Proper cytoskeletal architecture beneath the plasma membrane of red blood cells requires Ttll4.

Mammalian red blood cells (RBCs) circulate through blood vessels, including capillaries, for tens of days under high mechanical stress. RBCs tolerate this mechanical stress while maintaining their shape because of their elastic membrane skeleton. This membrane skeleton consists of spectrin-actin lattices arranged as quasi-hexagonal units beneath the plasma membrane. In this study, we found that the organization of the RBC cytoskeleton requires tubulin tyrosine ligase-like 4 (Ttll4). RBCs from Ttll4-knockout mice showed larger average diameters in smear test. Based on the rate of hemolysis, Ttll4-knockout RBCs showed greater vulnerability to phenylhydrazine-induced oxidative stress than did wild-type RBCs. Ultrastructural analyses revealed the macromolecular aggregation of cytoskeletal components in RBCs of Ttll4-knockout mice. Immunoprecipitation using the anti-glutamylation antibody GT335 revealed nucleosome assembly protein 1 (NAP1) to be the sole target of TTLL4 in the RBCs, and NAP1 glutamylation was completely lost in Ttll4-knockout RBCs. In wild-type RBCs, the amount of glutamylated NAP1 in the membrane was nearly double that in the cytosol. Furthermore, the absence of TTLL4-dependent glutamylation of NAP1 weakened the binding of NAP1 to the RBC membrane. Taken together, these data demonstrate that Ttll4 is required for proper cytoskeletal organization in RBCs.

SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

Functionalized magnetic nanoparticles as an in vivo delivery system.

We developed extremely small functionalized magnetic nanoparticles (MNPs) for use as an in vivo delivery system for pharmaceuticals and biomolecules. We functionalized the MNPs (d = 3 nm) by silanization of amino groups on the particles with (3-aminopropyl)triethoxysilane for subsequent cross-linking with pharmaceuticals and biomolecules. The MNPs were successfully introduced into living cells without any further modification, such as the use of cationic residues, to enhance endocytic internalization. The particles could be incorporated into the subcutaneous tissue of a mouse's ear through the skin of the ear and could be localized by application of an external magnetic field.We also developed a cell-specific delivery system that makes use of MNPs (d = 3 nm) conjugated with folic acid and a coumarin fluorophore for recognition by folate receptors on the cell surface. The modified MNPs were internalized by human pharyngeal cancer cells (KB cells) after an incubation time that was short compared with the time required for internalization of MNPs without folic acid. Cellular recognition of MNPs may lead to the development of other cell-specific delivery systems.These functionalized MNPs are expected to be useful as a new drug delivery tool.

A novel chalcone polyphenol inhibits the deacetylase activity of SIRT1 and cell growth in HEK293T cells.

SIRT1 is one of seven mammalian orthologs of yeast silent information regulator 2 (Sir2), and it functions as a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase. Recently, resveratrol and its analogues, which are polyphenols, have been reported to activate the deacetylase activity of SIRT1 in an in vitro assay and to expand the life-span of several species through Sir2 and the orthologs. To find activators or inhibitors to SIRT1, we examined thirty-six polyphenols, including stilbenes, chalcones, flavanones, and flavonols, with the SIRT1 deacetylase activity assay using the acetylated peptide of p53 as a substrate. The results showed that 3,2',3',4'-tetrahydroxychalcone, a newly synthesized compound, inhibited the SIRT1-mediated deacetylation of a p53 acetylated peptide and recombinant protein in vitro. In addition, this agent induced the hyperacetylation of endogenous p53, increased the endogenous p21CIP1/WAF1 in intact cells, and suppressed the cell growth. These results indicated that 3,2',3',4'-tetrahydroxychalcone had a stronger inhibitory effect on the SIRT1-pathway than sirtinol, a known SIRT1-inhibitor. Our results mean that 3,2',3',4'-tetrahydroxychalcone is a novel inhibitor of SIRT1 and produces physiological effects on organisms probably through inhibiting the deacetylation by SIRT1.

Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy.

