Histone deacetylase 6 (HDAC6) is a tubulin-specific deacetylase that regulates microtubule-dependent cell movement. In this study, we identify the F-actin-binding protein cortactin as a HDAC6 substrate. We demonstrate that HDAC6 binds cortactin and that overexpression of HDAC6 leads to hypoacetylation of cortactin, whereas inhibition of HDAC6 activity leads to cortactin hyperacetylation. HDAC6 alters the ability of cortactin to bind F-actin by modulating a "charge patch" in its repeat region. Introduction of charge-preserving or charge-neutralizing mutations in this cortactin repeat region correlates with the gain or loss of F-actin binding ability, respectively. Cells expressing a charge-neutralizing cortactin mutant were less motile than control cells or cells expressing a charge-preserving mutant. These findings suggest that, in addition to its role in microtubule-dependent cell motility, HDAC6 influences actin-dependent cell motility by altering the acetylation status of cortactin, which, in turn, changes the F-actin binding activity of cortactin.

The glucose transporters (GLUT/SLC2A) are members of the major facilitator superfamily. Here, we generated a three-dimensional model for Glut1 using a two-step strategy: 1), GlpT structure as an initial homology template and 2), evolutionary homology using glucose-6-phosphate translocase as a template. The resulting structure (PDB No. 1SUK) exhibits a water-filled passageway communicating the extracellular and intracellular domains, with a funnel-like exofacial vestibule (infundibulum), followed by a 15 A-long x 8 A-wide channel, and a horn-shaped endofacial vestibule. Most residues which, by mutagenesis, are crucial for transport delimit the channel, and putative sugar recognition motifs (QLS, QLG) border both ends of the channel. On the outside of the structure there are two positively charged cavities (one exofacial, one endofacial) delimited by ATP-binding Walker motifs, and an exofacial large side cavity of yet unknown function. Docking sites were found for the glucose substrate and its inhibitors: glucose, forskolin, and phloretin at the exofacial infundibulum; forskolin, and phloretin at an endofacial site next to the channel opening; and cytochalasin B at a positively charged endofacial pocket 3 A away from the channel. Thus, 1SUK accounts for practically all biochemical and mutagenesis evidence, and provides clues for the transport process.

Vitamin C is a wide spectrum antioxidant essential for humans, which are unable to synthesize the vitamin and must obtain it from dietary sources. There are two biologically important forms of vitamin C, the reduced form, ascorbic acid, and the oxidized form, dehydroascorbic acid. Vitamin C exerts most of its biological functions intracellularly and is acquired by cells with the participation of specific membrane transporters. This is a central issue because even in those species capable of synthesizing vitamin C, synthesis is restricted to the liver (and pancreas) from which is distributed to the organism. Most cells express two different transporter systems for vitamin C; a transporter system with absolute specificity for ascorbic acid and a second system that shows absolute specificity for dehydroascorbic acid. The dehydroascorbic acid transporters are members of the GLUT family of facilitative glucose transporters, of which at least three isoforms, GLUT1, GLUT3 and GLUT4, are dehydroascorbic acid transporters. Ascorbic acid is transported by the SVCT family of sodium-coupled transporters, with two isoforms molecularly cloned, the transporters SVCT1 y SVCT2, that show different functional properties and differential cell and tissue expression. In humans, the maintenance of a low daily requirement of vitamin C is attained through an efficient system for the recycling of the vitamin involving the two families of vitamin C transporters.

Until recently, the only facilitated hexose transporter GLUT proteins (SLC2A) known to transport fructose were GLUTs 2 and 5. However, the recently cloned GLUT7 can also transport fructose as well as glucose. Comparison of sequence alignments indicated that GLUTs 2, 5, and 7 all had an isoleucine residue at position "314" (GLUT7), whereas the non-fructose-transporting isoforms, GLUTs 1, 3, and 4, had a valine at this position. Mutation of Ile-314 to a valine in GLUT7 resulted in a loss of fructose transport, whereas glucose transport remained completely unaffected. Similar results were obtained with GLUTs 2 and 5. Energy minimization modeling of GLUT7 indicated that Ile-314 projects from transmembrane domain 7 (TM7) into the lumen of the aqueous pore, where it could form a hydrophobic interaction with tryptophan 89 from TM2. A valine residue at 314 appeared to produce a narrowing of the vestibule when compared with the isoleucine. It is proposed that this hydrophobic interaction across the pore forms a selectivity filter restricting the access of some hexoses to the substrate binding site(s) within the aqueous channel. The presence of a selectivity filter in the extracellular vestibule of GLUT proteins would allow for subtle changes in substrate specificity without changing the kinetic parameters of the protein.The facilitative glucose transporters (SLC2a) belong to the facilitated transporter super gene family, which all appear to have a core structure of 12 transmembrane helices clustered in two sets of six between which there is a central aqueous pore (1). The recent crystal structures solved for LacY and GlpT suggest that during the transport cycle, these two clusters undergo an alteration in their tilt such that their binding site moves from an outward-to an inward-facing conformation (2, 3). In the case of the GLUT 3 proteins, scanning mutagenesis studies and computer modeling indicate that their pore is formed by TMs 5, 7, 8, 10, and 11, whereas other helices may influence the pore structure, i.e. TMs 1, 2, 3, and 4 (4 -6). What is less clear is the shape of the outer-or innerfacing vestibules allowing entry of the substrates into the pore and access to the proposed substrate binding site. Mueckler et al. (7) initially reported that a naturally occurring conservative mutation of valine 197 to isoleucine (V197I) in GLUT2 resulted in a greatly reduced transport capacity when the protein was expressed in Xenopus oocytes. Subsequently, they also reported that a comparable mutation in GLUT1 (valine 165 to isoleucine) abolished glucose transport (8). Valine is a smaller residue than isoleucine, and this raised the possibility that, if these residues in TM5 faced into the pore, the larger isoleucine might hinder the passage of substrate. These results did not explain why such a conservative substitution could have such a profound effect on the function of these proteins. However, it has been proposed that one or more hydrophobic residues projecting into the pore can influence substrate access to the binding sit...

