Comparative kinetic analysis of AzgA and Fcy21p, prototypes of the two major fungal hypoxanthine-adenine-guanine transporter families

In fungi, uptake of salvageable purines is carried out by members of two evolutionarily distinct protein families, the Purine-Related Transporters (PRT/NCS1) and the AzgA-like Transporters. We carried out a comparative kinetic analysis of two prototypes of these transporter families. The first was Fcy21p, a herein characterized protein of Candida albicans, and the second was AzgA, a transporter of Aspergillus nidulans. Our results showed that: (i) AzgA and Fcy21p are equally efficient high-affinity, high-capacity, purine transporters, (ii) Fcy21p, but not AzgA, is an efficient cytosine and 5-fluorocytosine transporter, interacting with =O2 and C4-NH2 of the pyrimidine ring, (iii) the major interactions of AzgA and Fcy21p with the purine ring are similar, but not identical, involving in all cases positions 6 and 7, and for some substrates, positions 1 and 9 as well, and (iv) in AzgA, bulky groups at position N3 have a detrimental steric effect on substrate binding, while similar substitutions at C2 or N9 are fully or partially tolerated. In contrast, in Fcy21p, C2 and N9 bulky substitutions abolish substrate binding, while similar substitutions in N3 are fully tolerated. These results suggest that all fungal purine transporters might have evolved from a single ancestral protein, and show that fungal transporters use different substrate interactions compared to the analogous protozoan or mammalian proteins. Finally, results are also discussed in respect of the possibility of using fungal purine transporters as specific gateways for the development of targeted antifungal pharmacological therapies.     

The SLC6 orphans are forming a family of amino acid transporters

Transporters in the human genome are grouped in solute carrier families (SLC). The SLC6 family is one of the biggest transporter families in the human genome comprising 20 members. It is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters GABA, noradrenaline, serotonin and dopamine. The family also includes a number of 'orphan' transporters, the function of which has remained elusive until recently. Identification of the broadly specific neutral amino acid transporter SLC6A19 (also called B(0)AT1) suggested that all orphan transporters may in fact be amino acid transporters. This was subsequently confirmed by the identification of SLC6A20 as the long-sought IMINO system, a proline transporter found in kidney, intestine and brain. Very recently, SLC6A15 was identified as the neutral amino acid transporter B(0)AT2. All amino acid transporters appear to cotransport only 1Na(+) together with the amino acid substrate. Both, B(0)AT1 and B(0)AT2 are chloride independent, whereas IMINO is chloride dependent. The amino acid transporters of the SLC6 family are functionally and sequence related to the recently crystallized leucine transporter from Aquifex aeolicus. The structure elegantly explains many of the mechanistic features of the SLC6 amino acid transporters.     

Technologies for transporter drug discovery

Transporters represent attractive targets for drug discovery and are implicated in the pathophysiology of disorders across several therapeutic areas including asthma, cardiovascular disease, diabetes, and neuroscience. However, the intrinsic mechanistic properties of transporters present significant challenges to the development of high-throughput screening methodologies. This review provides an update on potential transporter targets and evaluates the impact of available technologies to enable transporter screening, lead optimization, and assessment of pharmacokinetics.     

Technologies for transporter drug discovery

Transporters represent attractive targets for drug discovery and are implicated in the pathophysiology of disorders across several therapeutic areas including asthma, cardiovascular disease, diabetes and neuroscience. However, the intrinsic mechanistic properties of transporters present significant challenges to the development of high-throughput screening methodologies. This review provides an update on potential transporter targets and evaluates the impact of available technologies to enable transporter screening, lead optimization and assessment of pharmacokinetics.     

Appointment of New Features Editor

Transporters are essential players in bacterial growth and survival, since they are key for uptake of nutrients on the one hand, and for defence against endogenous and environmental stresses on the other hand. Remarkably, in addition to their primary role in substrate translocation, it has become clear that some transporters have acquired a secondary function as sensors and information processors in signalling pathways. In this review, we describe recent advances in our understanding of the role of transporters in such signalling cascades, and discuss some of the emergent dynamic behaviour found in hallmark examples. A particular focus is placed on new insights into mechanistic details of information transfer between transporters and regulatory proteins. Quantitative considerations reveal that these signalling complexes can implement a remarkable diversity of regulatory logic functions, where the transporter can act as activity switch, as positive or negative reporter of transport flux, or as a signalling hub for the integration of multiple inputs. Such a dual use of transport proteins not only enables efficient substrate translocation but is also an elegant strategy to integrate important information about the cell's external conditions with its current physiological state.     

