Conserved Glutamate Residues Glu-343 and Glu-519 Provide Mechanistic Insights into Cation/Nucleoside Cotransport by Human Concentrative Nucleoside Transporter hCNT3

Human concentrative nucleoside transporter 3 (hCNT3) utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate pyrimidine and purine nucleosides within cells. We have employed radioisotope flux and electrophysiological techniques in combination with site-directed mutagenesis and heterologous expression in Xenopus oocytes to identify two conserved pore-lining glutamate residues (Glu-343 and Glu-519) with essential roles in hCNT3 Na⁺/nucleoside and H⁺/nucleoside cotransport. Mutation of Glu-343 and Glu-519 to aspartate, glutamine, and cysteine severely compromised hCNT3 transport function, and changes included altered nucleoside and cation activation kinetics (all mutants), loss or impairment of H⁺ dependence (all mutants), shift in Na⁺:nucleoside stoichiometry from 2:1 to 1:1 (E519C), complete loss of catalytic activity (E519Q) and, similar to the corresponding mutant in Na⁺-specific hCNT1, uncoupled Na⁺ currents (E343Q). Consistent with close-proximity integration of cation/solute-binding sites within a common cation/permeant translocation pore, mutation of Glu-343 and Glu-519 also altered hCNT3 nucleoside transport selectivity. Both residues were accessible to the external medium and inhibited by p-chloromercuribenzene sulfonate when converted to cysteine.Physiologic nucleosides and the majority of synthetic nucleoside analogs with antineoplastic and/or antiviral activity are hydrophilic molecules that require specialized plasma membrane nucleoside transporter (NT)³ proteins for transport into or out of cells (1–4). NT-mediated transport is required for nucleoside metabolism by salvage pathways and is a critical determinant of the pharmacologic actions of nucleoside drugs (3–6). By regulating adenosine availability to purinoreceptors, NTs also modulate a diverse array of physiological processes, including neurotransmission, immune responses, platelet aggregation, renal function, and coronary vasodilation (4, 6, 7). Two structurally unrelated NT families of integral membrane proteins exist in human and other mammalian cells and tissues as follows: the SLC28 concentrative nucleoside transporter (CNT) family and the SLC29 equilibrative nucleoside transporter (ENT) family (3, 4, 6, 8, 9). ENTs are normally present in most, possibly all, cell types (4, 6, 8). CNTs, in contrast, are found predominantly in intestinal and renal epithelia and other specialized cell types, where they have important roles in absorption, secretion, distribution, and elimination of nucleosides and nucleoside drugs (1–3, 5, 6, 9).The CNT protein family in humans is represented by three members, hCNT1, hCNT2, and hCNT3. Belonging to a CNT subfamily phylogenetically distinct from hCNT1/2, hCNT3 utilizes electrochemical gradients of both Na⁺ and H⁺ to accumulate a broad range of pyrimidine and purine nucleosides and nucleoside drugs within cells (10, 11). hCNT1 and hCNT2, in contrast, are Na⁺-specific and transport pyrimidine and purine nucleosides, respectively (11–13). Together, hCNT1–3 account for the three major concentrative nucleoside transport processes of human and other mammalian cells. Nonmammalian members of the CNT protein family that have been characterized functionally include hfCNT, a second member of the CNT3 subfamily from the ancient marine prevertebrate the Pacific hagfish Eptatretus stouti (14), CeCNT3 from Caenorhabditis elegans (15), CaCNT from Candida albicans (16), and the bacterial nucleoside transporter NupC from Escherichia coli (17). hfCNT is Na⁺- but not H⁺-coupled, whereas CeCNT3, CaCNT, and NupC are exclusively H⁺-coupled. Na⁺:nucleoside coupling stoichiometries are 1:1 for hCNT1 and hCNT2 and 2:1 for hCNT3 and hfCNT3 (11, 14). H⁺:nucleoside coupling ratios for hCNT3 and CaCNT are 1:1 (11, 16).Although much progress has been made in molecular studies of ENT proteins (4, 6, 8), studies of structurally and functionally important regions and residues within the CNT protein family are still at an early stage. Topological investigations suggest that hCNT1–3 and other eukaryote CNT family members