The in-silico evaluation of important GLUT9 residue for uric acid transport based on renal hypouricemia type 2.

Uric acid is the end product of purine metabolism. Uric acid transporters in the renal proximal tubule plays a key role in uric acid transport. Functional abnormalities in these transporters could lead to high or low levels of uric acid in the blood plasma, known as hyperuricemia and hypouricemia, respectively. GLUT9 has been reported as a key transporter for uric acid reuptake in renal proximal tubule. GLUT9 mutation is known as causal gene for renal hypouricemia due to defective uric acid uptake, with more severe cases resulting in urolithiasis and exercise induced acute kidney injury (EIAKI). However, the effect of mutation is not fully investigated and hard to predict the change of binding affinity. We comprehensively described the effect of GLUT9 mutation for uric acid transport using molecular dynamics and investigated the specific site for uric acid binding differences. R171C and R380W showed the significant disruption of the structure not affecting transport dynamics whereas L75R, G216R, N333S, and P412R showed the reduced affinity of the extracellular vestibular area towards urate. Interestingly, T125 M showed a significant increase in intracellular binding energy, associated with distorted geometries. We can use this classification to consider the effect mutations by comparing the transport profiles of mutants against those of chemical candidates for transport and providing new perspectives to urate lowering drug discovery using GLUT9.

Baicalein alleviates hyperuricemia by promoting uric acid excretion and inhibiting xanthine oxidase.

BACKGROUND: Insufficient renal urate excretion and/or overproduction of uric acid (UA) are the dominant causes of hyperuricemia. Baicalein (BAL) is widely distributed in dietary plants and has extensive biological activities, including antioxidative, anti-inflammatory and antihypertensive activities. PURPOSE: To investigate the anti-hyperuricemic effects of BAL and the underlying mechanisms in vitro and in vivo. METHODS: We investigated the inhibitory effects of BAL on GLUT9 and URAT1 in vitro through electrophysiological experiments and 14C-urate uptake assays. To evaluate the impact of BAL on serum and urine UA, the expression of GLUT9 and URAT1, and the activity of xanthine oxidase (XOD), we developed a mouse hyperuricemia model by potassium oxonate (PO) injection. Molecular docking analysis based on homology modeling was performed to explain the predominant efficacy of BAL compared with the other test compounds. RESULTS: BAL dose-dependently inhibited GLUT9 and URAT1 in a noncompetitive manner with IC50 values of 30.17 ± 8.68 µM and 31.56 ± 1.37 µM, respectively. BAL (200 mg/kg) significantly decreased serum UA and enhanced renal urate excretion in PO-induced hyperuricemic mice. Moreover, the expression of GLUT9 and URAT1 in the kidney was downregulated, and XOD activity in the serum and liver was suppressed. The docking analysis revealed that BAL potently interacted with Trp336, Asp462, Tyr71 and Gln328 of GLUT9 and Ser35 and Phe241 of URAT1. CONCLUSION: These results indicated that BAL exerts potent antihyperuricemic efects through renal UA excretal promotion and serum UA production. Thus, we propose that BAL may be a promising treatment for the prevention of hyperuricemia owing to its multitargeted inhibitory activity.

Asp22 drives the protonation state of the Staphylococcus epidermidis glucose/H+ symporter.

The Staphylococcus epidermidis glucose/H+ symporter (GlcPSe) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H+ symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcPSe has a loose H+/sugar coupling. Asp22 is part of the proton-binding site of GlcPSe and crucial for the glucose/H+ co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcPSe We found that Asp22 has a pKa of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcPSe A neutral replacement of the negatively charged Asp22 led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp22 We expected that the substitution of the residue Ile105 for a serine, located within hydrogen-bonding distance to Asp22, would change the microenvironment, but the pKa of Asp22 corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp22, with the latter now exhibiting a pKa of 6.4. These studies confirm that Asp22 is the proton-binding residue in GlcPSe and show that charged residues in its vicinity affect the pKa of glucose/H+ symport.

Glucose transporters in the small intestine in health and disease.

