RESUMO
BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) is the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19. Viral entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). Transcriptomic studies showed that children display lower ACE2 than adults, though gene expression levels do not always correlate with protein levels. We investigated the effect of age on ACE2 and TMPRSS2 protein expression in alveolar type II (AT2) cells in the lungs of children compared to adults. We also analysed the ratio of Ang-(1-7) to Ang II as a surrogate marker of ACE2 activity in the subjects' lung parenchyma. METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. RESULTS: The amount of ACE2-expressing AT2 cells and ACE2 protein content were lower in children than in adults. Ang II levels were higher in children compared to adults and inversely correlated with the amount of ACE2-expressing AT2 cells. Children presented lower Ang-(1-7)/Ang II ratio than adult suggesting lower ACE2 activity in children. TMPRSS2 protein expression was not influenced by age. CONCLUSIONS: These results expand on previous transcriptomic studies and may partially explain the low susceptibility of children to SARS-CoV-2 infection. CATEGORY OF STUDY: Clinical original research IMPACT: Children display lower ACE2 protein content and activity compared to adults. Ang II levels were higher in children compared to adults and inversely correlated with the amount of ACE2-expressing AT2 cells TMPRSS2 protein expression was not influenced by age. These results expand on previous transcriptomic studies and may partially explain the low susceptibility of children to SARS-CoV-2 infection.
Assuntos
COVID-19 , Adulto , Criança , Humanos , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Pulmão , Processamento de Proteína Pós-TraducionalRESUMO
BACKGROUND: Besides its counterbalancing role of the renin-angiotensin system (RAS), angiotensin-converting enzyme (ACE) 2 is the receptor for the type 2 coronavirus that causes severe acute respiratory syndrome, the etiological agent of COVID-19. COVID-19 is associated with increased plasmatic ACE2 levels, although conflicting results have been reported regarding angiotensin (Ang) II and Ang-(1-7) levels. We investigated plasmatic ACE2 protein levels and enzymatic activity and Ang II and Ang-(1-7) levels in normotensive and hypertensive patients hospitalized with COVID-19 compared to healthy subjects. METHODS: Ang II and Ang-(1-7), and ACE2 activity and protein levels were measured in 93 adults (58 % (n = 54) normotensive and 42 % (n = 39) hypertensive) hospitalized with COVID-19. Healthy, normotensive (n = 33) and hypertensive (n = 7) outpatient adults comprised the control group. RESULTS: COVID-19 patients displayed higher ACE2 enzymatic activity and protein levels than healthy subjects. Within the COVID-19 group, ACE2 activity and protein levels were not different between normotensive and hypertensive-treated patients, not even between COVID-19 hypertensive patients under RAS blockade treatment and those treated with other antihypertensive medications. Ang II and Ang-(1-7) levels significantly decreased in COVID-19 patients. When COVID-19 patients under RAS blockade treatment were excluded from the analysis, ACE2 activity and protein levels remained higher and Ang II and Ang-(1-7) levels lower in COVID-19 patients compared to healthy people. CONCLUSIONS: Our results support the involvement of RAS in COVID-19, even when patients under RAS blockade treatment were excluded. The increased circulating ACE2 suggest higher ACE2 expression and shedding.
Assuntos
COVID-19 , Hipertensão , Adulto , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2 , Humanos , Peptidil Dipeptidase A/metabolismo , Sistema Renina-AngiotensinaRESUMO
AIMS: Angiotensin-converting enzyme (ACE) 2 is the receptor for severe acute respiratory syndrome coronavirus 2 which causes coronavirus disease 2019 (COVID-19). Viral cellular entry requires ACE2 and transmembrane protease serine 2 (TMPRSS2). ACE inhibitors (ACEIs) or angiotensin (Ang) receptor blockers (ARBs) influence ACE2 in animals, though evidence in human lungs is lacking. We investigated ACE2 and TMPRSS2 in type II pneumocytes, the key cells that maintain lung homeostasis, in lung parenchymal of ACEI/ARB-treated subjects compared to untreated control subjects. MAIN METHODS: Ang II and Ang-(1-7) levels and ACE2 and TMPRSS2 protein expression were measured by radioimmunoassay and immunohistochemistry, respectively. KEY FINDINGS: We found that the ratio Ang-(1-7)/Ang II, a surrogate marker of ACE2 activity, as well as the amount of ACE2-expressing type II pneumocytes were not different between ACEI/ARB-treated and untreated subjects. ACE2 protein content correlated positively with smoking habit and age. The percentage of TMPRSS2-expressing type II pneumocytes was higher in males than females and in subjects under 60 years of age but it was not different between ACEI/ARB-treated and untreated subjects. However, there was a positive association of TMPRSS2 protein content with age and smoking in ACEI/ARB-treated subjects, with high TMPRSS2 protein levels most evident in ACEI/ARB-treated older adults and smokers. SIGNIFICANCE: ACEI/ARB treatment influences human lung TMPRSS2 but not ACE2 protein content and this effect is dependent on age and smoking habit. This finding may help explain the increased susceptibility to COVID-19 seen in smokers and older patients with treated cardiovascular-related pathologies.