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-β, Myxovirus resistance gene A (MxA) and RNaseL.

No abstract

Hypoxia is a key regulator of cancer progression and chemoresistance. Ambiguity remains about how cancer cells adapt to hypoxic microenvironments and transfer oncogenic factors to surrounding cells. In this study, we determined the effects of hypoxia on the bioactivity of sEVs in a panel of ovarian cancer (OvCar) cell lines. The data obtained demonstrate a varying degree of platinum resistance induced in OvCar cells when exposed to low oxygen tension (1% oxygen). Using quantitative mass spectrometry (Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra, SWATH) and targeted multiple reaction monitoring (MRM), we identified a suite of proteins associated with glycolysis that change under hypoxic conditions in cells and sEVs. Interestingly, we identified a differential response to hypoxia in the OvCar cell lines and their secreted sEVs, highlighting the cells’ heterogeneity. Proteins are involved in metabolic reprogramming such as glycolysis, including putative hexokinase (HK), UDP-glucuronosyltransferase 1–6 (UD16), and 6-phosphogluconolactonase (6 PGL), and their presence correlates with the induction of platinum resistance. Furthermore, when normoxic cells were exposed to sEVs from hypoxic cells, platinum-resistance increased significantly (p < 0.05). Altered chemoresistance was associated with changes in glycolysis and fatty acid synthesis. Finally, sEVs isolated from a clinical cohort (n = 31) were also found to be enriched in glycolysis-pathway proteins, especially in patients with recurrent disease. These data support the hypothesis that hypoxia induces changes in sEVs composition and bioactivity that confers carboplatin resistance on target cells. Furthermore, we propose that the expression of sEV-associated glycolysis-pathway proteins is predictive of ovarian cancer recurrence and is of clinical utility in disease management.

Na؉ -coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. The Na ϩ -coupled ascorbic acid cotransporters SVCT1 and SVCT2 transport ascorbic acid down the electrochemical sodium gradient (1-5). SVCT1 and SVCT2 have a 65% amino acid sequence identity and a similar hydropathy profile and are predicted to conform to a secondary structure model containing 12 transmembrane-spanning helices, with the hydrophilic amino and C-terminal domains located intracellularly (1-5). Most tissues express SVCT2, with SVCT1 showing a more restricted tissue distribution (3, 6). SVCT1 and SVCT2 show different kinetic properties; SVCT1 has an apparent ascorbic acid transport K m in the range of 50 -200 M, whereas the K m of SVCT2 is lower, in the range of 10 -30 M (1-3, 6 -9). Both transporters are activated by Na ϩ in a cooperative manner, with a Hill coefficient (n H ) near 2 (1-3, 6 -9). We showed that both substrates, ascorbic acid and sodium, modify the kinetic properties of SVCT2 in a reciprocal manner (7). When extracellular sodium increases, the K m for ascorbic acid transport decreases more than 100 times without affecting the transport V max , converting a low affinity form of the transporter into a high affinity transporter (7). In turn, ascorbic acid modifies the sodium cooperativity of SVCT2 in a complex concentrationdependent manner, with maximal Na ϩ cooperativity observed at 100 M ascorbic acid (7). Moreover, SVCT2 is completely dependent on the presence of calcium or magnesium ions for function, with the bivalent ions switching the transporter from an inactive into an active form by increasing the transport V max without affecting the K m or the sodium cooperativity. In contrast, SVCT1 is active in the complete absence of bivalent cations (7).Little is currently known about the functional-structural determinants that define the activity of SVCT1 and SVCT2. The available information is restricted to the effect of protein phosphorylation on the functional activity and subcellular localization of SVCT2 (10), with evidence indicating that the C-terminal region is fundamental for the differential sorting and apical localization of SVCT1 in polarized cells (9 -14) and that N-linked glycosylation sites are important for maintaining ascorbic transporter function (15).Evidence has been published indicating that the functional activity of SVCT2 is impaired at acid pH (3,6,16). This finding may have physiological implications for the regulation of ascorbic acid transport and metabolism in organs and tissues that