植物寡肽运输与硝酸根运输基因家族的研究进展

氮素是植物生长发育的重要营养元素,也是限制植物生物量尤其是经济产量的关键营养元素之一.植物不仅能从外界获取无机氮素(硝酸根、铵根和尿素等),还能以氨基酸、寡肽等形式获取有机氮素.植物已进化出复杂的运输系统来吸收与运输这些含氮化合物.硝酸根运输基因家族分为低亲和力硝酸根运输基因(low-affmity nitrate t...     

Pharmacogenomics and systems biology of membrane transporters

Membrane transporters are essential for fundamental cellular functions and normal physiological processes. These molecules influence drug absorption and distribution, and play key roles in drug therapeutic effects. A primary goal of current research in drug discovery and development is to fully understand the interaction between transporters and drugs at both system level and individual level for personalized therapy. Pharmacogenomics studies the genetic basis of the individual variations in response to drug therapy, whereas systems biology provides the understanding of biological processes at the system level. The integration of pharmacogenomics with systems biology in membrane transporter study is necessary to solve complex problems in diseases and drug effects. Such integration provides insight to key issues of pharmacogenomics and systems biology of membrane transporters. These key issues include the correlations between structure and function, genotype and phenotype, and systematic interactions between different transporters, between transporters and other proteins, and between transporters and drugs. The exploration in these key issues may ultimately contribute to the personalized medicine with high efficacy but less toxicity, which is the overall goal of pharmacogenomics and systems biology.     

Prediction of transporter family from protein sequence by support vector machine approach

Transporters play key roles in cellular transport and metabolic processes, and in facilitating drug delivery and excretion. These proteins are classified into families based on the transporter classification (TC) system. Determination of the TC family of transporters facilitates the study of their cellular and pharmacological functions. Methods for predicting TC family without sequence alignments or clustering are particularly useful for studying novel transporters whose function cannot be determined by sequence similarity. This work explores the use of a machine learning method, support vector machines (SVMs), for predicting the family of transporters from their sequence without the use of sequence similarity. A total of 10,636 transporters in 13 TC subclasses, 1914 transporters in eight TC families, and 168,341 nontransporter proteins are used to train and test the SVM prediction system. Testing results by using a separate set of 4351 transporters and 83,151 nontransporter proteins show that the overall accuracy for predicting members of these TC subclasses and families is 83.4% and 88.0%, respectively, and that of nonmembers is 99.3% and 96.6%, respectively. The accuracies for predicting members and nonmembers of individual TC subclasses are in the range of 70.7-96.1% and 97.6-99.9%, respectively, and those of individual TC families are in the range of 60.6-97.1% and 91.5-99.4%, respectively. A further test by using 26,139 transmembrane proteins outside each of the 13 TC subclasses shows that 90.4-99.6% of these are correctly predicted. Our study suggests that the SVM is potentially useful for facilitating functional study of transporters irrespective of sequence similarity.     

Towards a structural understanding of drug and peptide transport within the proton-dependent oligopeptide transporter (POT) family

One of the principal aims of modern drug design is the targeted delivery of drugs within the body, such as to the central nervous system, combined with their exclusion from the liver and kidneys, which break down foreign molecules and subsequently eliminate them. Many of the commonly prescribed drugs are transported into cells and across the plasma membrane via endogenous membrane transporters, whose principal roles are the uptake of essential nutrients for metabolism. In many cases, such drug transport is serendipitous as they are simply mistaken as 'natural' compounds. Many of these transporters could, however, be targeted more efficiently, improving drug absorption, distribution and retention. The molecular details of these drug-transporter interactions, however, are at best poorly understood, in large part through the absence of any high-resolution structural information. To address this issue, we recently determined the structure of a prokaryotic peptide transporter, PepTSo from Shewanella oneidensis, which shares a high degree of sequence similarity and functional characteristics with the human PepT1 and PepT2 proteins. PepT1 and PepT2 contribute significantly to the oral bioavailability and pharmacokinetic properties of a number of important drug families, including antibiotics, antivirals and anticancer agents. The crystal structure of PepTSo provides the first high-resolution model of a drug importer and provides the starting point for understanding drug and peptide transport within the human body.     