Absorption of monosaccharides is mainly mediated by Na+-D-glucose cotransporter SGLT1 and the facititative transporters GLUT2 and GLUT5. SGLT1 and GLUT2 are relevant for absorption of D-glucose and D-galactose while GLUT5 is relevant for D-fructose absorption. SGLT1 and GLUT5 are constantly localized in the brush border membrane (BBM) of enterocytes, whereas GLUT2 is localized in the basolateral membrane (BLM) or the BBM plus BLM at low and high luminal D-glucose concentrations, respectively. At high luminal D-glucose, the abundance SGLT1 in the BBM is increased. Hence, D-glucose absorption at low luminal glucose is mediated via SGLT1 in the BBM and GLUT2 in the BLM whereas high-capacity D-glucose absorption at high luminal glucose is mediated by SGLT1 plus GLUT2 in the BBM and GLUT2 in the BLM. The review describes functions and regulations of SGLT1, GLUT2, and GLUT5 in the small intestine including diurnal variations and carbohydrate-dependent regulations. Also, the roles of SGLT1 and GLUT2 for secretion of enterohormones are discussed. Furthermore, diseases are described that are caused by malfunctions of small intestinal monosaccharide transporters, such as glucose-galactose malabsorption, Fanconi syndrome, and fructose intolerance. Moreover, it is reported how diabetes, small intestinal inflammation, parental nutrition, bariatric surgery, and metformin treatment affect expression of monosaccharide transporters in the small intestine. Finally, food components that decrease D-glucose absorption and drugs in development that inhibit or downregulate SGLT1 in the small intestine are compiled. Models for regulations and combined functions of glucose transporters, and for interplay between D-fructose transport and metabolism, are discussed.

Structure, function and regulation of mammalian glucose transporters of the SLC2 family.

The SLC2 genes code for a family of GLUT proteins that are part of the major facilitator superfamily (MFS) of membrane transporters. Crystal structures have recently revealed how the unique protein fold of these proteins enables the catalysis of transport. The proteins have 12 transmembrane spans built from a replicated trimer substructure. This enables 4 trimer substructures to move relative to each other, and thereby alternately opening and closing a cleft to either the internal or the external side of the membrane. The physiological substrate for the GLUTs is usually a hexose but substrates for GLUTs can include urate, dehydro-ascorbate and myo-inositol. The GLUT proteins have varied physiological functions that are related to their principal substrates, the cell type in which the GLUTs are expressed and the extent to which the proteins are associated with subcellular compartments. Some of the GLUT proteins translocate between subcellular compartments and this facilitates the control of their function over long- and short-time scales. The control of GLUT function is necessary for a regulated supply of metabolites (mainly glucose) to tissues. Pathophysiological abnormalities in GLUT proteins are responsible for, or associated with, clinical problems including type 2 diabetes and cancer and a range of tissue disorders, related to tissue-specific GLUT protein profiles. The availability of GLUT crystal structures has facilitated the search for inhibitors and substrates and that are specific for each GLUT and that can be used therapeutically. Recent studies are starting to unravel the drug targetable properties of each of the GLUT proteins.

The molecular basis for sugar import in malaria parasites.

Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.

Comprehensive in silico Study of GLUT10: Prediction of Possible Substrate Binding Sites and Interacting Molecules.

OBJECTIVES: The Arterial Tortuosity Syndrome (ATS) is an autosomal recessive connective tissue disorder, mainly characterized by tortuosity and stenosis of the arteries with a propensity towards aneurysm formation and dissection. It is caused by mutations in the SLC2A10 gene that encodes the facilitative glucose transporter GLUT10. The molecules transported by and interacting with GLUT10 have still not been unambiguously identified. Hence, the study attempts to identify both the substrate binding site of GLUT10 and the molecules interacting with this site. METHODS: As High-resolution X-ray crystallographic structure of GLUT10 was not available, 3D homology model of GLUT10 in open conformation was constructed. Further, molecular docking and bioinformatics investigation were employed. RESULTS AND DISCUSSION: Blind docking of nine reported potential in vitro substrates with this 3D homology model revealed that substrate binding site is possibly made with PRO531, GLU507, GLU437, TRP432, ALA506, LEU519, LEU505, LEU433, GLN525, GLN510, LYS372, LYS373, SER520, SER124, SER533, SER504, SER436 amino acid residues. Virtual screening of all metabolites from the Human Serum Metabolome Database and muscle metabolites from Human Metabolite Database (HMDB) against the GLUT10 revealed possible substrates and interacting molecules for GLUT10, which were found to be involved directly or partially in ATS progression or different arterial disorders. Reported mutation screening revealed that a highly emergent point mutation (c. 1309G>A, p. Glu437Lys) is located in the predicted substrate binding site region. CONCLUSION: Virtual screening expands the possibility to explore more compounds that can interact with GLUT10 and may aid in understanding the mechanisms leading to ATS.

