RESUMO
Remodeling the erythrocyte membrane and skeleton by the malarial parasite Plasmodium falciparum is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not ß-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of P. falciparum, from the ring to the schizont stage. We further observed an upregulated expression of P. falciparum phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during P. falciparum development. We further revealed that inhibition of the activity of PfPI3K impaired P. falciparum development in vitro and Plasmodium berghei infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of Plasmodium species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria. IMPORTANCE: Plasmodium falciparum is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after P. falciparum invasion, while ß-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by P. falciparum phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in P. falciparum-infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.
Assuntos
Malária Falciparum , Plasmodium falciparum , Humanos , Animais , Camundongos , Plasmodium falciparum/metabolismo , Espectrina/metabolismo , Espectrina/farmacologia , Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Ubiquitina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, ßsp (1.4 MHz) and γ1sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the ßsp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the ßsp and γ1sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the ßsp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1sp relaxation.
Assuntos
Glicoforinas , Espectrina , Humanos , Espectrina/química , Espectrina/metabolismo , Espectrina/farmacologia , Glicoforinas/metabolismo , Glicoforinas/farmacologia , Ligação de Hidrogênio , Espectroscopia Dielétrica , Membrana Eritrocítica/metabolismo , Eritrócitos , Esqueleto/metabolismo , Lipídeos/farmacologia , Concentração de Íons de HidrogênioRESUMO
OBJECTIVE: To investigate the expression of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) in gastric cancer and its effect on gastric cancer cell invasion and metastasis. METHODS: The association of CAMSAP2 expression levels with progression and prognosis of gastric cancer was analyzed using public cancer data and in 106 patients receiving radical gastrectomy in our hospital from October, 2013 to October, 2017. The biological functions of CAMSAP2 were predicted using bioinformatics analysis. Gastric cancer MGC803 cells with CAMSAP2 overexpression and knockdown were observed for epithelial-mesenchymal transition (EMT), migration and invasion. A nude mouse model bearing orthotopic gastric cancer cell xenografts was established for verifying the results and exploring the underlying molecular mechanism. RESULTS: Gastric cancer tissues expressed high levels of CAMSAP2, which were positively correlated with CEA and CA19-9 (P<0.001). Cox regression analysis showed that CAMSAP2 expression level was an independent risk factor affecting the 5-year survival rate of gastric cancer patients (HR=2.969, 95% CI: 1.031-8.548). Enrichment analysis suggested that CAMSAP2 was involved in epithelialmesenchymal transition (EMT) and TGF-ß signaling. In gastric cancer cells, CAMSAP2 overexpression significantly increased the expressions of vimentin and N-cadherin, inhibited the expression of E-cadherin, and enhanced cell migration and invasion (P<0.05); CAMSAP2 knockdown produced the opposite effects in the cells (P<0.05). In the tumor- bearing mice, xenografts overexpressing CAMSAP2 showed enhanced metastasis (P<0.05), increased vimentin and N-cadherin expressions and lowered E-cadherin expression (P<0.05), and the xenografts with CAMSAP2 knockdown showed the opposite changes (P<0.05). Both the in vivo and in vitro experiments showed that CAMSAP2 overexpression increased and CAMSAP2 knockdown lowered the levels of TGF-ß and p-Smad2/3 in the gastric cancer cells (P<0.05). CONCLUSION: The high expression of CAMSAP2 contributes to disease progression and poor prognosis of gastric cancer possibly by upregulating TGF-ß signaling to promote EMT.
Assuntos
Neoplasias Gástricas , Humanos , Animais , Camundongos , Neoplasias Gástricas/genética , Vimentina/metabolismo , Espectrina/metabolismo , Espectrina/farmacologia , Linhagem Celular Tumoral , Invasividade Neoplásica , Fator de Crescimento Transformador beta/metabolismo , Caderinas/metabolismo , Transição Epitelial-Mesenquimal , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Protein isoforms are structural variants with changes in the overall flexibility predominantly at the tertiary level. For membrane associated proteins, such structural flexibility or rigidity affects membrane stability by playing modulatory roles in lipid-protein interaction. Herein, we investigate the protein chain flexibility mediated changes in the mechanistic behavior of phospholipid model membranes in the presence of two well-known isoforms, erythroid (ER) and nonerythroid (NER) spectrin. We show dramatic alterations of membrane elasticity and stability induced by spectrin in the Langmuir monolayers of phosphatidylocholine (PC) and phosphatidylethanolamine (PE) by a combination of isobaric relaxation, surface pressure-area isotherm, X-ray scattering, and microscopy measurements. The NER spectrin drives all monolayers to possess an approximately equal stability, and that required 25-fold increase and 5-fold decrease of stability in PC and PE monolayers, respectively. The untilting transition of the PC membrane in the presence of NER spectrin observed in X-ray measurements can explain better membrane packing and stability.
