The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms

The first examples of the equilibrative nucleoside transporter (ENT) family were characterized in human tissues at the molecular level only 4 years ago. Since that time, the identification of homologous proteins by functional cloning and genome analysis has revealed that the family is widely distributed in eukaryotes. Family members are predicted to possess 11 transmembrane helices (TMs), and recent investigations on the mammalian ENTs have implicated the TM 3-6 region in solute recognition. Whilst the name of the family reflects the properties ofits prototypical member hENT1, an equilibrative transporter of nucleosides, some family members can also transport nucleobases and some are proton-dependent, concentrative transporters. In addition to their role in nucleoside salvage, ENTs are targets for coronary vasodilator drugs and act as routes for uptake of cytotoxic drugs in humans and protozoa. This paper summarizes current knowledge of the family and reports on the identification of a novel mammalian ENT isoform, designated ENT3, from mouse and human tissues.     

The ENT family of eukaryote nucleoside and nucleobase transporters: recent advances in the investigation of structure/function relationships and the identification of novel isoforms

The first examples of the equilibrative nucleoside transporter (ENT) family were characterized in human tissues at the molecular level only 4 years ago. Since that time, the identification of homologous proteins by functional cloning and genome analysis has revealed that the family is widely distributed in eukaryotes. Family members are predicted to possess 11 transmembrane helices (TMs), and recent investigations on the mammalian ENTs have implicated the TM 3-6 region in solute recognition. Whilst the name of the family reflects the properties of its prototypical member hENT1, an equilibrative transporter of nucleosides, some family members can also transport nucleobases and some are proton-dependent, concentrative transporters. In addition to their role in nucleoside salvage, ENTs are targets for coronary vasodilator drugs and act as routes for uptake of cytotoxic drugs in humans and protozoa. This paper summarizes current knowledge of the family and reports on the identification of a novel mammalian ENT isoform, designated ENT3, from mouse and human tissues.     

Evolution, Homology Conservation, and Identification of Unique Sequence Signatures in GH19 Family Chitinases

The discovery of GH (Glycoside Hydrolase) 19 chitinases in Streptomyces sp. raises the possibility of the presence of these proteins in other bacterial species, since they were initially thought to be confined to higher plants. The present study mainly concentrates on the phylogenetic distribution and homology conservation in GH19 family chitinases. Extensive database searches are performed to identify the presence of GH19 family chitinases in the three major super kingdoms of life. Multiple sequence alignment of all the identified GH19 chitinase family members resulted in the identification of globally conserved residues. We further identified conserved sequence motifs across the major sub groups within the family. Estimation of evolutionary distance between the various bacterial and plant chitinases are carried out to better understand the pattern of evolution. Our study also supports the horizontal gene transfer theory, which states that GH19 chitinase genes are transferred from higher plants to bacteria. Further, the present study sheds light on the phylogenetic distribution and identifies unique sequence signatures that define GH19 chitinase family of proteins. The identified motifs could be used as markers to delineate uncharacterized GH19 family chitinases. The estimation of evolutionary distance between chitinase identified in plants and bacteria shows that the flowering plants are more related to chitinase in actinobacteria than that of identified in purple bacteria. We propose a model to elucidate the natural history of GH19 family chitinases.     

FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity,High Affinity,Nucleoside and Nucleobase Transporter

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [³H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [³H]adenosine or [³H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.     

Evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase family of the enolase superfamily

Understanding how proteins evolve to provide both exquisite specificity and proficient activity is a fundamental problem in biology that has implications for protein function prediction and protein engineering. To study this problem, we analyzed the evolution of structure and function in the o-succinylbenzoate synthase/N-acylamino acid racemase (OSBS/NAAAR) family, part of the mechanistically diverse enolase superfamily. Although all characterized members of the family catalyze the OSBS reaction, this family is extraordinarily divergent, with some members sharing <15% identity. In addition, a member of this family, Amycolatopsis OSBS/NAAAR, is promiscuous, catalyzing both dehydration and racemization. Although the OSBS/NAAAR family appears to have a single evolutionary origin, no sequence or structural motifs unique to this family could be identified; all residues conserved in the family are also found in enolase superfamily members that have different functions. Based on their species distribution, several uncharacterized proteins similar to Amycolatopsis OSBS/NAAAR appear to have been transmitted by lateral gene transfer. Like Amycolatopsis OSBS/NAAAR, these might have additional or alternative functions to OSBS because many are from organisms lacking the pathway in which OSBS is an intermediate. In addition to functional differences, the OSBS/NAAAR family exhibits surprising structural variations, including large differences in orientation between the two domains. These results offer several insights into protein evolution. First, orthologous proteins can exhibit significant structural variation, and specificity can be maintained with little conservation of ligand-contacting residues. Second, the discovery of a set of proteins similar to Amycolatopsis OSBS/NAAAR supports the hypothesis that new protein functions evolve through promiscuous intermediates. Finally, a combination of evolutionary, structural, and sequence analyses identified characteristics that might prime proteins, such as Amycolatopsis OSBS/NAAAR, for the evolution of new activities.     