Identification and characterisation of two high-affinity glucose transporters from the spoilage yeast Brettanomyces bruxellensis.

The yeast Brettanomyces bruxellensis (syn. Dekkera bruxellensis) is an emerging and undesirable contaminant in industrial low-sugar ethanol fermentations that employ the yeast Saccharomyces cerevisiae. High-affinity glucose import in B. bruxellensis has been proposed to be the mechanism by which this yeast can outcompete S. cerevisiae. The present study describes the characterization of two B. bruxellensis genes (BHT1 and BHT3) believed to encode putative high-affinity glucose transporters. In vitro-generated transcripts of both genes as well as the S. cerevisiae HXT7 high-affinity glucose transporter were injected into Xenopus laevis oocytes and subsequent glucose uptake rates were assayed using 14C-labelled glucose. At 0.1 mM glucose, Bht1p was shown to transport glucose five times faster than Hxt7p. pH affected the rate of glucose transport by Bht1p and Bht3p, indicating an active glucose transport mechanism that involves proton symport. These results suggest a possible role for BHT1 and BHT3 in the competitive ability of B. bruxellensis.

Targeted Degradation of Glucose Transporters Protects against Arsenic Toxicity.

The abundance of cell surface glucose transporters must be precisely regulated to ensure optimal growth under constantly changing environmental conditions. We recently conducted a proteomic analysis of the cellular response to trivalent arsenic, a ubiquitous environmental toxin and carcinogen. A surprising finding was that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure. Here we show that this downregulation reflects targeted arsenic-dependent degradation of glucose transporters. Degradation occurs in the vacuole and requires the E2 ubiquitin ligase Ubc4, the E3 ubiquitin ligase Rsp5, and K63-linked ubiquitin chains. We used quantitative proteomic approaches to determine the ubiquitinated proteome after arsenic exposure, which helped us to identify the ubiquitination sites within these glucose transporters. A mutant lacking all seven major glucose transporters was highly resistant to arsenic, and expression of a degradation-resistant transporter restored arsenic sensitivity to this strain, suggesting that this pathway represents a protective cellular response. Previous work suggests that glucose transporters are major mediators of arsenic import, providing a potential rationale for this pathway. These results may have implications for the epidemiologic association between arsenic exposure and diabetes.

Chemical biology probes of mammalian GLUT structure and function.

The structure and function of glucose transporters of the mammalian GLUT family of proteins has been studied over many decades, and the proteins have fascinated numerous research groups over this time. This interest is related to the importance of the GLUTs as archetypical membrane transport facilitators, as key limiters of the supply of glucose to cell metabolism, as targets of cell insulin and exercise signalling and of regulated membrane traffic, and as potential drug targets to combat cancer and metabolic diseases such as type 2 diabetes and obesity. This review focusses on the use of chemical biology approaches and sugar analogue probes to study these important proteins.

Large-scale whole-exome sequencing association studies identify rare functional variants influencing serum urate levels.

Elevated serum urate levels can cause gout, an excruciating disease with suboptimal treatment. Previous GWAS identified common variants with modest effects on serum urate. Here we report large-scale whole-exome sequencing association studies of serum urate and kidney function among ≤19,517 European ancestry and African-American individuals. We identify aggregate associations of low-frequency damaging variants in the urate transporters SLC22A12 (URAT1; p = 1.3 × 10-56) and SLC2A9 (p = 4.5 × 10-7). Gout risk in rare SLC22A12 variant carriers is halved (OR = 0.5, p = 4.9 × 10-3). Selected rare variants in SLC22A12 are validated in transport studies, confirming three as loss-of-function (R325W, R405C, and T467M) and illustrating the therapeutic potential of the new URAT1-blocker lesinurad. In SLC2A9, mapping of rare variants of large effects onto the predicted protein structure reveals new residues that may affect urate binding. These findings provide new insights into the genetic architecture of serum urate, and highlight molecular targets in SLC22A12 and SLC2A9 for lowering serum urate and preventing gout.

