RESUMO
Ca2+ plays a crucial role in cell signaling, cytosolic Ca2+ can change up to 10,000-fold in concentration due to the action of Ca2+-ATPases, including PMCA, SERCA and SCR. The regulation and balance of these enzymes are essential to maintain cytosolic Ca2+ homeostasis. Our laboratory has discovered a novel PMCA regulatory system, involving acetylated tubulin alone or in combination with membrane lipids. This regulation controls cytosolic Ca2+ levels and influences cellular properties such as erythrocyte rheology. This review summarizes the findings on the regulatory mechanism of PMCA activity by acetylated tubulin in combination with lipids. The combination of tubulin cytoskeleton and membrane lipids suggests a novel regulatory system for PMCA, which consequently affects cytosolic Ca2+ content, depending on cytoskeletal and plasma membrane dynamics. Understanding the interaction between acetylated tubulin, lipids and PMCA activity provides new insights into Ca2+ signaling and cell function. Further research may shed light on potential therapeutic targets for diseases related to Ca2+ dysregulation. This discovery contributes to a broader understanding of cellular processes and offers opportunities to develop innovative approaches to treat Ca2+-related disorders. By elucidating the complex regulatory mechanisms of Ca2+ homeostasis, we advance our understanding of cell biology and its implications for human health.
Assuntos
Cálcio , ATPases Transportadoras de Cálcio da Membrana Plasmática , Tubulina (Proteína) , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cálcio/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Membrana Celular/metabolismo , Acetilação , Lipídeos de Membrana/metabolismo , Sinalização do Cálcio , HomeostaseRESUMO
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. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton. (AU)
Assuntos
Humanos , Eritrócitos/metabolismo , Tubulinos/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Deformação Eritrocítica/fisiologiaRESUMO
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. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton.
Assuntos
Eritrócitos , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Eritrócitos/metabolismo , Deformação Eritrocítica/fisiologia , Citoesqueleto/metabolismo , Membrana Celular/metabolismo , Cálcio/metabolismoRESUMO
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. Based on these results, the existence of a new physiological mechanism is proposed, in which tubulin is a key regulator of aldose reductase activity. This mechanism can explain the incorrect functioning of aldose reductase and Na+,K+-ATPase, two key enzymes in the pathogenesis of diabetes mellitus. Moreover, we found that such alterations can be prevented by CAFs, which are able to dissociate tubulin/aldose reductase complex.
Assuntos
Diabetes Mellitus Experimental , Cristalino , Aldeído Redutase , Animais , Diabetes Mellitus Experimental/complicações , Ratos , Ratos Wistar , Tubulina (Proteína)RESUMO
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. In most cells, PS is transported from the exoplasmic to the cytoplasmic leaflet of the membrane by lipid flippases. Here, we report that PS is more exposed in erythrocytes from both hypertensive and diabetic patients than in healthy erythrocytes, which could be attributed to the inhibition of flippase activity by tubulin. This is supported by: (i) the translocation rate of a fluorescent PS analog in hypertensive and diabetic erythrocytes was slower than in healthy cells, (ii) the pharmacological variation of membrane tubulin in erythrocytes and K562 cells was linked to changes in PS translocation and (iii) the P-ATPase-dependent PS translocation in inside-out vesicles (IOVs) from human erythrocytes was inhibited by tubulin. These results suggest that tubulin regulates flippase activity and hence, the membrane phospholipid asymmetry.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Diabetes Mellitus/patologia , Eritrócitos/metabolismo , Hipertensão/patologia , Fosfatidilserinas/metabolismo , Tubulina (Proteína)/metabolismo , Adenosina Trifosfatases/metabolismo , Adulto , Estudos de Casos e Controles , Diabetes Mellitus/metabolismo , Feminino , Humanos , Hipertensão/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
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. Cytoskeleton components play an important role in this process, which explains why they and the interaction between them have been the focus of much recent research. Moreover, in mature erythrocytes, the cytoskeleton integrity is also essential, because the cytoskeleton confers remarkable deformability and stability on the erythrocytes, thus enabling them to undergo deformation in microcirculation. Defects in the cytoskeleton produce changes in erythrocyte deformability and stability, affecting cell viability and rheological properties. Such abnormalities are seen in different pathologies of special interest, such as different types of anemia, hypertension, and diabetes, among others. This review highlights the main findings in mammalian erythrocytes and their progenitors regarding the presence, conformation and function of the three main components of the cytoskeleton: actin, intermediate filaments, and tubulin.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Eritrócitos/citologia , Eritrócitos/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Humanos , ReologiaRESUMO