Bioinformatics of the glycoside hydrolase family 57 and identification of catalytic residues in amylopullulanase from Thermococcus hydrothermalis.

Fifty-nine amino acid sequences belonging to family 57 (GH-57) of the glycoside hydrolases were collected using the CAZy server, Pfam database and blast tools. Owing to the sequence heterogeneity of the GH-57 members, sequence alignments were performed using mainly manual methods. Likewise, five conserved regions were identified, which are postulated to be GH-57 consensus motifs. In the 659 amino acid-long 4-alpha-glucanotransferase from Thermococcus litoralis, these motifs correspond to 13_HQP (region I), 76_GQLEIV (region II), 120_WLTERV (region III), 212_HDDGEKFGVW (region IV), and 350_AQCNDAYWH (region V). The third and fourth conserved regions contain the catalytic nucleophile and the proton donor, respectively. Based on our sequence alignment, residues Glu291 and Asp394 were proposed as the nucleophile and proton donor, respectively, in a GH-57 amylopullulanase from Thermococcus hydrothermalis. To validate this prediction, site-directed mutagenesis was performed. The results of this work reveal that both residues are critical for the pullulanolytic and amylolytic activities of the amylopullulanase. Therefore, these data support the prediction and strongly suggest that the bifunctionality of the amylopullulanase is determined by a single catalytic centre. Despite this positive validation, our alignment also reveals that certain GH-57 members do not possess the Glu and Asp corresponding to the predicted GH-57 catalytic residues. However, the sequences concerned by this anomaly encode putative proteins for which no biochemical or enzymatic data are yet available. Finally, the evolutionary trees generated for GH-57 reveal that the entire family can be divided into several subfamilies that may reflect the different enzyme specificities.     

Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families.

Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes.     

Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface

Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.     

A multidomain TIGR/olfactomedin protein family with conserved structural similarity in the N-terminal region and conserved motifs in the C-terminal region

Based on the similarity between the TIGR (trabecular-meshwork inducible glucocorticoid response) (also known as myocilin) and olfactomedin protein families identified throughout the length of the TIGR protein, we have identified more distantly related proteins to determine the elements essential to the function/structure of the TIGR and olfactomedin proteins. Using a sequence walk method and the Shotgun program, we have identified a family including 31 olfactomedin domain-containing sequences. Multiple sequence alignments and secondary structure analyses were used to identify conserved sequence elements. Pairwise identity in the olfactomedin domain ranges from 8 to 64%, with an average pairwise identity of 24%. The N-terminal regions of the proteins fall into two subgroups, one including the TIGR and olfactomedin families and another group of apparently unrelated domains. The TIGR and olfactomedin sequences display conserved motifs including a residual leucine zipper region and maintain a similar secondary structure throughout the N-terminal region. The correlation between conserved elements and disease-associated mutations and apparent polymorphisms in human TIGR was also examined to evaluate the apparent importance of conserved residues to the function/structure of TIGR. Several residues have been identified as essential to the function and/or structure of the human TIGR protein based on their degree of conservation across the family and their implication in the pathogenesis of primary open-angle glaucoma. Additionally, we have identified a group of chitinase sequences containing several of the highly conserved motifs present in the C-terminal region of the olfactomedin domain-containing sequences.     

