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Regolazione della tirosin-fosforilazione della proteina Banda 3 Eritrocitaria
Get PDFIn human erythrocytes, Syk kinase is a key enzyme that triggers membrane protein band 3 Tyr-phosphorylation (Tyr-P). This enzyme, also called p72Syk, undergoes significant proteolysis in the absence of protease inhibitors, giving rise to p36Syk formation, which induces much greater band 3 Tyr-P then p72Syk. Besides, proteolysed Syk is capable to prepare membranes for further phosphorylation by p72Syk: when membranes are pre-phosphorylated by p36Syk, subsequent p72Syk-catalysed phosphorylation is much higher than that obtained with non-pretreated membranes. Since proteolytic enzymes are present in isolated membranes and p72Syk binds closely to the cytoskeleton, probably to the spanning domain of the anchored band 3, this binding could allow the activation of the enzyme by proteolysis.
In human erythrocytes, the P-Tyr level of proteins, mainly transmembrane band 3, is closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation of band 3 can be triggered, due to the inhibition of Tyr-phosphatase action and the reorganisation of erythrocyte membrane. SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell lines. We demonstrate that, in human erythrocytes, SHP-1 is present in membranes from resting cells, but in 5% of the protein amount. This amount increases up to three fold following NEM treatment of intact cells, whereas diamide and pervanadate do not alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane is not affected by band 3 P-Tyr level and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1, which dephosphorylates Tyr residues 8, 21 and 904.
From the cytosol of the red blood cells, through a DEAE-Sepharose ion-exchange chromatography followed by a Sephadex G-75 column, we purified an enzyme with phosphatasic activity on pNPP. Its characterization revealed that the enzyme is an acid phosphatase with a low molecular weight, and western blotting followed by immunostaining with the appropriate antibody confirmed that the enzyme was the Low Mr PTPase. The purified enzyme was able to dephosphorylate the four Tyr residues of band 3: 8, 21, 359 and 904, thus inducing for the first time the total dephosphorylaion of this protein.
Dapsone is a drug used in the treatment of leprosy, malaria or AIDS-related Pneumocystis pneumonia. N-hydroxylation of dapsone (DDS) leads to the formation of the toxic hydroxylamines responsible for the clinical methaemoglobinaemia associated with DDS therapy. In addition, the drug has been associated with shortening of the erythrocyte lifespan, with resulting potential clinical consequences such as anaemia and morbidity in areas where DDS is used to treat malaria.
We studied how DDS and/or its hydroxylamine (DDS-NHOH) induce erythrocyte membrane alterations leading to premature cell removal. Results indicate that the hydroxylamine, but not dapsone, is able to trigger Tyr-phosphorylation of membrane proteins, mainly of band 3. DDS-NHOH-induced band 3 Tyr-P peaked in 30’ (at 0.3 mM) and was already completely reversed after 45’ of incubation. However, when analysed for the enzymes involved in this process, Syk and SHP-2, membranes revealed dose- and time-dependent recruitment of both enzymes. Band 3 Tyr-phosphorylation is not due to an imbalance between enzymatic activities, since both Tyr-kinase and phosphatase activities were promptly inhibited by DDS-NHOH in both dose- and time-dependent manners, but more probably by a favoured substrate-kinase interaction.
The band 3 Tyr-phosphorylation process is very useful in detecting early DDS-NHOH-induced alterations. The membrane modifications continue, with the increase in DDS-NHOH incubation time, leading to aggregation of band 3. Band 3 high molecular weight aggregates (HMWA) location in the first 30’ of incubation was in the Triton-soluble fraction of the RBCs’membranes, while prolonging the incubation time, not only the content of band 3 HMWA further increased, but complete membrane reorganisation also occurred, the cytoskeleton containing almost all the band 3 HMWA complexes formed.