Assuntos
Fosfolipídeos , Espectrina , Espectrina/química , Espectrina/metabolismo , Espectrina/farmacologia , Fosfolipídeos/química , Proteínas de MembranaRESUMO
The aim of the current study was to assess the influence of embryonic exposure to cadmium on basic and derived erythrocyte indices, the morphology and morphometric properties of erythrocytes, as well as erythrocyte spectrin distribution in newly hatched Gallus gallus domesticus chicks. The eggs were injected with cadmium (Cd) at a dose of 2 µg, 4 µg, 6 µg, or 8 µg per egg on the sixth day of incubation. Blood samples were collected on the first day after hatching. Exposure to cadmium resulted in higher levels of red blood cell count, hemoglobin concentration, and hematocrit value, while derived erythrocyte indices were lower (mean corpuscular volume) or higher (mean corpuscular hemoglobin concentration) in comparison to the control. These changes occurred in animals exposed to higher doses of this toxic agent. In cadmium-treated individuals (2 and 8 µg of Cd), the percentage of erythrocytes which exhibited changed shape increased. Increases in the length (6 and 8 µg) and width (2, 6, and 8 µg) of erythrocytes and the length and width of the nucleus (2-8 µg) of red blood cells were observed. Changes in spectrin distribution were also observed, which indicate alterations at structural and molecular levels.
Assuntos
Galinhas , Índices de Eritrócitos , Animais , Cádmio/toxicidade , Índices de Eritrócitos/veterinária , Eritrócitos , Óvulo , Espectrina/farmacologiaRESUMO
AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.
Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo , Potenciais de Ação/fisiologia , Animais , Síndrome de Brugada/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Espectrina/metabolismo , Espectrina/farmacologiaRESUMO
Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs) has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS) was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes), dotU, or iglC (two genes encoding T6SS machinery) severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus), which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.
Assuntos
Eritrócitos/microbiologia , Eritrócitos/fisiologia , Francisella tularensis/patogenicidade , Tularemia/sangue , Tularemia/microbiologia , Actinas , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Endocitose , Eritrócitos/patologia , Feminino , Francisella tularensis/crescimento & desenvolvimento , Genes Bacterianos/genética , Interações Hospedeiro-Patógeno , Humanos , Concentração de Íons de Hidrogênio , Ixodes/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fagocitose , Espectrina/farmacologia , Doenças Transmitidas por Carrapatos/microbiologia , Carrapatos/microbiologia , Sistemas de Secreção Tipo VI/genéticaRESUMO
We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B(max)) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in deltapi change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The deltapi change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a "raft-like" mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.
Assuntos
Colesterol/farmacologia , Fluidez de Membrana , Fosfolipídeos/metabolismo , Espectrina/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Colesterol/metabolismo , Relação Dose-Resposta a Droga , Eritrócitos/citologia , Eritrócitos/metabolismo , Ácidos Graxos/química , Fluidez de Membrana/efeitos dos fármacos , Espectrina/farmacologiaRESUMO
Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, occurs in a variety of pathophysiological conditions, and is regulated by a balance of angiogenic activators and inhibitors. To identify novel angiogenic factors, we developed a gene screening method by combining the prediction analysis of transcription factor (TF) binding site and the chromosomal localization analysis. First, we analyzed the promoter sequences from known angiogenesis-related factors using the MATINSPECTOR program in TRANSFAC database. Interestingly, we found that the binding site of LMO2 complex is highly conserved in the promoter regions of these factors. Second, we analyzed chromosome loci based on the hypothesis that angiogenesis-related factors might be co-localized in a specific chromosomal band. We found that angiogenesis-related factors are localized in specific 14 chromosomal bands including 5q31 and 19q13 using AngioDB and LocusLink database mining. From these two approaches, we identified 32 novel candidates that have the LMO2 complex binding site in their promoter and are located on one of 14 chromosomal bands. Among them, human recombinant troponin T and spectrin markedly inhibited the neovascularization in vivo and in vitro. Collectively, we suggest that the combination of the prediction analysis of TF binding site and the chromosomal localization analysis might be a useful strategy for gene screening of angiogenesis.