Assuntos
Células Epiteliais Alveolares/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Enzima de Conversão de Angiotensina 2/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Sistema Renina-Angiotensina/fisiologia , Serina Endopeptidases/metabolismo , Adulto , Fatores Etários , Idoso , Células Epiteliais Alveolares/química , Células Epiteliais Alveolares/efeitos dos fármacos , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Enzima de Conversão de Angiotensina 2/análise , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Feminino , Humanos , Pulmão/química , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Estudos Retrospectivos , Serina Endopeptidases/análise , Fumar/metabolismo , Fumar/patologiaRESUMO
The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms "x" and "z". While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants "x" and "z", maintaining constant the most frequent splice variant "b" at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant "z" of the splicing site A.
RESUMO
The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the "arm-like" domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the "arm-like" domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the "arm-like" domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Hidrólise , FosforilaçãoRESUMO
The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Membrana Celular/enzimologia , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Biocatálise , Cloreto de Cálcio/metabolismo , Ensaios Enzimáticos , Hidrólise , Mutação , Naftóis , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , TriazinasRESUMO
Sphingolipids regulate several aspects of cell behavior and it has been demonstrated that cells adjust their sphingolipid metabolism in response to metabolic needs. Particularly, sphingosine-1-phosphate (S1P), a final product of sphingolipid metabolism, is a potent bioactive lipid involved in the regulation of various cellular processes, including cell proliferation, cell migration, actin cytoskeletal reorganization and cell adhesion. In previous work in rat renal papillae, we showed that sphingosine kinase (SK) expression and S1P levels are developmentally regulated and control de novo sphingolipid synthesis. The aim of the present study was to evaluate the participation of SK/S1P pathway in the triggering of cell differentiation by external hypertonicity. We found that hypertonicity evoked a sharp decrease in SK expression, thus activating the de novo sphingolipid synthesis pathway. Furthermore, the inhibition of SK activity evoked a relaxation of cell-cell adherens junction (AJ) with accumulation of the AJ complex (E-cadherin/ß-catenin/α-catenin) in the Golgi complex, preventing the acquisition of the differentiated cell phenotype. This phenotype alteration was a consequence of a sphingolipid misbalance with an increase in ceramide levels. Moreover, we found that SNAI1 and SNAI2 were located in the cell nucleus with impairment of cell differentiation induced by SK inhibition, a fact that is considered a biochemical marker of epithelial to mesenchymal transition. So, we suggest that the expression and activity of SK1, but not SK2, act as a control system, allowing epithelial cells to synchronize the various branches of sphingolipid metabolism for an adequate cell differentiation program.
Assuntos
Lisofosfolipídeos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Esfingolipídeos/biossíntese , Esfingosina/análogos & derivados , Junções Aderentes/metabolismo , Animais , Diferenciação Celular , Movimento Celular , Proliferação de Células , Cães , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Soluções Hipertônicas , Células Madin Darby de Rim Canino , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Esfingosina/metabolismoRESUMO
We studied the kinetics of extracellular ATP (ATPe) in Escherichia coli and their outer membrane vesicles (OMVs) stimulated with amphipatic peptides melittin (MEL) and mastoparan 7 (MST7). Real-time luminometry was used to measure ATPe kinetics, ATP release, and ATPase activity. The latter was also determined by following [32P]Pi released from [γ-32P]ATP. E. coli was studied alone, co-incubated with Caco-2 cells, or in rat jejunum segments. In E. coli, the addition of [γ-32P]ATP led to the uptake and subsequent hydrolysis of ATPe. Exposure to peptides caused an acute 3-fold (MST7) and 7-fold (MEL) increase in [ATPe]. In OMVs, ATPase activity increased linearly with [ATPe] (0.1-1⠵M). Exposure to MST7 and MEL enhanced ATP release by 3-7 fold, with similar kinetics to that of bacteria. In Caco-2 cells, the addition of ATP to the apical domain led to a steep [ATPe] increase to a maximum, with subsequent ATPase activity. The addition of bacterial suspensions led to a 6-7 fold increase in [ATPe], followed by an acute decrease. In perfused jejunum segments, exposure to E. coli increased luminal ATP 2 fold. ATPe regulation of E. coli depends on the balance between ATPase activity and ATP release. This balance can be altered by OMVs, which display their own capacity to regulate ATPe. E. coli can activate ATP release from Caco-2 cells and intestinal segments, a response which in vivo might lead to intestinal release of ATP from the gut lumen.