Screening and Scale-Up of GLUT Transporter Constructs Suitable for Biochemical and Structural Studies.

Identifying membrane proteins that can be produced and isolated in homogenous form in detergent is a lengthy trial-and-error process that can be facilitated by fluorescence-based screening approaches. We describe (1) the strategy and protocol of cloning by homologous recombination, (2) whole-cell and in-gel fluorescence measurements to estimate GLUT-GFP fusion protein yields, (3) use of size-exclusion chromatography monitored by fluorescence (FSEC) for assessing the homogeneity of the GLUT-GFP fusion proteins, and (4) the protocol for large-scale production and purification of the Bos taurus GLUT5 construct that enabled its crystal structure determination.

Glucose Transport Activity Measured in Giant Vesicles.

Incorporation of membrane proteins and internal reporter systems directly into giant vesicles, during their formation from a hydrogel surface, has emerged as a promising new concept in membrane protein characterization. Here, we provide the detailed protocol for a glucose transporter activity assay based on giant vesicles containing a fluorescent enzyme-linked reporter system internally. This assay is applicable for the functional analysis of a variety of hexose-transporting proteins. We furthermore believe that it can aid in the development of drugs targeting hexose transporters.

Design, Synthesis, and Evaluation of GLUT Inhibitors.

The Warburg effect describes how most cancer cells exhibit higher-than-normal glucose consumption, not only under hypoxic conditions, but also when normal oxygen levels are present. Although glucose transporter 1 (GLUT1) has been found to play a key role in the cellular uptake of glucose, especially in cancer cells, where it is generally overexpressed, it has not been given consideration as a suitable target for the development of anticancer drugs. In this chapter, an example of molecular design and realization of novel GLUT1 inhibitors, including in silico modeling, chemical synthesis, and biological characterization, is provided. This process started with the identification of a focused series of oxime derivatives, originally designed as estrogen receptor (ER) ligands, which were structurally optimized in order to direct their activity towards GLUT1 and to minimize their binding to the ERs, leading to the production of efficient and selective inhibitors of glucose uptake in cancer cells.

Identification of essential amino acids for glucose transporter 5 (GLUT5)-mediated fructose transport.

Intestinal fructose uptake is mainly mediated by glucose transporter 5 (GLUT5/SLC2A5). Its closest relative, GLUT7, is also expressed in the intestine but does not transport fructose. For rat Glut5, a change of glutamine to glutamic acid at codon 166 (p.Q166E) has been reported to alter the substrate-binding specificity by shifting Glut5-mediated transport from fructose to glucose. Using chimeric proteins of GLUT5 and GLUT7, here we identified amino acid residues of GLUT5 that define its substrate specificity. The proteins were expressed in NIH-3T3 fibroblasts, and their activities were determined by fructose radiotracer flux. We divided the human GLUT5 sequence into 26 fragments and then replaced each fragment with the corresponding region in GLUT7. All fragments that yielded reduced fructose uptake were analyzed further by assessing the role of individual amino acid residues. Various positions in the first extracellular loop, in the fifth, seventh, eighth, ninth, and tenth transmembrane domains (TMDs), and in the regions between the ninth and tenth TMDs and tenth and 11th TMDs were identified as being important for proper fructose uptake. Although the p.Q167E change did not render the human protein into a glucose transporter, molecular dynamics simulations revealed a drastic change in the dynamics and a movement of the intracellular loop connecting the sixth and seventh TMDs, which covers the exit of the ligand. Finally, we generated a GLUT7-GLUT5 chimera consisting of the N-terminal part of GLUT7 and the C-terminal part of GLUT5. Although this chimera was inactive, we demonstrate fructose transport after introduction of four amino acids derived from GLUT5.

Affinity and path of binding xylopyranose unto E. coli xylose permease.

