Tubulin–Na+ ,K +-ATPase interaction: Involvement in enzymatic regulation and cellular function

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na⁺,K ⁺-ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin–NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na ⁺. Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na ⁺ and K ⁺, and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na ⁺/K ⁺ homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.     

High glucose levels induce inhibition of Na,K-ATPase via stimulation of aldose reductase, formation of microtubules and formation of an acetylated tubulin/Na,K-ATPase complex

Our previous studies demonstrated that acetylated tubulin forms a complex with Na(+),K(+)-ATPase and thereby inhibits its enzyme activity in cultured COS and CAD cells. The enzyme activity was restored by treatment of cells with l-glutamate, which caused dissociation of the acetylated tubulin/Na(+),K(+)-ATPase complex. Addition of glucose, but not elimination of glutamate, led to re-formation of the complex and inhibition of the Na(+),K(+)-ATPase activity. The purpose of the present study was to elucidate the mechanism underlying this effect of glucose. We found that exposure of cells to high glucose concentrations induced: (a) microtubule formation; (b) activation of aldose reductase by the microtubules; (c) association of tubulin with membrane; (d) formation of the acetylated tubulin/Na(+),K(+)-ATPase complex and consequent inhibition of enzyme activity. Exposure of cells to sorbitol caused similar effects. Studies on erythrocytes from diabetic patients and on tissues containing insulin-insensitive glucose transporters gave similar results. Na(+),K(+)-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients as compared with normal subjects. Immunoprecipitation analysis showed that acetylated tubulin was a constituent of a complex with Na(+),K(+)-ATPase in erythrocyte membranes from diabetic patients. Based on these findings, we propose a mechanism whereby glucose triggers a synergistic effect of tubulin and sorbitol, leading to activation of aldose reductase, microtubule formation, and consequent Na(+),K(+)-ATPase inhibition.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila

In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Only a small fraction of avian erythrocyte histone is involved in ongoing acetylation.

We have studied histone acetylation in chicken erythrocytes. We find that about 30% of the histone in these cells is acetylated, however the majority of these histones are not in a dynamic steady state typical of other chicken cells and of mammalian cells, but rather are frozen in this state of modification. A very small fraction of erythrocyte histones are being modified normally but cannot be detected as shifting to higher levels of acetylation upon treatment with butyrate because the amount of histone so modified is small. Nonetheless, chicken erythrocytes incorporate 3H-acetate into histones about 40% as well as seen in the dynamically active HTC cells. This is most likely due to the formation of very high specific activity Acetyl CoA pools in erythrocytes which have very low levels of coenzyme A. We conclude that these genetically inactive cells are involved in only a minor way with histone acetylation.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.     

Reversible cross-linking and CO treatment as an approach in red cell stabilization

We explored the use of the reversible cross-linking reagent dimethyl 3,3-dithiobispropionimidate (DTBP) in combination with CO treatment as an approach to stabilizing erythrocyte structure and function. Erythrocytes were cross-linked with different concentrations of DTBP for different times. DTBP increased erythrocyte osmotic stability, blocked lysolecithin-induced echinocytosis, and decreased erythrocyte deformability in a concentration- and time-dependent manner. Reversal of the cross-linking with the reducing agent dithioerythritol (DTE) restored osmotic fragility and response to lysolecithin as well as deformability. Complete reversal, however, is a function of the DTBP concentration and the time of cross-linking. The effects of cross-linking with 5 mM DTBP for 1 h were completely reversible after treatment with 10 mM DTE for 20 min. Longer incubation times or higher concentrations of DTBP resulted in partial reversal by DTE of the effects produced by DTBP. Cross-linking and reversal only slightly reduced the ATP content. The hemoglobin contained in the cross-linked and reversed cells could still undergo reversible oxygenation and deoxygenation. Erythrocytes were pretreated with CO, cross-linked with 5 mM DTBP for 1 or 3 h, loaded with a solution containing 500 mM glucose for 24 h, and freeze-dried in a medium containing 15% (w/v) albumin. Rehydration followed in distilled water. Complete recovery, measured as the percentage of free hemoglobin, was achieved for cells cross-linked with 5 mM DTBP for 3 h and freeze-dried to a final water content of 10-15%. Non-cross-linked cells lysed 100% on rehydration in distilled water. No methemoglobin (MetHb) formation as a result of freeze-drying was detected in CO-treated cells. In non-CO-treated cells 20% of the Hb was converted to MetHb.     

Electron spin resonance, hematological, and deformability studies of erythrocytes from patients with Huntington's disease.

