Search

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.

Tryptophan fluorescence has shown the conformational state of a dynamic loop in recombinant porcine fructose-1,6-bisphosphatase. The kinetic and structural properties and conformational response of the Trp57 mutant of FBPase to various metal cations and physiologically relevant ligands have been found.

Crystal structures of fructose 1,6-bisphosphatase (FBPase) have been studied and a mechanism proposed for catalysis and allosteric inhibition seen in product complexes. Competition was seen between AMP and divalent cations in crystal structure of metal-product complexes of FBPase.

A study was conducted to analyze the crystal structures of wild-type and mutant forms of adenylosuccinate synthetase from Escherichia coli. The structures supported multiple roles for Asp13 and His41 in a proposed catalytic mechanism of the synthetase. Results indicated that the mutation of Asp13 to alanine eliminates measurable activity. Findings also showed that the structures can function together as catalytic acids in the formation of adenylosuccinate.

A study was conducted to characterize the crystallography of wild-type fructose-1,6-bisphosphatase (FBPase) in a complex of fructose 6-phosphate, phosphate and Zn2+. Escherichia coli was utilized to express ion recombinant wild-type porcine FBPase. Results indicated catalysis mechanisms when Asp121 serves as a catalytic base for the alpha-anomer while Glu280 functions as a catalytic base for the beta-anomer.

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]