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Introduction Macrophages are immune cells that play critical roles in both inflammation and tissue homeostasis. While macrophages appear to exhibit dynamic and complex functional phenotypes in vivo, the dichotomous model [...], The facilitative GLUT1 and GLUT3 hexose transporters are expressed abundantly in macrophages, but whether they have distinct functions remains unclear. We confirmed that GLUT1 expression increased after M1 polarization stimuli and found that GLUT3 expression increased after M2 stimulation in macrophages. Conditional deletion of Glut3 (LysM-Cre [Glut3.sup.fl/fl]) impaired M2 polarization of bone marrow-derived macrophages. Alternatively activated macrophages from the skin of patients with atopic dermatitis showed increased GLUT3 expression, and a calcipotriol-induced model of atopic dermatitis was rescued in LysM-Cre [Glut3.sup.fl/fl] mice. M2-like macrophages expressed GLUT3 in human wound tissues as assessed by transcriptomics and costaining, and GLUT3 expression was significantly decreased in nonhealing, compared with healing, diabetic foot ulcers. In an excisional wound healing model, LysM-Cre [Glut3.sup.fl/fl] mice showed significantly impaired M2 macrophage polarization and delayed wound healing. GLUT3 promoted IL-4/STAT6 signaling, independently of its glucose transport activity. Unlike plasma membrane-localized GLUT1, GLUT3 was localized primarily to endosomes and was required for the efficient endocytosis of IL-4R[alpha] subunits. GLUT3 interacted directly with GTP-bound RAS in vitro and in vivo through its intracytoplasmic loop domain, and this interaction was required for efficient STAT6 activation and M2 polarization. PAK activation and macropinocytosis were also impaired without GLUT3, suggesting broader roles for GLUT3 in the regulation of endocytosis. Thus, GLUT3 is required for efficient alternative macrophage polarization and function, through a glucose transport-independent, RAS-mediated role in the regulation of endocytosis and IL-4/STAT6 activation.

Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system. [ABSTRACT FROM AUTHOR]

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD. Author summary: Excitable cells, such as neurons and muscles, are essential for the movement and behavior of animals. These cells express a set of specific types of ion channels that allow the selective passage of ions across the plasma membrane. The alteration in the levels of these ion channels influences cell excitability and the function of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control the function of excitable cells. The BK SLO-1 channel is a calcium-activated potassium channel that reduces excitability at presynaptic axon terminals and sites of muscle excitation. In a C. elegans genetic study, authors found that the delayed exit of SLO-1 channels from the ER causes their degradation by a mechanism called ER-associated degradation (ERAD). Interestingly, the same components that directly mediate SLO-1 ERAD also process a key transcriptional factor that maintains proteasome levels, thus indirectly influencing SLO-1 degradation. These data show that the levels of SLO-1 channels are regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD. [ABSTRACT FROM AUTHOR]

Increased understanding of fructose metabolism, which begins with uptake via the intestine, is important because fructose now constitutes a physiologically significant portion of human diets and is associated with increased incidence of certain cancers and metabolic diseases. New insights in our knowledge of intestinal fructose absorption mediated by the facilitative glucose transporter GLUT5 in the apical membrane and by GLUT2 in the basolateral membrane are reviewed. We begin with studies related to structure as well as ligand binding, then revisit the controversial proposition that apical GLUT2 is the main mediator of intestinal fructose absorption. The review then describes how dietary fructose may be sensed by intestinal cells to affect the expression and activity of transporters and fructolytic enzymes, to interact with the transport of certain minerals and electrolytes, and to regulate portal and peripheral fructosemia and glycemia. Finally, it discusses the potential contributions of dietary fructose to gastrointestinal diseases and to the gut microbiome. [ABSTRACT FROM AUTHOR]

Infection by the parasite Plasmodium falciparum is the leading cause of malaria in humans. The parasite has a unique and essential plastid-like organelle called the apicoplast. The apicoplast contains a genome that undergoes replication and repair through the action of a replicative polymerase (apPOL). apPOL has no direct orthologs in mammalian polymerases and is therefore an attractive antimalarial drug target. No structural information exists for apPOL, and the Klenow fragment of Escherichia coli DNA polymerase I, which is its closest structural homolog, shares only 28% sequence identity. Here, conditions for the crystallization of and preliminary X-ray diffraction data from crystals of P. falciparum apPOL are reported. Data complete to 3.5 Å resolution were collected from a single crystal (2 × 2 × 5 µm) using a 5 µm beam. The space group P6522 (unit-cell parameters a = b = 141.8, c = 149.7 Å, α = β = 90, γ = 120°) was confirmed by molecular replacement. Refinement is in progress. [ABSTRACT FROM AUTHOR]

Presents a study which looked at fructose-1,6-bisphosphatase (FBPase) in a complex involving zinc cations and fructose 6-phosphate and phosphate, which revealed disordered loop (loop 52-72). Details on loop 52-72; Information on FBPase; Method used for the study; Discussion based on the study.

Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity. [ABSTRACT FROM AUTHOR]

The CaV2 voltage-gated calcium channel is the major conduit of calcium ions necessary for neurotransmitter release at presynaptic active zones (AZs). The CaV2 channel is a multimeric complex that consists of a pore-forming α 1 subunit and two auxiliary β and α 2 δ subunits. Although auxiliary subunits are critical for channel function, whether they are required for α 1 trafficking is unresolved. Using endogenously fluorescent protein-tagged CaV2 channel subunits in Caenorhabditis elegans , we show that UNC-2/α 1 localizes to AZs even in the absence of CCB-1/β or UNC-36/α 2 δ, albeit at low levels. When UNC-2 is manipulated to be trapped in the endoplasmic reticulum (ER), CCB-1 and UNC-36 fail to colocalize with UNC-2 in the ER, indicating that they do not coassemble with UNC-2 in the ER. Moreover, blocking ER-associated degradation does not further increase presynaptic UNC-2 channels in ccb-1 or unc-36 mutants, indicating that UNC-2 levels are not regulated in the ER. An unc-2 mutant lacking C-terminal AZ protein interaction sites with intact auxiliary subunit binding sites displays persistent presynaptic UNC-2 localization and a prominent increase of UNC-2 channels in nonsynaptic axonal regions, underscoring a protective role of auxiliary subunits against UNC-2 degradation. In the absence of UNC-2, presynaptic CCB-1 and UNC-36 are profoundly diminished to barely detectable levels, indicating that UNC-2 is required for the presynaptic localization of CCB-1 and UNC-36. Together, our findings demonstrate that although the pore-forming subunit does not require auxiliary subunits for its trafficking and transport to AZs, it recruits auxiliary subunits to stabilize and expand calcium channel signalosomes. SIGNIFICANCE STATEMENT Synaptic transmission in the neuron hinges on the coupling of synaptic vesicle exocytosis with calcium influx. This calcium influx is mediated by CaV2 voltage-gated calcium channels. These channels consist of one pore-forming α 1 subunit and two auxiliary β and α 2 δ subunits. The auxiliary subunits enhance channel function and regulate the overall level of channels at presynaptic terminals. However, it is not settled how these auxiliary subunits regulate the overall channel level. Our study in C. elegans finds that although the auxiliary subunits do not coassemble with α 1 and aid trafficking, they are recruited to α 1 and stabilize the channel complex at presynaptic terminals. Our study suggests that drugs that target the auxiliary subunits can directly destabilize and have an impact on CaV2 channels., (Copyright © 2023 the authors.)

Surface-enhanced infrared absorption spectroscopy (SEIRAS) is a powerful tool that allows studying the reactivity of protein monolayers at very low concentrations and independent from the protein size. In this study, we probe the surface's morphology of electroless gold deposition for optimum enhancement using two different types of immobilization adapted to two proteins. Independently from the mode of measurement (i.e., transmission or reflection) or type of protein immobilization (i.e., through electrostatic interactions or nickel-HisTag), the enhancement and reproducibility of protein signals in the infrared spectra critically depended on the gold nanostructured surface morphology deposited on silicon. Just a few seconds deviation from the optimum time in the nanoparticle deposition led to a significantly weaker enhancement. Scanning electron microscopy and atomic force microscopy measurements revealed the evolution of the nanostructured surface when comparing different deposition times. The optimal deposition time led to isolated gold nanostructures on the silicon crystal. Importantly, in the case of the immobilization using nickel-HisTag, the surface morphology is rearranged upon immobilization of linker and the protein. A complex three-dimensional (3D) network of nanoparticles decorated with the protein could be observed leading to the optimal enhancement. The electroless deposition of gold is a simple technique, which can be adapted to flow cells and used in analytical approaches.

Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Schmidl, Tamayo Rojas, Iancu, Choe and Oreb.)

Hexoses are the major source of energy and carbon skeletons for biosynthetic processes in all kingdoms of life. Their cellular uptake is mediated by specialized transporters, including glucose transporters (GLUT, SLC2 gene family). Malfunction or altered expression pattern of GLUTs in humans is associated with several widespread diseases including cancer, diabetes and severe metabolic disorders. Their high relevance in the medical area makes these transporters valuable drug targets and potential biomarkers. Nevertheless, the lack of a suitable high-throughput screening system has impeded the determination of compounds that would enable specific manipulation of GLUTs so far. Availability of structural data on several GLUTs enabled in silico ligand screening, though limited by the fact that only two major conformations of the transporters can be tested. Recently, convenient high-throughput microbial and cell-free screening systems have been developed. These remarkable achievements set the foundation for further and detailed elucidation of the molecular mechanisms of glucose transport and will also lead to great progress in the discovery of GLUT effectors as therapeutic agents. In this mini-review, we focus on recent efforts to identify potential GLUT-targeting drugs, based on a combination of structural biology and different assay systems.