脊椎动物ABCA基因亚家族研究进展

ABC（ATP-binding cassette）基因家族编码膜蛋白,其成员负责多种物质的跨膜运输。基于氨基酸序列的同源性,人的48个ABC成员被分为7个亚家族：ABCA~ABCG。与其他亚家族相比,ABCA基因编码的蛋白具有独特的拓扑结构,并且其家族成员在两栖动物和哺乳动物分化之后各发生过一次大的扩展（expanding）。基因结构分析发现这两次扩展均是通过基因倍增实现的,这些倍增的产物在啮齿目和食肉目中得到保留,而在灵长目中却有一半变成假基因或被删除。ABCA成员主要负责不同组织器官脂类和胆固醇的跨膜运输,部分成员的突变与疾病相关。     

Phylogenetic relationships within cation transporter families of Arabidopsis

Uptake and translocation of cationic nutrients play essential roles in physiological processes including plant growth, nutrition, signal transduction, and development. Approximately 5% of the Arabidopsis genome appears to encode membrane transport proteins. These proteins are classified in 46 unique families containing approximately 880 members. In addition, several hundred putative transporters have not yet been assigned to families. In this paper, we have analyzed the phylogenetic relationships of over 150 cation transport proteins. This analysis has focused on cation transporter gene families for which initial characterizations have been achieved for individual members, including potassium transporters and channels, sodium transporters, calcium antiporters, cyclic nucleotide-gated channels, cation diffusion facilitator proteins, natural resistance-associated macrophage proteins (NRAMP), and Zn-regulated transporter Fe-regulated transporter-like proteins. Phylogenetic trees of each family define the evolutionary relationships of the members to each other. These families contain numerous members, indicating diverse functions in vivo. Closely related isoforms and separate subfamilies exist within many of these gene families, indicating possible redundancies and specialized functions. To facilitate their further study, the PlantsT database (http://plantst.sdsc.edu) has been created that includes alignments of the analyzed cation transporters and their chromosomal locations.     

溶质转运蛋白超家族的功能及结构研究进展

溶质转运蛋白(solute carriers,SLC)超家族是人类细胞膜(含胞内膜)上最重要的膜转运蛋白家族之一,它参与了细胞间的物质运输、能量传递、营养代谢、信号传导等重要生理活动。SLC转运蛋白超家族包含52个亚家族,共有400多名成员。研究表明,人类基因突变所致SLC蛋白表达异常或功能缺陷与糖尿病、高血压、抑郁症等多种重大疾病密切相关,使得该家族蛋白的功能研究近年来备受关注。SLC转运家族蛋白三维结构的解析有助于阐述其底物选择性结合与转运的精确分子机制,为研究该家族功能相关疾病的分子机理以及针对理性药物研发奠定了精细的三维结构基础。本文对近年来溶质转运蛋白超家族的结构及功能研究进展进行了总结,试图对该家族的共性规律进行阐述。     

