Tubulin Regulates Plasma Membrane Ca-ATPase Activity in a Lipid Environment-dependent Manner.

Ca plays a crucial role in cell signaling, cytosolic Ca can change up to 10,000-fold in concentration due to the action of Ca-ATPases, including PMCA, SERCA and SCR. The regulation and balance of these enzymes are essential to maintain cytosolic Ca homeostasis. Our laboratory has discovered a novel PMCA regulatory system, involving acetylated tubulin alone or in combination with membrane lipids.

Tubulin-mediated anatomical and functional changes caused by Ca in human erythrocytes.

In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation.

Prevention of tubulin/aldose reductase association delays the development of pathological complications in diabetic rats.

In recent studies, we found that compounds derived from phenolic acids (CAFs) prevent the formation of the tubulin/aldose reductase complex and, consequently, may decrease the occurrence or delay the development of secondary pathologies associated with aldose reductase activation in diabetes mellitus. To verify this hypothesis, we determined the effect of CAFs on Na,K-ATPase tubulin-dependent activity in COS cells, ex vivo cataract formation in rat lenses and finally, to evaluate the antidiabetic effect of CAFs, diabetes mellitus was induced in Wistar rats, they were treated with different CAFs and four parameters were determinates: cataract formation, erythrocyte deformability, nephropathy and blood pressure. After confirming that CAFs are able to prevent the association between aldose reductase and tubulin, we found that treatment of diabetic rats with these compounds decreased membrane-associated acetylated tubulin, increased NKA activity, and thus reversed the development of four AR-activated complications of diabetes mellitus determined in this work.

Inhibition of flippase-like activity by tubulin regulates phosphatidylserine exposure in erythrocytes from hypertensive and diabetic patients.

Plasma membrane tubulin is an endogenous regulator of P-ATPases and the unusual accumulation of tubulin in the erythrocyte membrane results in a partial inhibition of some their activities, causing hemorheological disorders like reduced cell deformability and osmotic resistance. These disorders are of particular interest in hypertension and diabetes, where the abnormal increase in membrane tubulin may be related to the disease development. Phosphatidylserine (PS) is more exposed on the membrane of diabetic erythrocytes than in healthy cells.

Erythrocyte plasma membrane potential: past and current methods for its measurement.

The plasma membrane functions both as a natural insulator and a diffusion barrier to the movement of ions. A wide variety of proteins transport and pump ions to generate concentration gradients that result in voltage differences, while ion channels allow ions to move across the membrane down those gradients. Plasma membrane potential is the difference in voltage between the inside and the outside of a biological cell, and it ranges from ~- 3 to ~- 90 mV.

Human erythrocytes: cytoskeleton and its origin.

In the last few years, erythrocytes have emerged as the main determinant of blood rheology. In mammals, these cells are devoid of nuclei and are, therefore, unable to divide. Consequently, all circulating erythrocytes come from erythropoiesis, a process in the bone marrow in which several modifications are induced in the expression of membrane and cytoskeletal proteins, and different vertical and horizontal interactions are established between them.

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.

Regulation of aldose reductase activity by tubulin and phenolic acid derivates.

In this work we demonstrate that aldose reductase (AR) interacts directly with tubulin and, was subjected to microtubule formation conditions, enzymatic AR activity increased more than sixfold. Since AR interacts mainly with tubulin that has 3-nitro-tyrosine in its carboxy-terminal, we evaluated whether tyrosine and other phenolic acid derivatives could prevent the interaction tubulin/AR and the enzymatic activation. The drugs evaluated have two characteristics in common: the presence of an aromatic ring and a carboxylic substituent.

Tubulin is retained throughout the human hematopoietic/erythroid cell differentiation process and plays a structural role in sedimentable fraction of mature erythrocytes.

We investigated the properties of tubulin present in the sedimentable fraction ("Sed-tub") of human erythrocytes, and tracked the location and organization of tubulin in various types of cells during the process of hematopoietic/erythroid differentiation. Sed-tub was sensitive to taxol/nocodazole (drugs that modify microtubule assembly/disassembly), but was organized as part of a protein network rather than in typical microtubule form. This network had a non-uniform "connected-ring" structure, with tubulin localized in the connection areas and associated with other proteins.

Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects.

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes.

PMCA activity and membrane tubulin affect deformability of erythrocytes from normal and hypertensive human subjects.

Our previous studies demonstrated formation of a complex between acetylated tubulin and brain plasma membrane Ca(2+)-ATPase (PMCA), and the effect of the lipid environment on structure of this complex and on PMCA activity. Deformability of erythrocytes from hypertensive human subjects was reduced by an increase in membrane tubulin content. In the present study, we examined the regulation of PMCA activity by tubulin in normotensive and hypertensive erythrocytes, and the effect of exogenously added diacylglycerol (DAG) and phosphatidic acid (PA) on erythrocyte deformability.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS-like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published.

Effects of detyrosinated tubulin on Na+,K+-ATPase activity and erythrocyte function in hypertensive subjects.

Formation of tubulin/Na(+),K(+)-ATPase (NKA) complex in erythrocytes of hypertensive subjects results in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects.

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified.

New case of mitochondrial HMG-CoA synthase deficiency. Functional analysis of eight mutations.

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found.

Activation of H(+)-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease.

Background: Glucose induces H(+)-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H(+)-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity.

Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3.

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.

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

Objective: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub).

Methods And Results: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub.

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na+, K+-ATPase activity.

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na(+), K(+)-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS).

Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria.

3-Hydroxy-3-methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3-hydroxy-3-methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA.

Submembraneous microtubule cytoskeleton: regulation of ATPases by interaction with acetylated tubulin.

The ATP-hydrolysing enzymes (Na(+),K(+))-, H(+)- and Ca(2+)-ATPase are integral membrane proteins that play important roles in the exchange of ions and nutrients between the exterior and interior of cells, and are involved in signal transduction pathways. Activity of these ATPases is regulated by several specific effectors. Here, we review the regulation of these P-type ATPases by a common effector, acetylated tubulin, which interacts with them and inhibits their enzyme activity.

Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex.

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex.

Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model.

The ability of lactic acid bacteria (LAB) and Saccharomyces cerevisiae to remove aflatoxin B1 (AFB1) from liquid medium was tested. The experimental results indicated that (i) AFB1 binding to microorganisms was a rapid process (no more than 1 min); (ii) this binding involved the formation of a reversible complex between the toxin and microorganism surface, without chemical modification of the toxin; (iii) the amount of AFB1 removed was both toxin- and bacteria concentration-dependent; and (iv) quantitatively similar results were obtained with viable and nonviable (heat-treated) bacteria. According to these details, a physical adsorption model is proposed for the binding of AFB1 to LAB and S.