ATB(0,+) [SLC6A14 (solute carrier family 6 member 14)] is an Na(+)/Cl(-)-coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB(0,+) in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB(0,+). The transport process is Na(+)/Cl(-)-dependent with an Na(+)/Cl(-)/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of alpha-methyltryptophan as a blocker, not a transportable substrate, for ATB(0,+). ATB(0,+) can transport 18 of the 20 proteinogenic amino acids. alpha-Methyltryptophan blocks the transport function of ATB(0,+) with an IC(50) value of approximately 250 muM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that alpha-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB(0,+) is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB(0,+)-positive tumour cells with alpha-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB(0,+)-negative cell lines are not affected. The blockade of ATB(0,+) in these cells with alpha-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB(0,+) as a drug target for cancer chemotherapy.

Functionalized nano-magnetic particles for an in vivo delivery system.

Nanotechnologies to allow the nondisruptive introduction of carriers in vivo have wide potential for therapeutic delivery system. We have prepared functional nano-magnetic particles (d = 3 nm) by silanization with (3-aminopropyl) triethoxysilane. For the purpose of functionalizing the surface of the nanoparticles with amino groups for subsequent cross-linking with pharmaceuticals and biomolecules. The extremely small particles were successfully introduced into living cells without any further modification to enhance endocytic internalization, such as the use of a cationic help. The cells containing the internalized particles continued to thrive, indicating that the particles have no inhibition effect for mitosis. In addition, the particles could be incorporated into the subcutaneous tissue of mouse's ear from ear skin and were able to be localized upon application of an external magnetic field. The functionalized nano-magnetic particles are expected to be useful as a new delivery tool.

Amino acid transporter ATA2 is stored at the trans-Golgi network and released by insulin stimulus in adipocytes.

Recently, we cloned the ATA/SNAT transporters responsible for amino acid transport system A. System A is one of the major transport systems for small neutral and glucogenic amino acids represented by alanine and is involved in the metabolism of glucose and fat. Here, we describe the cellular mechanisms that participate in the acute translocation of ATA2 by insulin stimulus in 3T3-L1 adipocytes. We monitored this insulin-stimulated translocation of ATA2 using an expression system of enhanced green fluorescent protein-tagged ATA2. Studies in living cells revealed that ATA2 is stored in a discrete perinuclear site and that the transporter is released in vesicles from this site toward the plasma membrane. In immunofluorescent analysis, the storage site of ATA2 overlapped with the location of syntaxin 6, a marker of the trans-Golgi network (TGN), but not with that of EEA1, a marker of the early endosomes. The ATA2-containing vesicles on or near the plasma membrane were distinct from GLUT4-containing vesicles. Brefeldin A, an inhibitor of vesicular exit from the TGN, caused morphological changes in the ATA2 storage site along with the similar changes in the TGN. In non-transfected adipocytes, brefeldin A inhibited insulin-stimulated uptake of alpha-(methylamino)isobutyric acid more profoundly than insulin-stimulated uptake of 2-deoxy-d-glucose. These data demonstrate that the ATA2 storage site is specifically associated with the TGN and not with the general endosomal recycling system. Thus, the insulin-stimulated translocation pathways for ATA2 and GLUT4 in adipocytes are distinct, involving different storage sites.

Regulation of amino acid transporter ATA2 by ubiquitin ligase Nedd4-2.

We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of alpha-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface.

Transport of amino acid-based prodrugs by the Na+- and Cl(-) -coupled amino acid transporter ATB0,+ and expression of the transporter in tissues amenable for drug delivery.