When we analysed membranes from erythrocytes subjected to dose- and time-dependent DDS-NHOH treatments in the presence of autologous plasma, immunostaining with anti-human IgG revealed net enhancement of the autologous antibodies content. Since removal of human erythrocytes is mediated by antibody recognition, this can explain the shortening of the erythrocyte lifespan seen when dapsone is used clinically.Negli eritrociti umani la tirosin chinasi Syk è un enzima chiave nella tirosin fosforilazione della proteina di membrana banda 3. Questo enzima, chiamato anche p72Syk, va incontro ad una significante proteolisi in assenza di inibitori proteasici, portando alla formazione di p36Syk, capace di indurre con maggiore efficienza la tirosin fosforilazione della banda 3 rispetto al p72Syk. Inoltre, la fosforilazione innescata dal p36Syk prepara le membrane per la successiva fosforilazione da parte dell’oloenzima p72Syk. Infatti quando le membrane sono pre-fosforilate con p36Syk, la successiva fosforilazione catalizzata dal p72Syk è maggiore rispetto a quella ottenuta in assenza della pre-fosforilazione. Poiché gli enzimi proteolitici sono presenti nelle membrane isolate e il p72Syk si lega al citoscheletro della cellula, probabilmente al dominio transmembrana della banda 3 ancorata a questa frazione della membrana, questo legame potrebbe permettere l’attivazione dell’enzima p72Syk attraverso la sua proteolisi.
Negli eritrociti umani il livello di tirosin fosforilazione delle proteine, soprattutto della proteina transmembrana banda 3, è strettamente controllato dall’attività antitetica delle protein tirosin chinasi e fosfatasi, risultando in condizioni basali in uno stato di quasi completa defosforilazione. Solo stimoli particolari, come agenti ossidanti, diamide o pervanadato, o composti alchilanti, N-etilen maleimide (NEM), possono innescare la tirosin fosforilazione della banda 3, dovuta l’inibizione delle tirosin fosfatasi. SHP-1 è una protein tirosin fosfatasi contenente due domini SH2 espressa nelle cellule ematopoietiche. Noi dimostriamo che, negli eritrociti umani, SHP-1 è presente nelle membrane delle cellule in condizioni basali, ma solo il 5% della quantità totale dell’enzima. Questa percentuale incrementa fino a tre volte dopo il trattamento della cellula con NEM, mentre diamide e pervanadato, pur inducendo la tirosin fosforilazione della banda 3, non alterano la localizzazione basale dell’enzima. Ciò indica che la traslocazione della SHP-1 dal citoplasma alle membrane non dipende dalla tirosin fosforilazione della banda 3. Abbiamo dimostrato che la banda 3 è un substrato per l’enzima e che esso defosforila i residui tirosinici 8, 21 e 904 della proteina.
Dal citoplasma del globulo rosso abbiamo purificato un’enzima con attività fosfatasica su para-nitro fenilfosfato (pNPP) attraverso una cromatografia a scambio ionico su DEAE-Sepharose, seguita da una gel filtrazione su una colonna G-75 Sephadex. La caratterizzazione dell’enzima purificato ha portato alla sua identificazione con la fosfatasi acida a basso peso molecolare. Tale identificazione è stata confermata dal western blotting, seguito dalla immunorivelazione con l’anticorpo appropriato. La fosfatasi acida a basso peso molecolare purificata è in grado di defosforilare i quattro siti fosfo tirosinici della banda 3: 8, 21, 359 e 904. In questo modo si è ottenuta per la prima volta la totale defosforilazione della proteina.
Dapsone è un farmaco utilizzato nel trattamento della lebbra, malaria o pneumonia dovuta a Pneumocystis associata all’ AIDS. La sua N-idrossilazione porta alla formazione di idrossilamine tossiche responsabili della metaemoglobinemia associata al trattamento con dapsone. Inoltre, il farmaco è stato correlato alla riduzione della vita media dell’eritrocita, ottenendo come risultato conseguenze cliniche come anemia e morbidità nelle aree dove il dapsone è utilizzato per trattare la malaria.