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. Most of the most significant discoveries in this field have been made in excitable cells, such as nerve and muscle cells. Nevertheless, special attention has been paid to some events controlled by changes in membrane potential in non-excitable cells. The origins of several blood disorders, for instance, are related to disturbances at the level of plasma membrane in erythrocytes, the structurally simplest red blood cells. The high simplicity of erythrocytes, in particular, made them perfect candidates for the electrophysiological studies that laid the foundations for understanding the generation, maintenance, and roles of membrane potential. This article summarizes the methodologies that have been used during the past decades to determine Δψ in red blood cells, from seminal microelectrodes, through the use of nuclear magnetic resonance or lipophilic radioactive ions to quantify intra and extracellular ions, to continuously renewed fluorescent potentiometric dyes. We have attempted to highlight the advantages and disadvantages of each methodology, as well as to provide a description of the technical aspects involved.
RESUMO
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.
Assuntos
Eritrócitos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Sódio/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Membrana Celular/metabolismo , Fenômenos Fisiológicos Celulares/fisiologia , HumanosRESUMO
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. The 9 drugs tested were able to prevent both the interaction tubulin/AR and the enzymatic activation. In addition, we found that the induction of microtubule formation by high concentrations of glucose and the consequent activation of AR in cultured cells can be inhibited by phenolic acid derivates that prevent the interaction tubulin/AR. These results suggest that tubulin regulates the activation of AR through a direct interaction which can be controlled with phenolic derivates of carboxylic acids.
Assuntos
Aldeído Redutase/metabolismo , Hidroxibenzoatos/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Encéfalo/enzimologia , Células COS , Células Cultivadas , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Hidroxibenzoatos/química , Oxirredução , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
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. When tubulin was eliminated from Sed-tub fraction, this connected-ring structure disappeared. Spectrin, a major protein component in Sed-tub fraction, formed a complex with tubulin. During hematopoietic differentiation, tubulin shifts from typical microtubule structure (in pro-erythroblasts) to a disorganized structure (in later stages), and is retained in reticulocytes following enucleation. Thus, tubulin is not completely lost when erythrocytes mature; it continues to play a structural role in the Sed-tub fraction.
Assuntos
Eritrócitos/citologia , Eritrócitos/metabolismo , Hematopoese , Tubulina (Proteína)/metabolismo , Adulto , Sedimentação Sanguínea/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Feminino , Hematopoese/efeitos dos fármacos , Humanos , Masculino , Nocodazol/farmacologia , Paclitaxel/farmacologia , Espectrina/metabolismo , Tubulina (Proteína)/químicaRESUMO
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. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.
Assuntos
Diabetes Mellitus Tipo 1/patologia , Diabetes Mellitus Tipo 2/patologia , Deformação Eritrocítica , Eritrócitos/patologia , Tubulina (Proteína)/metabolismo , Adulto , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/patologia , Eletroforese em Gel de Poliacrilamida , Eritrócitos/citologia , Feminino , Humanos , Masculino , Ratos , Ratos WistarRESUMO
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. Some of the key findings were that: (i) PMCA was associated with tubulin in normotensive and hypertensive erythrocytes, (ii) PMCA enzyme activity was directly correlated with erythrocyte deformability, and (iii) when tubulin was present in the erythrocyte membrane, treatment with DAG or PA led to increased deformability and associated PMCA activity. Taken together, our findings indicate that PMCA activity is involved in deformability of both normotensive and hypertensive erythrocytes. This rheological property of erythrocytes is affected by acetylated tubulin and its lipid environment because both regulate PMCA activity.
