside drugs. The human concentrative nucleoside transporters cotranslocate nucleosides and sodium unidirectionally against the nucleoside concentration gradients and play a vital role in certain tissues. The regulation of nucleoside and nucleobase transporters is being studied more intensely now that more tools are available. This review provides an overview of recent advances in the molecular biology and regulation of the nucleoside and nucleobase transporters.Key words: nucleoside transporter, nucleoside transport, nucleobase transporter, nucleobase transport, regulation of nucleoside and nucleobase transport, nucleoside drugs.

Stable expression of a recombinant sodium-dependent, pyrimidine-selective nucleoside transporter (CNT1) in a transport-deficient mouse leukemia cell line

1998 ◽  
Vol 76 (5) ◽  
pp. 843-851 ◽  
Author(s):  
Charles R Crawford ◽  
Carol E Cass ◽  
James D Young ◽  
Judith A Belt
Keyword(s):  
Cell Line ◽  
Nucleoside Transporters ◽  
Stable Expression ◽  
Nucleoside Transport ◽  
Leukemia Cell Line ◽  
Nucleoside Transporter ◽  
Mouse Leukemia ◽  
Concentrative Nucleoside Transporter ◽  
Sodium Dependent ◽  
Cloned Gene

Previous studies of nucleoside transport in mammalian cells have identified two types of activities: the equilibrative nucleoside transporters and concentrative, Na+-nucleoside cotransporters. Characterization of the concentrative nucleoside transporters has been hampered by the presence in most cells and tissues of multiple transporters with overlapping permeant specificities. With the recent cloning of cDNAs encoding rat and human members of the concentrative nucleoside transporter (CNT) family, it is now possible to study the concentrative transporters in isolation by use of functional expression systems. We report here the isolation of a nucleoside transport-deficient subline of L1210 mouse leukemia (L1210/DNC3) that is a suitable recipient for stable expression of cloned nucleoside transporter cDNAs. We have used L1210/DNC3 as the recipient in gene transfer studies to develop a stable cell line (L1210/DU5) that produces the recombinant concentrative nucleoside transporter with selectivity for pyrimidine nucleosides (CNT1) that was initially identified in rat intestine (Q.Q. Huang, S.Y. Yao, M.W. Ritzel, A.R.P. Paterson, C.E. Cass, and J.D. Young. 1994. J. Biol. Chem. 269: 17 757 - 17 760). L1210/DU5 was used to examine the permeant selectivity of recombinant rat CNT1 by comparing a series of nucleoside analogs with respect to (i) inhibition of inward fluxes of [3H]thymidine, (ii) initial rates of transport of 3H-analog, and (iii) cytotoxicity to L1210/DU5 versus the parental transport-deficient cell line. By all three criteria, recombinant CNT1 transported 5-fluoro-2prime-deoxyuridine and 5-fluorouridine well and cytosine arabinoside poorly. Although some purine nucleosides (2prime-deoxyadenosine, 2-chloro-2prime-deoxyadenosine, 7-deazaadenosine) were potent inhibitors of CNT1, they were poor permeants when uptake was measured directly by analysis of isotopic fluxes or indirectly by comparison of cytotoxicity ratios. We conclude that comparison of analog cytotoxicity to L1210/DU5 versus L1210/DNC3 is a reliable indirect predictor of transportability, suggesting that cytotoxicity assays with a panel of such cell lines, each with a different recombinant nucleoside transporter, would be a valuable tool in the development of antiviral and antitumor nucleoside analogs.Key words: nucleoside transporter, CNT1, rat, sodium-dependent, recombinant, cloned gene, transfection, stable transfectant.

Allosteric and transport modulation of human concentrative nucleoside transporter 3 at the atomic scale

2021 ◽  
Author(s):  
Huaichuan Duan ◽  
Zhou Yanxia ◽  
XiaoDong Shi ◽  
Qing Luo ◽  
Gao Jiaxing ◽  
...  
Keyword(s):  
Nucleoside Transporters ◽  
Atomic Scale ◽  
Nucleoside Transport ◽  
Transmembrane Transport ◽  
Nucleoside Transporter ◽  
Nucleotide Synthesis ◽  
Physiological Processes ◽  
Concentrative Nucleoside Transporter ◽  
Transport Modulation ◽  
In Cells

Nucleosides are important precursors of nucleotide synthesis in cells, and nucleoside transporters play an important role in many physiological processes by mediating its transmembrane transport and absorption. During nucleoside transport,...

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