Noi abbiamo studiato il processo attraverso il quale il dapsone ed il suo metabolita idrossilamindapsome (DDS-NHOH) inducono alterazioni a livello della membrana eritrocitaria che portano alla rimozione prematura dell’eritrocita. I risultati indicano che DDS-NHOH, ma non il dapsone, è capace di innescare la tirosin fosforilazione delle proteine di membrana, soprattutto della banda 3. L’indotta tirosin fosforilazione raggiunge un massimo in 30’ di trattamento (con 0.3 mM) poi essa comincia a calare e dopo 45’ dall’inizio del processo sparisce completamente. Il reclutamento alla membrana degli enzimi coinvolti in questo processo, Syk e SHP-2, avviene in maniera dose e tempo dipendente, per entrambi gli enzimi. Inoltre la tirosin fosforilazione della banda 3 non è dovuta ad uno sbilanciamento tra le attività enzimatiche, dal momento che sia l’attività tirosin chinasica che quella tirosin fosfatasica sono inibite dal DDS-NHOH in maniera dose e tempo dipendente, ma più probabilmente è dovuta ad una favorevole interazione substrato-chinasi.
Le modifiche che avvengono nella membrana indotte da DDS-NHOH continuano anche dopo che la la fosforilazione è annulata (dopo 45’) e portano all’aggregazione della banda 3. La localizzazione degli aggregati ad alto peso molecolare di banda 3 (HMWA) nei primi 30’ di incubazione è prevalentemente nella frazione solubile in Triton X-100. Con il prolungamento del tempo d’incubazione si ha non solo l’ulteriore incremento della quantità di aggregati, ma anche un loro definitivo spostamento nel citoscheletro della membrana eritrocitaria. Questo è un indizio di una completa riorganizzazione della membrana.
L’analisi delle membrane ottenute da eritrociti incubati con concentrazioni crescenti di DDS-NHOH a vari tempi d’incubazione in presenza del plasma autologo ha rivelato un netto aumento nel contenuto di anticorpi autologhi. Poiché la rimozione dell’eritrocita è mediata dal riconoscimento da parte dei macrofagi degli anticorpi autologhi, la presenza di quest’ultimi sulla membrana del globulo rosso trattato con DDS-NHOH spiega la riduzione della vita media degli eritrociti nei pazienti trattati con dapsone
Effect of phospholipase-A2 on Syk-mediated phosphorylation of human erythrocyte membrane
No full textSHP-1 Tyrosine phosphatase in human erythrocytes
No full textAbstract: SHP-1 is a SH2-domain containing protein Tyr-phosphatase expressed in hematopoietic cell lines, which is hypothesized to play a negative role in signal transduction. In human erythrocytes, the phospho-Tyr level of proteins, mainly transmembrane band 3, is closely controlled by the antithetic activity of Tyr-protein kinases and phosphatases, resulting in a dephosphorylated state. Only after particular stimuli, as with oxidizing agents, diamide or pervanadate, or thiol alkylating compound, N-ethyl maleimide (NEM), Tyr-phosphorylation of band 3 can be triggered, inhibiting Tyr-phosphatase action and inducing erythrocyte membrane reorganization. We demonstrate that, in human erythrocytes, SHP-1 is present in membranes from resting cells, but in 5% of the protein amount. Interestingly, this amount increases up to threefold following NEM treatment of intact cells, whereas diamide and pervanadate do not alter the normal protein location. In addition, SHP-1 translocation from cytosol to membrane is not affected by band 3 P-Tyr level, because it is not mediated by the SH2-P-Tyr recruitment mechanism, and localizes into the cytoskeletal compartment. Band 3 is the target of SHP-1, which dephosphorylates Tyr8, 21, and 904. These findings support the idea that, in human erythrocytes, the normal level of Tyr-phosphorylation of membrane protein, mainly band 3, must be downregulated. We hypothesize that the presence of both SHP-2 and SHP-1 ensures band 3 dephosphorylation in different conditions: SHP-2, through interaction of its SH2 domain/s to P-Tyr protein, is regulated by the band 3 Tyr-phosphorylation level; SHP-1 may be involved by simple membrane rearrangement
Effector-induced Syk-mediated phosphorylation in human erythrocytes
Get PDFAbstract: Band 3 (AEI), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72(syk)) and Lyn (p53/56(lyn)). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane
High-protein goat's milk diet identified through newborn screening: clinical warning of a potentially dangerous dietetic practice
No full textObjective Breast-feeding is an unequalled way of providing optimal food for infants' healthy growth and development and the WHO recommends that infants should be exclusively breast-fed for the first 6 months of life. For mothers who are unable to breast-feed or who decide not to, infant formulas are the safest alternative. Despite recommendations, it is possible that parents make potentially harmful nutritional choices for their children because of cultural beliefs or misinformation on infant nutrition. We describe a possible health risk of not breast-feeding, highlighting a potentially dangerous dietetic practice. Design/Setting/Subjects We report the case of a newborn who was fed with undiluted goat's milk because her mother could not breast-feed and was not aware of infant formulas. Results The dietary mistake was detected because of a positive expanded newborn screening result, characterized by severe hypertyrosinaemia with high methionine and phenylalanine levels, a pattern suggestive of severe liver impairment. The pattern of plasma amino acids was related to a goat's milk diet, because of its very different composition compared with human milk and infant formula. Conclusions Our experience demonstrates that, when breast-feeding is not possible or is not exclusive, infants may be at risk of dangerous nutritional practices, including diets with very high protein content, such as a goat's milk diet. Families of not breast-fed infants may need appropriate advice on safe alternatives for infant nutrition to avoid the risks of inappropriate diets
Human sperm express cannabinoid receptor Cb1, the activation of which inhibits motility, acrosome reaction, and mitochondrial function.