Assuntos
Trifosfato de Adenosina/metabolismo , Enterócitos/metabolismo , Escherichia coli/fisiologia , Vesículas Extracelulares/metabolismo , Jejuno/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Células CACO-2 , Técnicas de Cocultura , Enterócitos/ultraestrutura , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Vesículas Extracelulares/ultraestrutura , Interações Hospedeiro-Patógeno , Humanos , Hidrólise , Peptídeos e Proteínas de Sinalização Intercelular , Jejuno/ultraestrutura , Cinética , Luminescência , Meliteno/metabolismo , Microscopia Eletrônica , Peptídeos , Monoéster Fosfórico Hidrolases/metabolismo , Ratos WistarRESUMO
P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was â¼1 µmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 µmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/genética , Fosforilação/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.
Assuntos
Cloretos/toxicidade , Compostos Férricos/toxicidade , Lisossomos/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , Lisossomos/patologia , Mutação , Tamanho das Organelas/efeitos dos fármacos , Permeabilidade , ATPases Translocadoras de Prótons/genética , TransfecçãoRESUMO
Aberrations in the ubiquitin-proteasome system (UPS) are implicated in the pathogenesis of various diseases. Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamines biosynthesis, is involved in hypertension development. In this study we investigated whether UPS regulated TH turnover in PC12 cells and hypothalamic and brainstem neurons from spontaneously hypertensive rats (SHR) and whether this system was impaired in hypertension. PC12 cells were exposed to proteasome or lysosome inhibitors and TH protein level evaluated by Western blot. Lactacystin, a proteasome inhibitor, induced an increase of 86 ± 15% in TH levels after 30 min of incubation, then it started to decrease up to 6 h to reach control levels and finally it rose up to 35.2 ± 8.5% after 24 h. Bafilomycin, a lysosome inhibitor, did not alter TH protein levels during short times, but it increased TH by 92 ± 22% above basal after 6 h treatment. Before degradation proteasome substrates are labeled by conjugation with ubiquitin. Efficacy of proteasome inhibition on TH turnover was evidenced by accumulation of ubiquitinylated TH after 30 min. Further, the inhibition of proteasome increased the quantity of TH phosphorylated at Ser40, which is essential for TH activity, by 2.7 ± 0.3 fold above basal. TH protein level was upregulated in neurons from hypothalami and brainstem of SHR when the proteasome was inhibited during 30 min, supporting that neuronal TH is also short-term regulated by the proteasome. Since the increased TH levels reported in hypertension may result from proteasome dysfunction, we evaluate proteasome activity. Proteasome activity was significantly reduced by 67 ± 4% in hypothalamic and brainstem neurons from SHR while its protein levels did not change. Present findings show that TH is regulated by the UPS. The impairment in proteasome activity observed in SHR neurons may be one of the causes of the increased TH protein levels reported in hypertension.