have a 13 (or possibly 15)-transmembrane helix (TM) architecture, and multiple alignments reveal strong sequence similarities within the C-terminal half of the proteins (18). Prokaryotic CNTs lack the first three TMs of their eukaryotic counterparts, and functional expression of N-terminally truncated human and rat CNT1 in Xenopus oocytes has established that these three TMs are not required for Na⁺-dependent uridine transport activity (18). Consistent with this finding, chimeric studies involving hCNT1 and hfCNT (14) and hCNT1 and hCNT3 (19) have demonstrated that residues involved in Na⁺- and H⁺-coupling reside in the C-terminal half of the protein. Present in this region of the transporter, but of unknown function, is a highly conserved (G/A)XKX₃NEFVA(Y/M/F) motif common to all eukaryote and prokaryote CNTs.By virtue of their negative charge and consequent ability to interact directly with coupling cations and/or participate in cation-induced and other protein conformational transitions, glutamate and aspartate residues play key functional and structural roles in a broad spectrum of mammalian and bacterial cation-coupled transporters (20–30). Little, however, is known about their role in CNTs. This study builds upon a recent mutagenesis study of conserved glutamate and aspartate residues in hCNT1 (31) to undertake a parallel in depth investigation of corresponding residues in hCNT3. By employing the multifunctional capability of hCNT3 as a template for these studies, this study provides novel mechanistic insights into the molecular mechanism(s) of CNT-mediated cation/nucleoside cotransport, including the role of the (G/A)XKX₃NEFVA(Y/M/F) motif.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Substituted Cysteine Accessibility Method Analysis of Human Concentrative Nucleoside Transporter hCNT3 Reveals a Novel Discontinuous Region of Functional Importance within the CNT Family Motif (G/A)XKX3NEFVA(Y/M/F)

The human SLC28 family of integral membrane CNT (concentrative nucleoside transporter) proteins has three members, hCNT1, hCNT2, and hCNT3. Na⁺-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 mediates transport of both pyrimidine and purine nucleosides utilizing Na⁺ and/or H⁺ electrochemical gradients. These and other eukaryote CNTs are currently defined by a putative 13-transmembrane helix (TM) topology model with an intracellular N terminus and a glycosylated extracellular C terminus. Recent mutagenesis studies, however, have provided evidence supporting an alternative 15-TM membrane architecture. In the absence of CNT crystal structures, valuable information can be gained about residue localization and function using substituted cysteine accessibility method analysis with thiol-reactive reagents, such as p-chloromercuribenzene sulfonate. Using heterologous expression in Xenopus oocytes and the cysteineless hCNT3 protein hCNT3C−, substituted cysteine accessibility method analysis with p-chloromercuribenzene sulfonate was performed on the TM 11–13 region, including bridging extramembranous loops. The results identified residues of functional importance and, consistent with a new revised 15-TM CNT membrane architecture, suggest a novel membrane-associated topology for a region of the protein (TM 11A) that includes the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F).Specialized nucleoside transporter proteins are required for passage of nucleosides and hydrophilic nucleoside analogs across biological membranes. Physiologically, nucleosides serve as nucleotide precursors in salvage pathways, and pharmacologically nucleoside analogs are used as chemotherapeutic agents in the treatment of cancer and antiviral diseases (1, 2). Additionally, adenosine modulates numerous cellular events via purino-receptor cell signaling pathways, including neurotransmission, vascular tone, immune responses, and other physiological processes (3, 4).Human nucleoside transporter proteins are divided into two families: the SLC29 ENT (equilibrative nucleoside transporter) family and the SLC28 CNT (concentrative nucleoside transporter) family (3, 5–7). hENTs³ mediate bidirectional fluxes of purine and pyrimidine nucleosides down their concentration gradients and are