Assuntos
Indutores da Angiogênese/antagonistas & inibidores , Sítios de Ligação , Células Cultivadas , Mapeamento Cromossômico/métodos , Biologia Computacional , Simulação por Computador , Bases de Dados de Ácidos Nucleicos , Bases de Dados de Proteínas , Humanos , Regiões Promotoras Genéticas , Espectrina/farmacologia , Fatores de Transcrição/fisiologia , Troponina T/farmacologiaRESUMO
Ca2+ store depletion activates both Ca2+ selective and non-selective currents in endothelial cells. Recently, considerable progress has been made in understanding the molecular make-up and regulation of an endothelial cell thapsigargin-activated Ca2+ selective current, I(SOC). Indeed, I(SOC) is a relatively small inward Ca2+ current that exhibits an approximate +40mV reversal potential and is strongly inwardly rectifying. This current is sensitive to organization of the actin-based cytoskeleton. Transient receptor potential (TRP) proteins 1 and 4 (TRPC1 and TRPC4, respectively) each contribute to the molecular basis of I(SOC), although it is TRPC4 that appears to be tethered to the cytoskeleton through a dynamic interaction with protein 4.1. Activation of I(SOC) requires association between protein 4.1 and the actin-based cytoskeleton (mediated through spectrin), suggesting protein 4.1 mediates the physical communication between Ca2+ store depletion and channel activation. Thus, at present findings indicate a TRPC4-protein 4.1 physical linkage regulates I(SOC) activation following Ca2+ store depletion.
Assuntos
Canais de Cálcio/fisiologia , Sinalização do Cálcio , Cálcio/metabolismo , Endotélio Vascular/citologia , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Eletrofisiologia , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Espectrina/metabolismo , Espectrina/farmacologiaRESUMO
Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC). In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site. In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.
Assuntos
Química Encefálica , Lipídeos de Membrana/química , Membranas Artificiais , Fosfolipídeos/química , Espectrina/farmacologia , Anquirinas/química , Eritrócitos/química , Humanos , Pressão , Espectrina/antagonistas & inibidores , Espectrina/isolamento & purificação , Tensão SuperficialRESUMO
Mechanism(s) underlying activation of store-operated Ca2+ entry currents, ISOC, remain incompletely understood. F-actin configuration is an important determinant of channel function, although the nature of interaction between the cytoskeleton and ISOC channels is unknown. We examined whether the spectrin membrane skeleton couples Ca2+ store depletion to Ca2+ entry. Thapsigargin activated an endothelial cell ISOC (-45 pA at -80 mV) that reversed at +40 mV, was inwardly rectifying when Ca2+ was the charge carrier, and was inhibited by La3+ (50 microM). Disruption of the spectrin-protein 4.1 interaction at residues A207-V445 of betaSpIISigma1 decreased the thapsigargin-induced global cytosolic Ca2+ response by 50% and selectively abolished the endothelial cell ISOC, without altering activation of a nonselective current through cyclic nucleotide-gated channels. In contrast, disruption of the spectrin-actin interaction at residues A47-K186 of betaSpIISigma1 did not decrease the thapsigargin-induced global cytosolic Ca2+ response or inhibit ISOC. Results indicate that the spectrin-protein 4.1 interaction selectively controls ISOC, indicating that physical coupling between calcium release and calcium entry is reliant upon the spectrin membrane skeleton.