Assuntos
Deformação Eritrocítica/fisiologia , Eritrócitos/fisiologia , Hipertensão/sangue , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Tubulina (Proteína)/metabolismo , Idoso , Células Cultivadas , Diglicerídeos/farmacologia , Deformação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feminino , Humanos , Hipertensão/fisiopatologia , Immunoblotting , Masculino , Microscopia de Fluorescência , Pessoa de Meia-Idade , Ácidos Fosfatídicos/farmacologia , Ligação ProteicaRESUMO
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. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development.
Assuntos
Eritrócitos/enzimologia , Hipertensão/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tubulina (Proteína)/metabolismo , Adulto , Deformação Eritrocítica/genética , Eritrócitos/patologia , Feminino , Humanos , Hipertensão/genética , Hipertensão/patologia , Masculino , Pessoa de Meia-Idade , ATPase Trocadora de Sódio-Potássio/genética , Tirosina/metabolismoRESUMO
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. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B'. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.
Assuntos
Síndrome de Cornélia de Lange/genética , Proteínas/genética , Splicing de RNA , Adolescente , Adulto , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Síndrome de Cornélia de Lange/patologia , Feminino , Mutação da Fase de Leitura , Humanos , Lactente , Masculino , Fenótipo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/metabolismo , Adulto JovemRESUMO
Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.
Assuntos
Síndrome de Cornélia de Lange/diagnóstico , Síndrome de Cornélia de Lange/genética , Anormalidades Musculoesqueléticas/genética , Mutação , Fenótipo , Proteínas/genética , Alelos , Sequência de Aminoácidos , Proteínas de Ciclo Celular , Hibridização Genômica Comparativa , Exoma , Ordem dos Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Conformação Proteica , Proteínas/química , Alinhamento de SequênciaRESUMO
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. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).
Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Hipoglicemia/enzimologia , Hipoglicemia/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Doenças Mitocondriais/enzimologia , Doenças Mitocondriais/genética , Mutação , Sequência de Aminoácidos , Sequência de Bases , Análise Mutacional de DNA , Ativação Enzimática , Éxons , Ordem dos Genes , Humanos , Hidroximetilglutaril-CoA Sintase/química , Lactente , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação ProteicaRESUMO
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. METHODS: We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H(+)-ATPase activation after glucose treatment. RESULTS: We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35-40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H(+)-ATPase complex did not undergo dissociation, and H(+)-ATPase activation was significantly delayed. CONCLUSION: Our findings indicate that the mechanism of H(+)-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H(+)-ATPase complex dissociation and the activation of the enzyme. GENERAL SIGNIFICANCE: Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H(+)-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H(+)-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.
Assuntos
Adenosina Trifosfatases/metabolismo , Glucose/farmacologia , Proteínas de Membrana/metabolismo , Fosfolipases A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Serina Proteases/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação/efeitos dos fármacos , Adenosina Trifosfatases/genética , Membrana Celular/enzimologia , Membrana Celular/genética , Ativação Enzimática/efeitos dos fármacos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosfolipases A/genética , Proteínas de Saccharomyces cerevisiae/genética , Serina Proteases/genética , Tubulina (Proteína)/genéticaRESUMO
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. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.
Assuntos
Citosol/enzimologia , Retículo Endoplasmático/enzimologia , Oxo-Ácido-Liases/análise , Oxo-Ácido-Liases/química , Sequência de Aminoácidos , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Oxo-Ácido-Liases/genética , Peroxissomos/enzimologia , Peroxissomos/metabolismo , Processamento de ProteínaRESUMO
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.
Assuntos
Aldeído Redutase/metabolismo , Glucose/farmacologia , Microtúbulos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Adulto , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Diabetes Mellitus/enzimologia , Diabetes Mellitus/metabolismo , Relação Dose-Resposta a Droga , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Feminino , Humanos , Immunoblotting , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos Wistar , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Sorbitol/farmacologia , Tubulina (Proteína)/farmacologiaRESUMO
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. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.