The transportome of the malaria parasite

Membrane transport proteins, also known as transporters, control the movement of ions, nutrients, metabolites, and waste products across the membranes of a cell and are central to its biology. Proteins of this type also serve as drug targets and are key players in the phenomenon of drug resistance. The malaria parasite has a relatively reduced transportome, with only approximately 2.5% of its genes encoding transporters. Even so, assigning functions and physiological roles to these proteins, and ascertaining their contributions to drug action and drug resistance, has been very challenging. This review presents a detailed critique and synthesis of the disruption phenotypes, protein subcellular localisations, protein functions (observed or predicted), and links to antimalarial drug resistance for each of the parasite's transporter genes. The breadth and depth of the gene disruption data are particularly impressive, with at least one phenotype determined in the parasite's asexual blood stage for each transporter gene, and multiple phenotypes available for 76% of the genes. Analysis of the curated data set revealed there to be relatively little redundancy in the Plasmodium transportome; almost two‐thirds of the parasite's transporter genes are essential or required for normal growth in the asexual blood stage of the parasite, and this proportion increased to 78% when the disruption phenotypes available for the other parasite life stages were included in the analysis. These observations, together with the finding that 22% of the transportome is implicated in the parasite's resistance to existing antimalarials and/or drugs within the development pipeline, indicate that transporters are likely to serve, or are already serving, as drug targets. Integration of the different biological and bioinformatic data sets also enabled the selection of candidates for transport processes known to be essential for parasite survival, but for which the underlying proteins have thus far remained undiscovered. These include potential transporters of pantothenate, isoleucine, or isopentenyl diphosphate, as well as putative anion‐selective channels that may serve as the pore component of the parasite's ‘new permeation pathways’. Other novel insights into the parasite's biology included the identification of transporters for the potential development of antimalarial treatments, transmission‐blocking drugs, prophylactics, and genetically attenuated vaccines. The syntheses presented herein set a foundation for elucidating the functions and physiological roles of key members of the Plasmodium transportome and, ultimately, to explore and realise their potential as therapeutic targets.     

节肢动物ABC转运蛋白及其介导的杀虫剂抗性

腺苷三磷酸结合盒转运蛋白（ATP-binding cassette transporter),简称ABC转运蛋白（ABC transporter）,是继细胞色素P450单加氧酶、羧酸酯酶、谷胱甘肽S-转移酶之后又一类参与解毒作用的重要蛋白家族,因其在杀虫剂解毒等方面起着非常重要的作用,近年来逐渐受到广泛关注。ABC转运蛋白是一大类跨膜蛋白,其核心结构通常由4个结构域组成,包括2个高度疏水的跨膜结构域（transmembrane domains , TMD）和2个核苷酸结合域（nucleotide binding domains, NBD）。根据序列相似性和保守结构域,可以把ABC转运蛋白家族分为8个亚家族,每个亚家族的成员数及功能不同。这类蛋白在各种生物体内均有分布,其主要功能包括转运物质、信号传导、细胞表面受体及参与细胞内DNA修复,转录及调节基因的表达过程等。此外,近年来的研究表明,ABC转运蛋白的突变或过表达不仅与节肢动物对化学农药的抗药性密切相关,而且在抗Bt毒素方面也起着非常重要的作用,对转Bt作物造成严重威胁。本文综述了节肢动物ABC转运蛋白的结构,ATP水解介导的作用机制,亚家族的分类、结构及生理功能,以及由ABC转运蛋白介导的抗药性研究进展,旨在深入了解ABC转运蛋白的研究现状及其在节肢动物抗药性方面的作用,为阐明节肢动物抗药性机制提供新的理论依据,对改进农业害虫的抗性监测和治理策略也具有一定的指导意义。     

Expression of human glucose transporters in Xenopus oocytes: kinetic characterization and substrate specificities of the erythrocyte, liver, and brain isoforms

We describe the functional expression of three members of the family of human facilitative glucose transporters, the erythrocyte-type transporter (GLUT 1), the liver-type transporter (GLUT 2), and the brain-type transporter (GLUT 3), by microinjection of their corresponding mRNAs into Xenopus oocytes. Expression was determined by the appearance of transport activity, as measured by the transport of 3-O-methyl-D-glucose or 2-deoxy-D-glucose. We have measured the Km for 3-O-methyl-D-glucose of GLUTs 1, 2, and 3, and the results are discussed in light of the possible roles for these different transporters in the regulation of blood glucose. The substrate specificity of these transporter isoforms has also been examined. We show that, for all transporters, the transport of 2-deoxy-D-glucose is inhibited by D-but not by L-glucose. In addition, both D-galactose and D-mannose are transported by GLUTs 1-3 at significant rates; furthermore, GLUT 2 is capable of transporting D-fructose. The nature of the glucose binding sites of GLUTs 1-3 was investigated by using hexose inhibition of 2-deoxy-D-glucose uptake. We show that the characteristics of this inhibition are different for each transporter isoform.     

ABC transporters: how small machines do a big job

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes. ATP binding and ATP hydrolysis have distinct roles in this cycle: binding favors the outward-facing orientation, whereas hydrolysis returns the transporter to an inward-facing conformation.     