Assuntos
Canais de Cálcio/efeitos dos fármacos , Proteínas do Citoesqueleto , Citoesqueleto , Endotélio Vascular/citologia , Neuropeptídeos , Espectrina/farmacologia , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células , Eletrofisiologia , Endotélio Vascular/ultraestrutura , Humanos , Cinética , Lantânio/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/farmacologia , Proteínas de Membrana/fisiologia , Técnicas de Patch-Clamp , Ratos , Espectrina/metabolismo , Espectrina/fisiologia , Tapsigargina/farmacologiaRESUMO
Brain fodrin inhibited in a dose dependent manner the GTPgammaS-stimulated cytosolic PLA2 (cPLA2), PLC, and PLD activities in differentiated HL-60 cells permeabilized with streptolysin O. cPLA2 and PLD were inhibited by the same concentrations of fodrin (IC50=1.5-2 nM) but PLC was inhibited by lower concentrations (IC50=0.3 nM). Moreover, the rates of inhibition were different between the phospholipases. Spectrin, which shares 50% homology with fodrin, had similar effects on the three phospholipases. However, using cytosol-depleted cells or recombinant PLD1, we showed that fodrin was not a direct inhibitor. Studying the potential mechanisms of these inhibitions, we demonstrated that a major decrease in membrane phosphatidylinositol 4-monophosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) amounts was induced by fodrin. Exogenous PtdIns(4,5)P2 partly reversed fodrin inhibition of GTPgammaS-stimulated phospholipase C activity. Hence, inhibition of PLC, cPLA2, and PLD activities observed with fodrin could be related to the decrease of PtdIns(4,5)P2, substrate of PLC, a cofactor of PLD and an enhancer of cPLA2 activity.
Assuntos
Proteínas de Transporte/farmacologia , Proteínas dos Microfilamentos/farmacologia , Fosfolipase D/antagonistas & inibidores , Fosfolipases A/antagonistas & inibidores , Fosfolipases Tipo C/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Guanosina Trifosfato/farmacologia , Células HL-60/enzimologia , Humanos , Proteínas do Tecido Nervoso/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipases A2 , Proteínas Recombinantes/metabolismo , Espectrina/farmacologiaRESUMO
The cluster patterns of multilamellar vesicles (MLV) of dimyristoylphosphatidylcholine (DMPC) were analyzed using a combination of fractal analysis and lattice simulation. Self-assembly of DMPC MLVs resulted in two types of microscopically observable clusters. The clusters were classified on the basis of their mass fractal dimension, two-dimensional porosity, and the light scattering properties. Spectrin, a cytoskeletal protein, well known for its role in determining the cellular morphology, was used to perturb such spontaneously formed clusters. The fragmentation of the clusters by hydrodynamic perturbation followed a power law, implying again a fractal behavior. A lattice-based simulation was performed generating different class of cluster patterns. The observed correspondence between the cluster patterns and their stability was discussed in the framework of the proposed lattice simulation.
Assuntos
Dimiristoilfosfatidilcolina/química , Fractais , Lipossomos/ultraestrutura , Espectrina/farmacologia , Processamento de Imagem Assistida por Computador , Tamanho da Partícula , Porosidade , Espalhamento de RadiaçãoRESUMO
The calcium-dependent protease calpain may contribute to neuronal death in acute neurological insults and may be activated very early in the neuronal injury cascade. We assessed the role of calpain in a model of rapid, reversible dendritic injury in murine cortical cultures. Brief sublethal NMDA exposure (10-30 microM for 10 min) resulted in focal swellings, or varicosities, along the length of neuronal dendrites as visualized with the lipophilic membrane tracer Dil or with immunostaining using antibodies to the somatodendritic protein MAP2. These varicosities appeared within minutes of NMDA exposure and recovered spontaneously within 2 hr after NMDA removal. Addition of the calpain inhibitors MDL28,170, calpain inhibitors I and II, and leupeptin (all 1-100 microM) had little effect on the development of NMDA-induced dendrite injury. However, the resolution of varicosities was substantially delayed by addition of calpain inhibitors after sublethal excitotoxic exposure. Using Western blots and immunocytochemistry, we observed reactivity for a calpain-specific spectrin proteolytic fragment during the period of recovery from dendritic swelling, but not during its formation. Spectrin breakdown product immunoreactivity could be blocked by the calpain inhibitor MDL28,170 and appeared in neuronal cell bodies and neurites in a time course that paralleled dendritic recovery. These observations suggest that calcium-dependent proteolysis contributes to recovery of dendritic structure after NMDA exposure. Calpain activation is not necessarily detrimental and may play a role in dendritic remodeling after neuronal injury.