Glucose transporters (GLUTs), expressed in all types of human cells, are responsible for the uptake of sugars as the primary energy source for the normal functions of good cells and for the abnormal growth of cancer cells. The E. coli xylose permease (XylE), a homologue of human GLUTs, has been investigated more thoroughly than other major facilitator proteins in the current literature. In this paper, we present a molecular dynamics (MD) study of an all-atom model system to elucidate the atomistic details and the free-energy landscape along the path of binding a xylopyranose (XYP) from the extracellular space to the inside of the transporter protein XylE. From the MD simulations, the Gibbs free energy of binding was found to be -4.4kcal/mol in agreement with the experimental value of -4.7kcal/mol. The accuracy of our study is further shown in the computed hydration energy of XYP of -14.6kcal/mol in comparison with the experimental data of -15.0kcal/mol. Along the binding path, the Gibbs free energy of the XYP-XylE complex first rises from zero in the dissociated state to approximately 4 kcal/mol in the transition state (when XylE slightly increases its opening toward the extracellular side to accommodate XYP) before dropping down to -9.0 kcal/mol in the bound state. These quantitative insights indicate the fast equilibration between the bound and the unbound states of XylE and XYP. They also serve as an atomistic-dynamic corroboration of the experimental conclusion that XylE is a high-affinity sugar transporter.

A Glimpse of Membrane Transport through Structures-Advances in the Structural Biology of the GLUT Glucose Transporters.

The cellular uptake of glucose is an essential physiological process, and movement of glucose across biological membranes requires specialized transporters. The major facilitator superfamily glucose transporters GLUTs, encoded by the SLC2A genes, have been a paradigm for functional, mechanistic, and structural understanding of solute transport in the past century. This review starts with a glimpse into the structural biology of membrane proteins and particularly membrane transport proteins, enumerating the landmark structures in the past 25years. The recent breakthrough in the structural elucidation of GLUTs is then elaborated following a brief overview of the research history of these archetypal transporters, their functional specificity, and physiological and pathophysiological significances. Structures of GLUT1, GLUT3, and GLUT5 in distinct transport and/or ligand-binding states reveal detailed mechanisms of the alternating access transport cycle and substrate recognition, and thus illuminate a path by which structure-based drug design may be applied to help discover novel therapeutics against several debilitating human diseases associated with GLUT malfunction and/or misregulation.

Electron crystallography reveals that substrate release from the PTS IIC glucose transporter is coupled to a subtle conformational change.

The phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) is a structurally and functionally complex system that mediates sugar uptake in bacteria. Besides several soluble subunits, the glucose-specific PTS includes the integral membrane protein IICB that couples the transmembrane transport of glucose to its phosphorylation. Here, we used electron crystallography of sugar-embedded tubular crystals of the glucose-specific IIC transport domain from Escherichia coli (ecIICglc) to visualize the structure of the transporter in the presence and absence of its substrate. Using an in vivo transport assay and binding competition experiments, we first established that, while it transports d-glucose, ecIICglc does not bind l-glucose. We then determined the projection structure of ecIICglc from tubular crystals embedded in d- and l-glucose and found a subtle conformational change. From comparison of the ecIICglc projection maps with crystal structures of other IIC transporters, we can deduce that the transporter adopts an inward-facing conformation, and that the maps in the presence and absence of the substrate reflect the transporter before and after release of the transported glucose into the cytoplasm. The transition associated with substrate release appears to require a subtle structural rearrangement in the region that includes hairpin 1.

Identification of Key Residues for Urate Specific Transport in Human Glucose Transporter 9 (hSLC2A9).

Human glucose transporter 9 (hSLC2A9) is critical in human urate homeostasis, for which very small deviations can lead to chronic or acute metabolic disorders. Human SLC2A9 is unique in that it transports hexoses as well as the organic anion, urate. This ability is in contrast to other homologous sugar transporters such as glucose transporters 1 and 5 (SLC2A1 &SLC2A5) and the xylose transporter (XylE), despite the fact that these transporters have similar protein structures. Our in silico substrate docking study has revealed that urate and fructose bind within the same binding pocket in hSLC2A9, yet with distinct orientations, and allowed us to identify novel residues for urate binding. Our functional studies confirmed that N429 is a key residue for both urate binding and transport. We have shown that cysteine residues, C181, C301 and C459 in hSLC2A9 are also essential elements for mediating urate transport. Additional data from chimæric protein analysis illustrated that transmembrane helix 7 of hSLC2A9 is necessary for urate transport but not sufficient to allow urate transport to be induced in glucose transporter 5 (hSLC2A5). These data indicate that urate transport in hSLC2A9 involves several structural elements rather than just a unique substrate binding pocket.