No full textCannabinoids and endocannabinoids negatively influence sperm functions. These substances have been demonstrated in many mammalian tissues, including male and female reproductive tracts, and previous studies have shown the presence of functional receptors for cannabinoids in human sperm. The present study, by means of RT-PCR and Western blot techniques, demonstrates that human sperm express the CB(1), but not CB(2), cannabinoid receptor (CB-R) subtype located in the head and middle piece of the sperm. The activation of this receptor by anandamide reduces sperm motility and inhibits capacitation-induced acrosome reaction. Activation of the CB(1)-R did not induce any variation in sperm intracellular calcium concentrations, but produced a rapid plasma membrane hyperpolarization that was reduced by the K(+) channel blocker tetraethylammonium. The effects of anandamide on human sperm motility were dependent on the reduction of sperm mitochondrial activity as determined by rhodamine 123 fluorescence. The specificity of anandamide effects in human sperm were confirmed by the effects of the CB(1)-R antagonist SR141716. These findings provide additional evidence that human sperm express functional CB(1)-R, the activation of which negatively influences important sperm functions, and suggest a possible role for the cannabinoid system in the pathogenesis of some forms of male infertility
Band 3 tyr-phosphorylation in normal and glucose-6-dehydrogenase-defcient huamn erythrocytes
No full textAbstract: Haemolysis is usually episodic in glucose-6-phosphate dehydrogenase (G6PD) deficiency, often triggered by a period of oxidative stress. In the present work, we investigate a possible biochemical mechanism underlying the enhanced susceptibility of G6PD deficient red blood cells (RBC) to oxidative stress. We analysed eight male subjects with Mediterranean glucose-6P-dehydrogenase deficiency (G6PDd), class II, for their ability in phosphorylating erythrocyte membrane band 3 following oxidative and osmotic stress. Our findings show that this sensitivity is connected to an early membrane band 3 Tyr-phosphorylation in the presence of diamide. However, since both Syk, and Lyn kinases, and SHP-2 phosphatase, mostly implicated in the band 3 P-Tyr level regulation, are alike in content and activity in normal and patient erythrocytes, an alteration in the membrane organization is likely the cause of the anomalous response to the oxidant. We report, in fact, that hypertonic-induced morphological change in G6PDd erythrocyte induces a higher membrane band 3 Tyr-phosphorylation, suggesting a pre-existing membrane alteration, likely due to the chronic lowering of the redox systems in patients. We also report that 1-chloro-2,4-dinitrobenzene-pre-treatment of normal red cells can alter the normal protein-protein and protein-membrane interaction under hypertonic rather than oxidative stress, thus partially resembling the response in patients, and that RBC may utilize a wider range of redox defence, under oxidative conditions, including, but not exclusively, NADPH and glutathione. On the whole, these results would encourage a different approach to the evaluation of the effects of pharmacological administration to patients, giving more attention to the possible drug-induced membrane alteration evidenced by the abnormal band 3 Tyr-phosphorylation
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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