Comparative genomic analysis of equilibrative nucleoside transporters suggests conserved protein structure despite limited sequence identity

Equilibrative nucleoside transporters (ENTs) are a recently characterized and poorly understood group of membrane proteins that are important in the uptake of endogenous nucleosides required for nucleic acid and nucleoside triphosphate synthesis. Despite their central importance in cellular metabolism and nucleoside analog chemotherapy, no human ENT gene has been described and nothing is known about gene structure and function. To gain insight into the ENT gene family, we used experimental and in silico comparative genomic approaches to identify ENT genes in three evolutionarily diverse organisms with completely (or almost completely) sequenced genomes, Homo sapiens, Caenorhabditis elegans and Drosophila melanogaster. We describe the chromosomal location, the predicted ENT gene structure and putative structural topologies of predicted ENT proteins derived from the open reading frames. Despite variations in genomic layout and limited ortholog protein sequence identity (≤27.45%), predicted topologies of ENT proteins are strikingly similar, suggesting an evolutionary conservation of a prototypic structure. In addition, a similar distribution of protein domains on exons is apparent in all three taxa. These data demonstrate that comparative sequence analyses should be combined with other approaches (such as genomic and proteomic analyses) to fully understand structure, function and evolution of protein families.     

Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs

The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs. We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter. hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular. Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7. Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.     

Evidence for a second conserved arginine residue in the integrase family of recombination proteins.

This study was designed to search for new regions of similarity in the integrase family of recombination proteins which consists of 28 members found in bacteria and yeast. A computer method based on an information content analysis has been used to align local regions of homology in the set of unaligned protein sequences from this family. Among the aligned regions with high information content were those containing the known conserved histidine, arginine and tyrosine residues. In addition, a new region was identified containing another arginine residue that appears to be conserved in all members of the family. To test further the importance of this newly identified arginine residue, mutants in the Cre protein from phase P1, a member of this integrase family, have been constructed which alter this residue. The mutations which change arginine to lysine and arginine to cysteine depress catalytic activity but not site-specific binding to the lox site. This result is expected for a conserved active site residue. This computer analysis also provides a means for searching for new members of the integrase family.     

Equilibrative nucleoside transporters—A review

Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that mediate the transport of nucleosides, nucleobases, and therapeutic analogs. The best-characterized ENTs are the human transporters hENT1 and hENT2. However, non-mammalian eukaryotic ENTs have also been studied (e.g., yeast, parasitic protozoa). ENTs are major pharmaceutical targets responsible for modulating the efficacy of more than 30 approved drugs. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. This review highlights findings on the characterization of ENTs by surveying studies on genetics, permeant and inhibitor interactions, mutagenesis, and structural models of ENT function.     

The integrase family of tyrosine recombinases: evolution of a conserved active site domain.

The integrases are a diverse family of tyrosine recombinases which rearrange DNA duplexes by means of conservative site-specific recombination reactions. Members of this family, of which the well-studied lambda Int protein is the prototype, were previously found to share four strongly conserved residues, including an active site tyrosine directly involved in transesterification. However, few additional sequence similarities were found in the original group of 27 proteins. We have now identified a total of 81 members of the integrase family deposited in the databases. Alignment and comparisons of these sequences combined with an evolutionary analysis aided in identifying broader sequence similarities and clarifying the possible functions of these conserved residues. This analysis showed that members of the family aggregate into subfamilies which are consistent with their biological roles; these subfamilies have significant levels of sequence similarity beyond the four residues previously identified. It was also possible to map the location of conserved residues onto the available crystal structures; most of the conserved residues cluster in the predicted active site cleft. In addition, these results offer clues into an apparent discrepancy between the mechanisms of different subfamilies of integrases.     

Evolution of the Rab family of small GTP-binding proteins.

Rab proteins are small GTP-binding proteins that form the largest family within the Ras superfamily. Rab proteins regulate vesicular trafficking pathways, behaving as membrane-associated molecular switches. Here, we have identified the complete Rab families in the Caenorhabditis elegans (29 members), Drosophila melanogaster (29), Homo sapiens (60) and Arabidopsis thaliana (57), and we defined criteria for annotation of this protein family in each organism. We studied sequence conservation patterns and observed that the RabF motifs and the RabSF regions previously described in mammalian Rabs are conserved across species. This is consistent with conserved recognition mechanisms by general regulators and specific effectors. We used phylogenetic analysis and other approaches to reconstruct the multiplication of the Rab family and observed that this family shows a strict phylogeny of function as opposed to a phylogeny of species. Furthermore, we observed that Rabs co-segregating in phylogenetic trees show a pattern of similar cellular localisation and/or function. Therefore, animal and fungi Rab proteins can be grouped in "Rab functional groups" according to their segregating patterns in phylogenetic trees. These functional groups reflect similarity of sequence, localisation and/or function, and may also represent shared ancestry. Rab functional groups can help the understanding of the functional evolution of the Rab family in particular and vesicular transport in general, and may be used to predict general functions for novel Rab sequences.