Electron spin resonance, hematologic, and deformability studies of erythrocytes from patients with Huntington's disease have been performed A decreased deformability of Huntington's disease erythrocytes compared to normal controls was demonstrated. No difference in erythrocyte hematologic indices, osmotic fragility, reticulocyte counts, or intracellular Na+ concentration was found. Huntington's disease serum had no demonstrable effect on electron spin resonance parameters of a protein-specific spin label attached to membrane proteins in control erythrocytes compared to the effect of control serum. This finding suggests that under the conditions employed no serum component or circulating factor is responsible for the changes in the physical state of membrane proteins in Huntington's disease erythrocytes (Butterfield, D.A., Oeswein, J.Q. and Markesbery, W.R. (1977) Nature 267, 453--455). No alteration in lipid fluidity of Huntington's disease erythrocyte membranes could be discerned suggesting that the underlying molecular defect in Huntington's disease involves a membrane protein. The results of the present studies on erythrocytes strongly support the concept that Huntington's disease is associated with a generalized membrane abnormality.     

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.     

Pathogenesis of neonatal hyperbilirubinaemia after induction of labour with oxytocin.

To determine the pathogenesis of neonatal hyperbilirubinaemia after oxytocin-induced labour venous cord blood from 95 healthy newborn infants was examined. Of these, 15 were delivered by elective caesarean section, 40 after spontaneous labour, and 40 after oxytocin-induced labour. There was no significant difference in any haematological or biochemical variable between the first two groups. Infants born after oxytocin-induced labour, however, showed clear evidence of increased haemolysis associated with significantly decreased erythrocyte deformability (P less than 0.001). In-vitro studies showed a time- and dose-related reduction in erythrocyte deformability in response to oxytocin. The findings suggest that the vasopressin-like action of oxytocin causes osmotic swelling of erythrocytes leading to decreased deformability and hence more rapid destruction with resultant hyperbilirubinaemia in the neonate.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.     

Beneficial influence of dietary curcumin, capsaicin and garlic on erythrocyte integrity in high-fat fed rats

In rats rendered hyperlipidemic by maintaining them on a high-fat diet (30%) for 8 weeks, inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet produced significant hypotriglyceridemic effect. Plasma cholesterol remained unaffected in high-fat treatment. Hepatic triglyceride content was significantly higher in high-fat fed rats, and this increase was effectively countered by inclusion of the hypolipidemic spice agents -- curcumin, capsaicin or garlic in the diet. The lipid profile of erythrocyte membranes of hyperlipidemic rats was similar to basal controls. An examination of the osmotic fragility of erythrocytes in various groups indicated that the red blood cells of hyperlipidemic rats display a slight resistance to osmotic lysis. Inclusion of spice principles [curcumin (0.2%) or capsaicin (0.015%)] or garlic (2.0%) in the diet, which produced the hypotriglyceridemic effect, appeared to beneficially correct this altered osmotic fragility of erythrocytes. Activities of ouabain-sensitive Na(+),K(+)-ATPase as well as acetylcholinesterase of erythrocyte membranes in high-fat fed rats remained unaltered. Activity of Ca(2+),Mg(2+)-ATPase in erythrocyte membrane was significantly decreased in high-fat fed animals, whereas dietary spice principles and garlic countered this reduction in enzyme activity. In the absence of any change in the cholesterol/phospholipid molar ratio in the erythrocyte membrane, a decreased activity of membrane-bound Ca(2+),Mg(2+)-ATPase could have probably contributed to the accumulation of intracellular calcium leading to the diminished deformability of the erythrocytes in high-fat fed rats.     

Activation of the plasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H(+)-ATPase-acetylated tubulin complex

In the yeast Saccharomyces cerevisiae, plasma membrane H(+)-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H(+)-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H(+)-ATPase-tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H(+)-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H(+)-pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H(+)-ATPase) immunoglobulins indicated a physical interaction between H(+)-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H(+)-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H(+)-ATPase-tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H(+)-ATPase activity by acetylated tubulin and concomitant increase of the H(+)-ATP ase-tubulin complex.     

Effect of pH on the velocity of erythrocyte aggregation

The effect of pH on the velocity of aggregation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer, in relation to the morphological changes of erythrocytes and their aggregates. (i) With increasing pH of the medium, the velocity of erythrocyte aggregation increased. (ii) The rouleaux formed at high pH were longer in shape and more stable against the increase of shear rate than those formed at low pH. (iii) With increasing pH, the diameter of erythrocyte increased, the (maximum) thickness decreased, and the cell volume decreased. The pH dependency of erythrocyte aggregation may be mainly due to the morphological change of erythrocytes, and partly due to the changes of erythrocyte deformability and of interaction with macromolecules.