Assuntos
Calpaína/fisiologia , Dendritos/fisiologia , Animais , Calpaína/antagonistas & inibidores , Células Cultivadas , Dendritos/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Camundongos , N-Metilaspartato/farmacologia , Espectrina/farmacologia , Fatores de TempoRESUMO
To facilitate functional studies of novel myosins, we have developed a strategy for characterizing the mechanochemical properties of motors isolated by immunoadsorption directly from small amounts of crude tissue extracts. In this initial study, silica beads coated with an antibody that specifically recognizes the tail of myosin-V were used to immunoadsorb this motor protein from brain extracts. The myosin-containing beads were then positioned with optical tweezers onto actin filaments nucleated from Limulus sperm acrosomal processes and observed for motility using high resolution video DIC microscopy. The addition of brush border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were approximately 4-fold longer than filaments grown in the absence of this actin crosslinking protein. The velocity of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or assessed using the sliding actin filament assay. Motile beads containing myosin-V immunoadsorbed from brain extracts bound poorly to nucleated actin filaments and were incapable of linear migrations following the addition of a different antibody that specifically recognizes the motor-containing head domain of myosin-V. Myosin-V motility was most robust in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-V mechanochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenously added actin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acrossomo/fisiologia , Encéfalo/fisiologia , Proteínas de Ligação a Calmodulina/fisiologia , Miosina Tipo V , Proteínas do Tecido Nervoso/fisiologia , Animais , Proteínas de Ligação a Calmodulina/química , Proteínas de Ligação a Calmodulina/isolamento & purificação , Galinhas , Caranguejos Ferradura , Técnicas de Imunoadsorção/instrumentação , Lasers , Masculino , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodos , Microvilosidades/fisiologia , Músculo Esquelético/fisiologia , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Espectrina/farmacologia , Tropomiosina/farmacologiaRESUMO
Proteins that react with anti-human spectrin antibodies raised in rabbit were found in pea seedlings and leaves. The immunoreactive proteins seem to be associated with the membranes and can be extracted with low ionic strength solutions.
Assuntos
Anticorpos , Fabaceae/química , Proteínas de Plantas/imunologia , Plantas Medicinais , Espectrina/imunologia , Reações Antígeno-Anticorpo , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Membrana Eritrocítica/química , Humanos , Peso Molecular , Proteínas de Plantas/isolamento & purificação , Protoplastos/química , Espectrina/farmacologiaRESUMO
Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain on its interaction with cytoskeletal elements. In contrast, these barrier properties seem to be rather insensitive to perturbations of integral proteins.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Proteínas de Membrana/metabolismo , Espectrina/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Eletrólitos , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Manitol/metabolismo , Proteínas de Membrana/efeitos dos fármacos , Floretina/farmacologia , Sacarose/metabolismo , Temperatura , Tripsina/metabolismoRESUMO
Human spectrin, when isolated, purified and stored in such conditions that preserve its tetrameric form, is able to associate with human hemoglobin as it is clearly shown by gel filtration. However, this hemoglobin-spectrin association does not seem to have a significant effect on hemoglobin oxygenation as indicated by equilibrium and rapid kinetics measurements.
Assuntos
Hemoglobinas , Espectrina , Adesividade , Ligação Competitiva/efeitos dos fármacos , Cromatografia em Gel , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Oxigênio/fisiologia , Espectrina/farmacologiaRESUMO
Actin in the human erythrocyte forms short protofilaments which are only long enough to accommodate tropomyosin monomers (Shen, B.W., Josephs, R. and Steck, T.L. (1986) J. Cell Biol. 102, 997-1006). This interaction between actin and tropomyosin monomers is predicted to be weak, since tropomyosin polymerization parallels its affinity for F-actin. We examine the binding of human erythrocyte tropomyosin to actin in the presence and absence of spectrin and its ability to polymerize. The binding of human erythrocyte tropomyosin to F-actin is not affected appreciably by the present of spectrin. Saturating F-actin with erythrocyte tropomyosin, however, weakens the binding of spectrin dimers to actin. Although tropomyosin from human erythrocyte and rabbit cardiac muscle have similar affinity for F-actin, the polymerizability of erythrocyte tropomyosin as determined by viscosity measurements is much reduced relative to muscle tropomyosin. This unusual property of erythrocyte tropomyosin is likely due to differences in its primary structure from other known tropomyosin at the amino and carboxyl terminal regions which are responsible for its head-to-tail polymerization and cooperative binding to F-actin. Analysis of the distribution of tyrosine by 2-dimensional tryptic mapping of 125I-labelled erythrocyte tropomyosin shows that tyrosine at positions 162, 214, 221, 261 and 267 in rabbit cardiac tropomyosin are conserved in human erythrocyte tropomyosin but Tyr-60 is absent. This observation suggests that erythrocyte tropomyosin has a carboxyl terminal region similar to its muscle counterparts but its amino terminal region resembles that of platelet tropomyosin which also lacks Tyr-60.