Carbohydrate Hydrolysis and Transport in the Extreme Thermoacidophile Sulfolobus solfataricus

Extremely thermoacidophilic microbes, such as Sulfolobus solfataricus, are strict chemoheterotrophs despite their geologic niche. To clarify their ecophysiology, the overlapping roles of endoglucanases and carbohydrate transporters were examined during growth on soluble cellodextrins as the sole carbon and energy source. Strain-specific differences in genome structure implied a unique role for one of three endogenous endoglucanases. Plasmid-based endoglucanase expression promoted the consumption of oligosaccharides, including cellohexaose (G6) through cellonanaose (G9). Protein transporters required for cellodextrin uptake were identified through mutagenesis and complementation of an ABC transporter cassette, including a putative oligosaccharide binding protein. In addition, ablation of the binding protein compromised growth on glucose and alpha-linked oligosaccharides while inactivation of a previously described glucose transporter had no apparent impact. These data demonstrate that S. solfataricus employs a redundant mechanism for soluble cellodextrin catabolism having both substrate uptake and extracytoplasmic hydrolytic components.     

A transporter for phenolamine uptake in the arthropod CNS

Biogenic monoamines play central roles in the nervous control of physiological processes in both vertebrates and invertebrates, each using a suite of neurotransmitters tailored through evolution. Among the ancillary proteins necessary for the deployment of monoamine transmitters are membrane-bound transporters that enable the reuptake of synaptically released transmitters. Transporters responsible for monoamine uptake include a novel transporter discovered in a pest insect, the cabbage looper Trichoplusia ni, which has high affinity for the phenolamines octopamine and tyramine. Sequence analysis suggests that this transporter has no direct ortholog in the sequenced genomes of model invertebrates. We report here a preliminary investigation into the true extent of the distribution of this type of transporter using RT-PCR with a set of degenerate primers selective for monoamine transporters on cDNAs made from the nervous systems of a range of arthropods. PCR products encoding the N-terminal region of orthologs of this transporter were detected in a variety of insect orders, as well as in a crustacean, but were not found in representatives of either the Diptera or the Hymenoptera. Thus, although this transporter is widely expressed in invertebrates, there are various invertebrates that appear to have evolved alternate ways of recycling phenolamine neurotransmitters released at the nerve synapse.     

Progress in study on organic anion transporters

Organic anion transporters (OATs) belong to a family of poly-specific transporters mainly located in barrier epithelia such as renal proximal tubule, brain, liver and placenta. OATs interact with endogenous metabolic end products such as urate and acidic neutrotransmitter metabolites, as well as with a multitude of widely used drugs, including antibiotics, diuretics, antihypertensives and anti-inflammatory drugs. Thereby, OATs play an important role in renal drug elimination and have an impact on pharmacokinetics. This review summarizes current knowledge of the properties and functional roles of the cloned OAT family members detailed in tissue differences in expression and physiologic function, drug-drug interactions, and finally, gender-dependent regulation in health and diseased states.     

Characterization of 5(6)-carboxy-2,'7'-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4

MRP2 (ABCC2) is an efflux transporter expressed on the apical membrane of polarized cells. This protein has a major role in the biliary elimination of toxic compounds from the liver. As MRP2 transports many endogenous compounds, including LTC4 as well as xenobiotics and toxic phase II metabolites, blockade of this transporter may cause the accumulation of these compounds in the hepatocyte, resulting in hepatotoxicity. The vesicular transport assay is a great tool to study drug-drug and drug-endogenous compound interactions of ABC transporters. In this assay, inside-out membrane vesicles are used, so the test compound can readily access the transporter. As MRP2 transports many ionic compounds that are difficult to investigate in a whole-cell system because of permeability reasons, the vesicular transport assay is a good choice for screening MRP2-mediated interactions. LTC4 is not an optimal substrate for high-throughput screening for MRP2 interactors, even though it is an important MRP2 substrate. Therefore, the transport of a drug surrogate, 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), by MRP2 was characterized using the vesicular transport assay. The data indicate that CDCF proves to be an ideal substrate for MRP2 vesicular transport assay with its optimal detection and transport properties.