ubiquitously found in most, possibly all, cell types (8). Additionally, the hENT2 isoform is capable of nucleobase transport (9). hCNTs, in contrast, are inwardly directed Na⁺-dependent nucleoside transporters found predominantly in intestinal and renal epithelial and other specialized cell types (10, 11). hCNT1 and hCNT2 are pyrimidine and purine nucleoside-selective, respectively, and couple Na⁺/nucleoside cotransport with 1:1 stoichiometry (12–18). In contrast, hCNT3 is broadly selective for both pyrimidine and purine nucleosides and couples Na⁺/nucleoside cotransport with 2:1 stoichiometry (10, 18, 19). hCNT3 is also capable of H⁺/nucleoside cotransport with a coupling stoichiometry of 1:1, whereby one of the two Na⁺ binding sites also functionally interacts with H⁺ (18, 19).Current models of CNT topology have 13 putative transmembrane helices (TMs) (10, 14, 16, 20). Two additional TMs (designated 5A and 11A) are weakly predicted by computer algorithms (20), and immunocytochemical experiments with site-specific antibodies and studies of native and introduced glycosylation sites have confirmed an intracellular N terminus and an extracellular C terminus (20). Chimeric studies involving hCNTs and hfCNT, a CNT from the ancient marine prevertebrate, the Pacific hagfish Eptatretus stouti, have revealed that the functional domains responsible for CNT nucleoside selectivity and cation coupling reside within the C-terminal TM 7–13 half of the protein (19, 21). NupC, an H⁺-coupled CNT family member from Escherichia coli, lacks TMs 1–3 but otherwise shares a topology similar to that of its eukaryote counterparts (22, 23).A functional cysteineless version of hCNT3 has been generated by mutagenesis of endogenous cysteine residues to serine, resulting in the cysteineless construct hCNT3C− employed originally in a yeast expression system for substituted cysteine accessibility method (SCAM) analysis of TMs 11, 12, and 13 using methanethiosulfonate (MTS) reagents (24). Subsequently, we have also characterized hCNT3C− in the Xenopus oocyte expression system (25) and have initiated SCAM analyses with the alternative thiol-specific reagent p-chloromercuribenzene sulfonate (PCMBS) (26). Measured by transport inhibition, reactivity of introduced cysteine residues with PCMBS, which is both membrane-impermeant and hydrophilic, indicates pore-lining status and access from the extracellular medium; the ability of a permeant to protect against this inhibition denotes location within, or closely adjacent to, the permeant-binding pocket (27, 28). Continuing the investigation of hCNT3 C-terminal membrane topology and function, the present study reports results of PCMBS SCAM analyses of TMs 11–13, including loop regions linking the putative TMs not previously studied using MTS reagents.In earlier structure/function studies of hCNT3, we identified a cluster of conformationally sensitive residue positions in TM 12 (Ile⁵⁵⁴, Tyr⁵⁵⁸, and Cys⁵⁶¹) that exhibit H⁺-activated inhibition by PCMBS, with uridine protection evident for Tyr⁵⁵⁸ and Cys⁵⁶¹ (26). Located deeper within the plane of the membrane, other uridine-protectable residue positions in TM 12 were PCMBS-sensitive in both H⁺- and Na⁺-containing media (26). hCNT3 Glu⁵¹⁹ and the corresponding residue in hCNT1 (Glu⁴⁹⁸) in region TM 11A were also identified as having key roles in permeant and cation binding and translocation (29, 30), and hCNT3 E519C showed inhibition of uridine uptake by PCMBS (30). Centrally positioned within the highly conserved CNT family motif (G/A)XKX₃NEFVA(Y/M/F), residue 519 is proposed to be a direct participant in cation coupling via the common hCNT3 Na⁺/H⁺-binding site that, in other CNTs, is either Na⁺-specific (e.g. hCNT1) or H⁺-specific (e.g. NupC) (30).Building upon the prior work with MTS reagents and other structure/function studies of hCNT3, the present study identified new residues of functional importance in the C-terminal one-third of hCNT3, established the orientations and α-helical structures of TMs 11–13, and determined a novel membrane-associated topology for the TM 11A region of the protein. A revised CNT membrane architecture is proposed.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.