Assuntos
Tronco Encefálico/metabolismo , Hipertensão/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Ubiquitina/metabolismo , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Animais , Tronco Encefálico/citologia , Hipotálamo/citologia , Masculino , Células PC12 , Inibidores de Proteassoma/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
Lecithins, mainly composed of the phospholipids phosphatidylcholines (PC), have many different uses in the pharmaceutical and clinical field. PC are involved in structural and biological functions as membrane trafficking processes and cellular signaling. Considering the increasing applications of lecithin-based nanosystems for the delivery of therapeutic agents, the aim of the present work was to determine the effects of phosphatidylcholine nanoparticles over breast cancer cellular proliferation and signaling. PC dispersions at 0.01 and 0.1% (w/v) prepared in buffer pH 7.0 and 5.0 were studied in the MCF-7 breast cancer cell line. Neutral 0.1% PC-derived nanoparticles induced the activation of the MEK-ERK1/2 pathway, increased cell viability and induced a 1.2 fold raise in proliferation. These biological effects correlated with the increase of epidermal growth factor receptor (EGFR) content and its altered cellular localization. Results suggest that nanoparticles derived from PC dispersion prepared in buffer pH 7.0 may induce physicochemical changes in the plasma membrane of cancer cells which may affect EGFR cellular localization and/or activity, increasing activation of the MEK-ERK1/2 pathway and inducing proliferation. Results from the present study suggest that possible biological effects of delivery systems based on lecithin nanoparticles should be taken into account in pharmaceutical formulation design.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mitógenos , Nanopartículas/química , Fosfatidilcolinas , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Humanos , Mitógenos/química , Mitógenos/farmacologia , Proteínas de Neoplasias/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacologiaRESUMO
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes, ATP13A (ATPase type 13A) 1-ATP13A5, that belong to the P5-ATPase group have been identified in humans. Mutations of the human gene ATP13A2 underlie a form of PD (Parkinson's disease). Previous studies have suggested a relation between polyamines and P5B-ATPases. We have recently shown that the cytotoxicity induced by the polyamine analogue paraquat (1,1'-dimethyl-4,4'-bipyridinium), which is an environmental agent related to PD development, was increased in ATP13A2-expressing CHO (Chinese-hamster ovary) cells. In the present study we showed that ATP13A2-expressing CHO cells exhibit a 2-fold higher accumulation of spermidine. Increasing concentrations of spermidine reduced the viability of CHO cells stably expressing ATP13A2. The higher levels of spermidine attained by the ATP13A2-expressing CHO cells were correlated with an increase in the ATP-dependent spermidine uptake in an isolated subcellular fraction containing lysosomes and late endosomes. The results of the present study support the idea that the human P5B-ATPase ATP13A2 is involved in polyamine uptake.
Assuntos
Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espermidina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/metabolismo , Humanos , Cinética , Lisossomos/metabolismo , Doença de Parkinson/genética , ATPases Translocadoras de Prótons/genética , TemperaturaRESUMO
The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of ~0.3-1 µmol of P(i)/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg(2+). No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg(2+) dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg(2+) acts as a cofactor. The addition of Mn(2+) to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/imunologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/química , Magnésio/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.
Assuntos
Ativação Enzimática , Fosfolipídeos/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Animais , Calmodulina/química , Cromatografia de Afinidade , Eritrócitos/enzimologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/isolamento & purificação , Ligação Proteica , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Coloração e Rotulagem , TitulometriaRESUMO
P-ATPases are membrane transporters energized by ATP. The subfamily of P5-ATPases is the least studied P-ATPases and the ion substrate specificity of the P5 subfamily is not known. Mutations of the human P5ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.
Assuntos
Herbicidas/toxicidade , Paraquat/toxicidade , ATPases Translocadoras de Prótons/fisiologia , Laranja de Acridina , Animais , Células CHO , Cloreto de Cádmio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cloretos/farmacologia , Cricetinae , Cricetulus , Espaço Extracelular/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Compostos de Manganês/farmacologia , Microscopia de Fluorescência , Oxidantes/metabolismo , Oxidantes/farmacologia , ATPases Translocadoras de Prótons/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Espermidina/farmacologiaRESUMO
Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(1-7). For this purpose, we generated a chimera between the MasR and the yellow fluorescent protein (YFP; MasR-YFP). MasR-YFP-transfected HEK 293T cells were incubated with Ang-(1-7), and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 minutes. Following the time course of [(125)I]Ang-(1-7) endocytosis, we observed that half of MasR-YFP underwent endocytosis after 10 minutes, and this was blocked by a MasR antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1, and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin 1, suggesting that at some point MasR-YFP traverses caveolin 1-positive compartments. In conclusion, MasR undergoes endocytosis on stimulation with Ang-(1-7), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell.
Assuntos
Angiotensina I/farmacologia , Endocitose/fisiologia , Endossomos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina I/metabolismo , Células Cultivadas , Clatrina/metabolismo , Células HEK293 , Humanos , Fragmentos de Peptídeos/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidoresRESUMO
The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E(2)-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Membrana Celular/metabolismo , Ácido Egtázico/metabolismo , Eritrócitos/enzimologia , Hidrólise , SuínosRESUMO
The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.