We evaluated the potential of the Na(+)- and Cl(-)-coupled amino acid transporter ATB(0,+) as a delivery system for amino acid-based prodrugs. Immunofluorescence analysis indicated that ATB(0,+) is expressed abundantly on the luminal surface of cells lining the lumen of the large intestine and the airways of the lung and in various ocular tissues, including the conjunctival epithelium, the tissues easily amenable for drug delivery. We screened a variety of beta-carboxyl derivatives of aspartate and gamma-carboxyl derivatives of glutamate as potential substrates for this transporter using heterologous expression systems. In mammalian cells expressing the cloned ATB(0,+), several of the aspartate and glutamate derivatives inhibited glycine transport via ATB(0,+). Direct evidence for ATB(0,+)-mediated transport of these derivatives was obtained in Xenopus laevis oocytes using electrophysiological methods. Exposure of oocytes, which express ATB(0,+) heterologously, to aspartate beta-benzyl ester as a model derivative induced inward currents in a Na(+)- and Cl(-)-dependent manner with a Na(+)/Cl(-)/aspartate beta-benzyl ester stoichiometry of 2:1:1. ATB(0,+) transported not only the beta-carboxyl derivatives of aspartate and the gamma-carboxyl derivatives of glutamate but also valacyclovir, which is an alpha-carboxyl ester of acyclovir with valine. The transport of valacyclovir via ATB(0,+) was demonstrable in both heterologous expression systems. This process was dependent on Na(+) and Cl(-). The ability of ATB(0,+) to transport valacyclovir was comparable with that of the peptide transporter PEPT1. These findings suggest that ATB(0,+) has significant potential as a delivery system for amino acid-based drugs and prodrugs.

A study of the substrate specificity of Na+-dependent and Na+-independent neutral amino acid transport systems in dog intestinal brush-border membrane vesicles using L-alanine analogues.

Neutral amino acids are mainly transported across the intestinal brush-border membrane by two Na+-dependent systems (system B0 and system B0+) and one Na+-independent system (system b0,+). To investigate potential differences in substrate specificity between these systems, we screened ten different alanine analogues for their ability to inhibit the transport of L-alanine in dog intestinal brush-border membrane vesicles. The results suggested that a phenyl group directly attached to the alpha-carbon has different effects on the Na+-dependent and Na+-independent transport systems, with an increased affinity for the former and a decreased affinity for the latter. Based on these inhibition studies, we investigated [14C]L-phenylglycine transport kinetics in comparison with L-alanine. Similar to L-alanine, L-phenylglycine transport followed at least three routes, however, the Km of the Na+-dependent transport system was lower and the K'm of the Na+-independent system was higher than the corresponding values for L-alanine. These results corroborated the conclusions drawn from the inhibition studies. Based on these data, we conclude that different sizes of immediate parts to the alpha-carbon in functional groups of amino acid analogues have differential effects on the interaction of these amino acid analogues with the Na+-dependent and Na+-independent transport systems for neutral amino acids.

The long-lasting anti-anginal effects of CP-060S in a rat model of arginine vasopressin-induced myocardial ischaemia.

The anti-anginal effect of CP-060S, a new cardioprotective agent that prevents myocardial Na+-, Ca2+-overload and has Ca2+-channel blocking activity, was evaluated in a rat model of arginine8-vasopressin (AVP)-induced cardiac ischaemia. Infusion of AVP (0.2 IU kg(-1)) depressed the electrocardiogram (ECG) ST segment, an index of myocardial ischaemia. Vehicle, CP-060S and diltiazem were given orally 1, 2, 4, 8, 12 and 24 h before the administration of AVP. CP-060S, at 3 mg kg(-1) and 10 mg kg(-1), suppressed AVP-induced ST-segment depression for 2 h and 12 h, respectively. In contrast, diltiazem, at 10 and 30 mg kg(-1), suppressed AVP-induced ST-segment depression for only 1 h. The persistent suppression of the AVP-induced ST-segment depression by CP-060S correlated with the time course of changes in its plasma concentration. The minimum effective concentration of CP-060S was estimated to be 30 ng mL(-1) (approximately 50 nM), consistent with its vasorelaxant potency in rat isolated aortic strips (concentration producing 50% relaxation of KCl contraction, IC50 = 32.6+/-8.3 nM). Intravenously administered CP-060S, at 300 microg kg(-1) and diltiazem at 500 microg kg(-1) showed similar haemodynamic changes, whereas CP-060S, at 300 microg kg(-1), significantly suppressed AVP-induced ST-segment depression and diltiazem, at 500 microg kg(-1), had no effect on AVP-induced ST-segment depression. In summary, orally administered CP-060S exerted a long-lasting anti-anginal effect proportionate to the time course of changes in its plasma concentration in a rat model of AVP-induced ischaemia.

Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon.

D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.