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

Salicylic acid (SA) is a plant hormone involved in a number of physiological responses including both local and systemic resistance of plants to pathogens. In Arabidopsis, SA is glucosylated to form either SA 2-O-β-d-glucose (SAG) or SA glucose ester (SGE). In this study, we show that SAG accumulates in the vacuole of Arabidopsis, while the majority of SGE was located outside the vacuole. The uptake of SAG by vacuolar membrane-enriched vesicles isolated from Arabidopsis was stimulated by the addition of MgATP and was inhibited by both vanadate (ABC transporter inhibitor) and bafilomycin A 1 (vacuolar H + -ATPase inhibitor), suggesting that SAG uptake involves both an ABC transporter and H + -antiporter. Despite its absence in the vacuole, we observed the MgATP-dependent uptake of SGE by Arabidopsis vacuolar membrane-enriched vesicles. SGE uptake was not inhibited by vanadate but was inhibited by bafilomycin A 1 and gramicidin D providing evidence that uptake was dependent on an H + -antiporter. The uptake of both SAG and SGE was also inhibited by quercetin and verapamil (two known inhibitors of multidrug efflux pumps) and salicin and arbutin. MgATP-dependent SAG and SGE uptake exhibited Michaelis-Menten-type saturation kinetics. The vacuolar enriched-membrane vesicles had a 46-fold greater affinity and a 10-fold greater transport activity with SGE than with SAG. We propose that in Arabidopsis, SAG is transported into the vacuole to serve as a long-term storage form of SA while SGE, although also transported into the vacuole, is easily hydrolyzed to release the active hormone which can then be remobilized to other cellular locations., (© 2017 Scandinavian Plant Physiology Society.)

Human GLUT5 is a fructose-specific transporter in the glucose transporter family (GLUT, SLC2 gene family). Its substrate-specificity and tissue-specific expression make it a promising target for treatment of diabetes, metabolic syndrome and cancer, but few GLUT5 inhibitors are known. To identify and characterize potential GLUT5 ligands, we developed a whole-cell system based on a yeast strain deficient in fructose uptake, in which GLUT5 transport activity is associated with cell growth in fructose-based media or assayed by fructose uptake in whole cells. The former method is convenient for high-throughput screening of potential GLUT5 inhibitors and activators, while the latter enables detailed kinetic characterization of identified GLUT5 ligands. We show that functional expression of GLUT5 in yeast requires mutations at specific positions of the transporter sequence. The mutated proteins exhibit kinetic properties similar to the wild-type transporter and are inhibited by established GLUT5 inhibitors N-[4-(methylsulfonyl)-2-nitrophenyl]-1,3-benzodioxol-5-amine (MSNBA) and (-)-epicatechin-gallate (ECG). Thus, this system has the potential to greatly accelerate the discovery of compounds that modulate the fructose transport activity of GLUT5.

Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed.

Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H(+) symporter in an inward-facing conformation at 3.2-Å resolution. The Staphylococcus epidermidis glucose/H(+) symporter is homologous to human glucose transporters, is very specific and has high avidity for glucose, and is inhibited by the human glucose transport inhibitors cytochalasin B, phloretin, and forskolin. On the basis of the crystal structure in conjunction with mutagenesis and functional studies, we propose a mechanism for glucose/H(+) symport and discuss the symport mechanism versus facilitated diffusion.

AMP triggers a 15° subunit-pair rotation in fructose-1,6-bisphosphatase (FBPase) from its active R state to its inactive T state. During this transition, a catalytically essential loop (residues 50-72) leaves its active (engaged) conformation. Here, the structures of Ile(10) → Asp FBPase and molecular dynamic simulations reveal factors responsible for loop displacement. The AMP/Mg(2+) and AMP/Zn(2+) complexes of Asp(10) FBPase are in intermediate quaternary conformations (completing 12° of the subunit-pair rotation), but the complex with Zn(2+) provides the first instance of an engaged loop in a near-T quaternary state. The 12° subunit-pair rotation generates close contacts involving the hinges (residues 50-57) and hairpin turns (residues 58-72) of the engaged loops. Additional subunit-pair rotation toward the T state would make such contacts unfavorable, presumably causing displacement of the loop. Targeted molecular dynamics simulations reveal no steric barriers to subunit-pair rotations of up to 14° followed by the displacement of the loop from the active site. Principal component analysis reveals high-amplitude motions that exacerbate steric clashes of engaged loops in the near-T state. The results of the simulations and crystal structures are in agreement: subunit-pair rotations just short of the canonical T state coupled with high-amplitude modes sterically displace the dynamic loop from the active site.

A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.